Epr

www.europeanpharmaceuticalreview.
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To oat or
not to oat?
Sue Staunton from James Cowper
Kreston weighs up the global IPO
opportunities for life science rms
Issue 6 2015
Screening
focus
With contributions from Angela Panarella
and Jeremy C. Simpson, UCD Cell
Screening Laboratory, University College
Dublin, and Horst Flotow from Hit
Discovery Constance
Monitoring, understanding
and assessing quality
Experts from the United States Food and Drug Administration review the major
achievements of the PAT movement in our In-Depth Focus
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INTRODUCTION
EDITORIAL BOARD
Sheraz Gul
Vice President and Head of Biology,
FraunhoferIME SP
Matthew Moran
Director, PharmaChemical Ireland
Don Clark
Pfizer Global Supply
Michael J. Miller
President, Microbiology Consultants
Jumping on the
M&A bandwagon
Michael H. Elliott
CEO, Atrium Research & Consulting
David Elder
Director Externalisation Group, GlaxoSmithKline
Andrew Teasdale
Principal Scientist Chair of Impurities
Advisory Group, AstraZeneca
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The pharmaceutical industry has a knack of bouncing back in times of adversity, and as the
challenges facing them rage on: drugs pricing pressures and loss of key patent exclusivities
to name but two, the number of mergers and acquisitions that have taken place lately have
rocketed, with companies forging relationships in a frenzy of deal-making as they seek to
maintain revenue growth. The most notable purchase recently has to be that of Irish firm
Allergan by global giant Pfizer in a deal valued at a record-breaking $160 billion. Other big
names have also been at it. For example, Teva recently announced plans to acquire Allergans
generic business, Actavis Generics; Valeant Pharmaceuticals purchased Sprout in a $1 billion
cash deal, and Swiss company Cytos Biotechnology announced that it will tie the knot
(so to speak) with orthobiologic player Kuros Biosurgery.
But will the resulting firms, especially the super companies emerging from deals such
as Pfizers, succeed in delivering increased value for shareholders, as well as more efficient
drugs for patients and payers? Some would say not, with the Pfizer deal believed to have
been a cunning tax-evasion ploy (Pfizer will move its headquarters to Ireland which has
a lower tax jurisdiction) shedding a reasonable amount of concern. Investors say that
mega-mergers can actually dampen value, the challenge of large-scale integration disrupting
the company and its R&D productivity.
On the other hand, whereas M&D activity used to be focused more on late-stage
pipelines, deals now take place earlier in the process at Phase 2 trials and sooner. It is
certainly a riskier strategy, financially, but arguably better for patients: it is logical to assume
that the effort of two companies, sharing their differing expertise to progress a less mature
candidate, is going to lead to a better outcome for that drug product, increasing its chances
of success.
Whatever your views on the latest headline-grabbing pharma news, I hope you
enjoy this issue of European Pharmaceutical Review our final edition of the year. If you
would like to get in touch, you can contact me by email on crichards@russellpublishing.com,
and to ensure you receive every issue of the magazine, please subscribe at
(www.europeanpharmaceuticalreview.com/subscribe); its free to do so. On our website, you
can also find details of current and future issues, sector news and event details.
Caroline Richards
Editor, European Pharmaceutical Review
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Published December 2015
VOLUME 20 ISSUE 6 2015
European Pharmaceutical Review
Pharma&Biotech
Endotoxin Expertise
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Contents
1
INTRODUCTION
Jumping on the M&A bandwagon
37 CONTINUOUS MANUFACTURING
Caroline Richards, Editor,

Designing a novel continuous

manufacturing plant with superior
monitoring and control
FOREWORD
Ravendra Singh, Jun Zhang, Marianthi Ierapetritou and

Rohit Ramachandran, State University of New Jersey
Use of the purge tool in

assessing mutagenic impurities
David Elder, GlaxoSmithKline and JPAG
NEWS
EVENTS
11 MICROBIOLOGY SERIES
The limitations of the colonyforming unit in microbiology

Tony Cundell, Microbiological Consulting LLC
43 IN-DEPTH FOCUS: PAT

With articles from David P. Elder, GlaxoSmithKline and
JPAG, and Huiquan Wu and colleagues from the United
States Food and Drug Administration. Kaiser Optical
Systems discusses its opinions on PAT and Raman on
page 48, and a PAT roundtable starts on page 57
60 SHOW PREVIEW
SLAS 2016
61 REGULATORY INSIGHT
To float or not to float?

15 IN-DEPTH FOCUS: SCREENING
Featuring articles from Angela Panarella and
Jeremy C. Simpson, UCD Cell Screening Laboratory
at the University College Dublin, and Horst Flotow,
Hit Discovery Constance. A Screening Roundtable
can be found on page 25
29 WEBINAR REVIEW
31 PRODUCT HUB
Chemspeed
32 PCR
MIQE compliance in expression

profiling and clinical biomarker
discovery
Irmgard Riedmaier, Melanie Spornraft, Benedikt Kirchner
and Michael W. Pfaffl, Technical University of Munich
Sue Staunton, James Cowper Kreston
64 UNDER THE MICROSCOPE

With Eurofins Lancaster Laboratories
COMING UP IN THE NEXT ISSUE:
NIR In-Depth Focus
Raman In-Depth Focus
Nanotechnology
Formulation
Published February 2016. Dont miss out on

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Innovation with Integrity
EPR
FOREWORD
Use of the purge

tool in assessing
mutagenic impurities
Dave Elder
GlaxoSmithKline and JPAG
The International Conference on Harmonization M7 text provides guidance on establishing acceptable levels of
mutagenic impurities (MIs)1. It also outlines the safety and quality risk management processes that manufacturers
need to undertake to control MIs that may potentially affect the drug substance or drug product. Over the past
decade, some of the most significant challenges facing the pharmaceutical industry have been linked to performing
genotoxic risk assessments (GRAs) and implementing a control strategy, including the analysis of these MIs and
potentially mutagenic impurities (PMIs) at very low levels (ppm) in drug substances and products.
Historically, industry has responded to regulatory concerns by
the medium and liquid/liquid extraction. Finally, chromatography is
generating significant amounts of supporting analytical data. This
allocated values of 10-100 based on the efficiency of the final separa-
approach exemplifies a quality by testing (QbT) paradigm it is very
tion. For each stage, the individual purge factors are multiplied to attain
resource-intensive (especially when applied to every PMI/MI that could
a stage purge factor. Then each stage purge factor is multiplied to
occur in a synthetic process), it can be technically challenging, and it
produce the overall purge factor2,3.
runs contrary to the underlying principles of quality by design (QbD).
Lhasa recently initiated a cross-industry collaboration (Mirabilis)
Tellingly, this QbT approach fails to acknowledge that these reactive
to further develop the purge tool to generate a semi-quantitative and
MIs will likely be purged by prevailing downstream chemistry conditions.
reproducible approach to predicting the probable purge factor for these
As a consequence, impurity fate mapping (or purging capability),
impurities at each stage within a synthetic process. One of the key
analytical testing and control strategies for MIs/PMIs in drug sub-
goals of this consortium is to make the process consistent, open and
stances and products have received significant focus during the
transparent, so that regulators can easily view and interrogate the
evolution of this guidance.
findings, increasing the chances of regulatory success and providing
One of the areas of particular interest to Industry is purging of MIs,
regulators with increased assurance of the output. The initial version of
because of the huge analytical resource burden required to support the
the Mirabilis software was released to the existing consortium
current GRA process. Significant reduction in the analytical resource
(comprising Lhasa and eight pharmaceutical companies) in late 20144.
burden can be achieved by analysing the purge factor , i.e., the ability of
3
a synthetic process to purge or remove a particular MI. The underlying
Conclusion
principles supporting the purge factor are straightforward and involve
The implementation of the purge factor and the activities of the
identifying those intrinsic physicochemical factors affecting the
Mirabilis consortium have the potential to significantly decrease
processes capability to remove a specified MI. The most important
analytical testing of MI/PMIs without any additional impact on patient
parameters are reactivity, solubility, volatility, ionisability, as well as any
safety. This approach is clearly aligned with the guidance provided in
other physical processes that remove impurities, e.g., preparative
ICH M7, i.e., as part of the control section (option 4). Additionally, there
chromatography. These parameters are then allocated a relative weight
is an emerging view that the purge tool may also be applicable to non-
(based on a pre-defined scoring system) in the overall purge factor .
mutagenic impurities, i.e., ICH Q3A5 impurities.
2,3
The chemical reactivity (R) of the MIs/PMIs is the reactivity towards

other reagents that will be encountered during the downstream
References
1.
ICH M7. 2014. Assessment and control of DNA reactive (mutagenic) impurities in
pharmaceuticals to limit potential carcinogenic risk. Step 4. June 2014
2.
Teasdale A, Elder DP, Chang S-J, Wang S, Thompson R, Benz N, Sanchez Flores, IH.
2013. Risk assessment of genotoxic impurities in new chemical entities: Strategies to
demonstrate control. Org Proc. Res. Dev. 17, 221-230
3.
Teasdale A, Elder, DP, Fenner, S. 2011. Chapter 9. Strategies for the evaluation of
Genotoxic Impurity Risk. In Genotoxic Impurities: Strategies for identification and
control, Teasdale, A. Ed. Willey, New York
4.
Lhasa Limited Mirabilis. 2015. http://www.lhasalimited.org/products/mirabilis.htm.

Accessed on 15th August 2015
5.
ICH Q3A(R2). 2006. Impurities in new drug substances. Step 4, October 2006
chemistry. The reactivity is classified as high (R=100), moderate (R=10) or

low (R=1). The solubility (S) of the specific MI/PMI is determined in the
designated process solvents and can be classified as freely (S=10),
moderately (S=3) or sparingly soluble (S=1). The volatility (V) of the
MI/PMI is defined relative to the temperature of the reaction process
and is allocated values of V=10, 3 or 1. Ionisability (I) is based on the
potential to reduce levels of MI/PMIs based on manipulating the pH of
Everett Historical / Shutterstock.com
NEWS
EMERGENT BIOSOLUTIONS
Label expansion for anthrax exposure

vaccine BioThrax
Emergent Biosolutions BioThrax (Anthrax
Vaccine Adsorbed) can now be used to prevent
anthrax following suspected or confirmed
exposure to Bacillus anthracis in the United
States market, following approval for this
additional indication by the US Food and Drug
Administration (FDA).
BioThrax was initially approved by the
FDA in 1970 for the prevention of anthrax
disease in those at high risk of exposure.
The vaccines additional indication means it
can be used in people 18 until 65 years of
age in conjunction with recommended antibiotic treatment.
BioThrax is the first vaccine to receive

approval based on the Animal Rule. The
Animal Rule allows animal efficacy data
to be used as a basis for approval when
human efficacy studies are not ethical or feasible. Protective antibody levels, which were
determined in rabbit and monkey studies,
were used to predict efficacy in humans based
on an assessment of the extent of antibody
response achieved in human study participants.
A 70% probability of survival in animal models
from inhalational anthrax disease was deemed
a reasonable level of protection and likely to
provide reasonable benefit in humans.
CHEMSPEED TECHNOLOGIES
SWILE automated workstation: step change

in solid compound management and more!
The compound library is at the heart of every
pharmaceutical company. Built from years of
synthetic work, most potential blockbusters
start and are stored here, since it is the source of
all screening activities. These compounds
are extremely precious: often difficult to
synthesise or extract, and sometimes the
worlds entire resource is in that little vial.
Compound management groups from
pharmaceutical companies typically handle
more than tens to hundreds of thousands of
compounds in their libraries, and have to
deliver small fractions (in milligram scale) for
biological tests. The conventional way to
automate these one-to-one dispenses with an
extruder has hit its limit. It requires higher
amounts of flowable crystalline material, is
prone to cross-contamination/ compound loss,
and simply does not meet the required

accuracy. More and more compounds are
unavailable in the required amounts, are too
sticky or oily, and the required manual
dispensing creates a bottle-neck.
Chemspeed Technologies launched a
paradigm shift in solid compound management
(SMILE WITH SWILE) an efficient and
widely applicable automated solution for true
one-to-one solid handling down to the sub-mg
scale. The use of economical, disposable
positive displacement tips enables the user to
handle an unparalleled variety of physical
consistencies, eliminating any cross con tamination at the same time. The technology
plugs into the overall workflow and handles
standard source vials, and virtually any
recipient, including Matrix TM tubes.
NICE
Cancer drugs fund

gets a revamp
Plans for a reformed cancer drugs fund have
been unveiled by the National Institute for
Health and Care Excellence (NICE), with the
aim of enabling patients access to the most
promising cancer drugs while NICE collects
more data on their cost-effectiveness.
The original cancer drugs fund, established
in 2011, has helped more than 72,000 cancer
patients in England access drugs not routinely
funded, but it is now widely acknowledged that a
new system is needed.
The new model will mean that cancer drugs
in line to receive marketing authorisation will be
evaluated by NICE as part of its appraisal
process, before NICE decides whether to
recommend the drug for NHS use, reject it,
or recommend it for including in the cancer
drugs fund. For drugs that seem clinically
promising, but which lack evidence to support
the go-ahead from NICE, the body will collect
evidence over two years before pulling
them from the fund and either recommending
their use in the NHS or advising against
them. The proposals can be consulted until
11th February 2016.
MERCK KGaA
Merck receives
disappointment
with evofosfamide
failure
Merck KGaA and its partner Threshold
Pharmaceuticals have announced that they
are not planning to file for approval of
evofosfamide in advanced soft tissue
sarcoma and advanced pancreatic adenocarcinoma after it failed to offer life
extensions in two Phase III studies.
The Phase 3 studies were being
conducted under Thresholds collaboration with Merck KGaA. In the Phase 3
MAESTRO study, patients with previously
untreated, locally advanced unresectable or
metastatic pancreatic adenocarcinoma
treated with evofosfamide in combination
with gemcitabine did not demonstrate a
statistically significant improvement in
overall survival (OS) compared with
gemcitabine plus placebo.
In the Phase 3 TH-CR-406/SARC021
trial, patients with locally advanced unresectable or metastatic soft tissue sarcoma
treated with evofosfamide in combination
with doxorubicin did not demonstrate a statistically significant improvement in OS
compared with doxorubicin alone.
Merck KGaA has said it will now be
redeploying its resources into high-profile
future products, such as avelumab.
Catch up with daily news at www.europeanpharmaceuticalreview.com
TAKARA BIO EUROPE
Takara Bio Europe

AB launches the
Cellartis iPS Cell
to Hepatocyte
Differentiation
System
Takara Bio Europe AB, a wholly-owned
subsidiary of Takara Bio Inc., recently
announced that it is launching the Cellartis
iPS Cell to Hepatocyte Differentiation
System and related ancillary products.
This system provides a complete solution for
directed differentiation of disease-specific or
patient-specific human inducible pluripotent
stem (iPS) cells to hepatocytes by combining
a standardised protocol and optimised, readyto-use media and coating reagents.
Takara Bio Europe AB has been offering
human iPS cell-derived hepatocytes
Cellartis Enhanced hiPS-HEP cells since
2010. These cells are a highly homogeneous
population of hepatocytes derived from
human iPS cells. But now, for the first time,
Takara Bio Europe ABs proven hepatocyte
differentiation protocol will be available
in a do-it-yourself kit format, enabling
researchers to differentiate their own human
iPS cells into definitive endoderms and
further into hepatocytes using a highly robust
and reproducible workflow. The hepatocyte
differentiation workflow was developed by
Takara Bio Europe AB scientists following
extensive internal testing and validation on
more than 25 lines, including three
commercially-available human iPS cellderived hepatocyte products prepared from
different donors.
This novel differentiation system is the
first commercially-available product to
support the complete differentiation workflow from human iPS cells to definitive
endoderm and further to hepatocytes, said
Kristina Runeberg, Site Head and Senior
Director, Business Development, at Takara
Bio Europe AB. Were excited to expand
our product offering with a complete
hepatocyte differentiation system that can
allow the creation of large panels of human
iPSC-derived hepatocytes with specific
genotypes/phenotypes, providing an ideal
solution for disease modelling, in vitro drug
discovery, drug metabolism and toxicologyrelated studies.
For more information about Cellartis iPS
Cell to Hepatocytes Differentiation System,
please visit www.clontech.com/hepatocytes.
Milena Ugrinova / Shutterstock.com
NEWS
BIOETHICS INTERNATIONAL
Study finds big pharma inconsistent with

disclosure of clinical trial data
A third of clinical trial results reviewed by the
United States Food and Drug Administration
(FDA) in 2012 had not been publically disclosed
by large pharmaceutical companies, according
to a new report from Bioethics International.
Almost half of all reviewed drugs had at
least one undisclosed Phase II or III trial. In
addition, the investigators found that only
57% of trials per drug were properly registered;
only 20% of final results were reported on
ClinicalTrials.gov; just 56% were published in
academic journals; and, only 65% were published or had their results reported in some
meaningful way.
Although the lack of publicly-available
clinical trial information has been widely

acknowledged as a decades-long problem, the
researchers believe this is the first report
that ranks specific drugs based on their
sponsors legally-required disclosure of clinical
trial information and their ethical obligation
to share information. The study also suggests
that US law is both narrow and unenforced in
this area. Information was gathered from a
variety of publicly-available documents,
including Drugs@FDA, a publicly accessible database containing records of drug
approvals and medical and scientific reviews
of approved drugs; ClinicalTrials.gov; and
journals indexed in Medline.
WORLD HEALTH ORGANIZATION
Over half of people globally believe

antibiotics can treat colds
That antibiotics can effectively treat colds and
flu is a misconception held by 64% of the global
population, a multi-country survey released
by the World Health Organization (WHO),
has found.
Almost two thirds of some 10,000 people
who were surveyed across 12 countries say they
know antibiotic resistance is an issue that could
affect them and their families, but how it affects
them and what they can do to address it are
clearly not well understood.
Close to one third of people surveyed
believe they should stop taking antibiotics
when they feel better, rather than completing
the prescribed course of treatment. Three
quarters think that antibiotic resistance happens

when the body (rather than bacteria) becomes
resistant to antibiotics. Nearly half of people
surveyed think antibiotic resistance is only a
problem for people who take antibiotics
regularly. More than half of respondents feel
there is not much they can do to stop antibiotic
resistance, while nearly two thirds believe
medical experts will solve the problem before it
becomes too serious.
The survey findings coincide with the launch
of a new WHO campaign Antibiotics: Handle
with care a global initiative to improve
understanding of the problem and change the
way antibiotics are used.
Julia Sudnitskaya / Shutterstock.com
NEWS
JOHN HOPKINS MEDICINE
Researchers say orphan drug loophole

needs to be addressed
Researchers at Johns Hopkins Medicine are
calling on lawmakers and regulators to close
loopholes in the Orphan Drug Act which, they
claim, give drug companies millions of dollars
in unintended and misplaced subsidies and
tax breaks.
In a commentary that appears in the
American Journal of Clinical Oncology,
the authors argue that pharmaceutical
companies are exploiting gaps in the law by
claiming orphan status a designation meant
to encourage the development of drugs for rare
diseases that affect fewer than 200,000 people
in the United States. Yet many of these drugs,
the authors say, end up being marketed for
other, more common conditions, generating
billions in profits.
Under the terms of the act, companies can
receive federal taxpayer subsidies of up to half
a million dollars a year for up to four years per

drug, large tax credits and waivers of marketing
application fees that can cost more than
$2 million. In addition, the US Food and
Drug Administration (FDA) can grant
companies seven years of marketing exclusivity for an orphan drug to ensure they recoup
the costs of research and development. The
authors believe companies exploit the law by
initially listing only a single indication for a
drugs use one narrow enough to qualify for
orphan disease benefits. After FDA approval,
however, some such drugs are marketed and
used off label far more broadly, thus turning
large profits.
One solution posed is that once a drug
exceeds the basic tenets of the act to treat fewer
than 200,000 people it should no longer receive
government support or marketing exclusivity.
ROCHE
Cotellic combination with Zelboraf

approved in EU
The European Commission has approved Roches Cotellic (cobimetinib) for use in combination
with Zelboraf (vemurafenib) for the treatment of adult patients with unresectable or metastatic
melanoma with a BRAF V600 mutation.
The EU approval is based on data from the Phase III coBRIM study, which showed that people
with previously untreated BRAF V600 mutation-positive advanced melanoma who were
being treated with the MEK inhibitor Cotellic in combination with Zelboraf lived a median of
12.3 months without their disease worsening, compared with 7.2 months with Zelborage alone.
Also in the coBRIM study, the objective response rate with the combination was 70%, compared
with 50% in the Zelboraf arm.
Additional data presented at this years Society for Melanoma Research congress
demonstrated that the combination of Cotellic plus Zelboraf met its secondary endpoint of
improving overall survival compared to Zelboraf alone. Roche plans to submit these data to the
European Medicines Agency for consideration and inclusion on the label.
In the United States, the combination is approved for the treatment of people with BRAF
V600E or V600K mutation-positive advanced melanoma. Further country approvals are
anticipated in 2016.
PFIZER
Mega-merger
between Pfizer and
Allergan costs $160bn
Pfizer is purchasing Allergan for a whopping $160 billion, in what is being heralded as
the most expensive pharmaceutical deal ever
to be made.
Under the terms of the transaction, the
businesses of Pfizer and Allergan will be
combined under Allergan plc, which will
be renamed Pfizer plc. The companies
expect that shares of the combined company
will be listed on the New York Stock
Exchange and trade under the PFE ticker.
However, the move will also see Pfizer
relocating its headquarters from the US to
Ireland, and thus it will benefit from a lower
corporate tax rate such an inversion deal is
increasingly attracting criticism in the US
and could have a knock-on effect on tax
inversion rules, leading to them being
tightened even further, with new legislation
brought in to prevent this practice.
Pfizer says the combined company will
benefit from a broader innovative portfolio of
leading medicines in key categories and a
platform for sustainable growth with
diversified payer groups. The combined
pipeline will contain over 100 mid-to-late
stage pipeline programmes.
VERTEX PHARMACEUTICALS
Orkambi approved
as first drug to treat
underlying cause
of cystic fibrosis
Vertex Pharmaceuticals cystic fibrosis treatment
Orkambi (lumacaftor/ivacaftor) has been
granted approval by the European Commission,
making it the first medicine to treat the underlying cause of cystic fibrosis.
Orkambi is authorised for use in patients
aged 12 and older who have two copies of the
F508del mutation, for which is was approved in
the United States in July 2015, however the
company is also currently conducting Phase III
trials in children aged six to 11 years.
The EU approval is based on previously
announced data from two 24-week global Phase
3 studies, TRAFFIC and TRANSPORT, and
additional interim 24-week data from the
subsequent extension study, PROGRESS. In the
TRAFFIC and TRANSPORT studies, which
enrolled more than 1,100 patients, those treated
with the combination Orkambi experienced
significant improvements in lung function.
Patients also experienced improvements in body
mass index (BMI) and reductions in pulmonary
exacerbations (acute lung infections) including
those requiring hospitalisations and intravenous
antibiotic use. Interim data from PROGRESS
showed that these improvements were sustained
through 48 total weeks of treatment.
PHARMA EVENTS
JANUARY 2016
FEBRUARY 2016
Festival of Genomics
11th Annual
Biomarkers Congress
Date: 19 21 January
Location: London, UK
e: eleanor@frontlinegenomics.com
w: www.festivalofgenomicslondon.com
PepTalk: The Protein

Information Week
Date: 18 22 January
Location: San Diego, CA, USA
e: nproulx@healthtech.com
w: www.CHI-PepTalk.com
Pharmaceutical
Microbiology 2016
Date: 20 21 January
e: events@smi-online.co.uk
w: www.pharma-microbiology.com/epr
SLAS 2016
Date: 23 27 January
Location: San Diego, CA, USA
w: www.slas2016.org
IFPAC 2016:
International Forum &
Exhibition Process
Analytical Technology
Quality by Design
Date: 17 18 March
e: hplc@confereneseries.com
w: www.hplc.conferenceseries.com
Date: 25 26 February
Location: Manchester, UK
e: d.dalby@oxfordglobal.co.uk
w: www.biomarkers-congress.com
APRIL 2016
MARCH 2016
3rd Annual
Peptides Congress
Pittcon 2016:
Where Innovations
in Pharmaceutical
Science go to Play
Date: 18 19 April
e: g.alonso@oxfordglobal.co.uk
w: www.peptides-congress.com
Date: 6 10 March
Location: Atlanta, Georgia, USA
e: info@pittcon.org
w: www.pittcon.org
4th Annual Biosimilars

& Biobetters UK
Date: 18 19 April
e: s.punfield@oxfordglobal.co.uk
w:www.biosimilars-congress.com
Continued And On-Going

Process Verification
Date: 29 February 2 March
Location: Berlin, Germany
e: sabine.gross@chem-academy.com
w: www.chem-academy.com/cpv
POWTECH 2016
Date: 19 21 April
Location: Nuremberg, Germany
w: www.powtech.de
PDA Annual 2016

Date: 13 16 March
Location: San Antonio
w: www.pda.org
MAY 2016
Genetics in Forensics
Date: 10 13 May
Location: Munich, Germany
w: www.analytica.de
Analytica 2016
Date: 14 15 March
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Date: 24 27 January
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(Washington, DC), USA
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HPLC
Congress 2016
2nd Formulation
& Drug Delivery
Congress 2016
Date: 18 19 May
w: www.formulation-congress.com
JUNE 2016
5th European
Biosimilars Congress
Date: 27 29 June
Location: Valencia, Spain
e: biosimilars@conferenceseries.net
w: www.biosimilars-biologics.pharmaceutical
conferences.com/europe
SLAS High
Content Screening
Conference
Date: 27 29 June
Location: Dresden, Germany
e: psantiago@slas.org
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high-content-screening-conference.htm
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MICROBIOLOGY SERIES
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brought to you in association with Charles River Laboratories
The limitations of the colonyforming unit in microbiology

Tony Cundell
Microbiological Consulting LLC
The recent revision to USP General Informational Chapter <1223> Validation of Alternative Microbiological Methods
that became official on December 1, 2015 contained a section discussing the limitations of the colony-forming unit
(CFU) in terms of enumerating only those microorganisms that readily grow on solid microbiological media.
The section highlights its inappropriateness as a gold standard for method validation when there are many signals
available other than CFUs for the detection, enumeration and identification of microorganisms in water, air,
pharmaceutical ingredients and drug products.
The advent of ribosomal RNA gene sequence analysis thirty years ago
of the pigmented bacteria was consistent when transferred to another
has significantly expanded our awareness of microbial diversity in
potato slice, and inspired the development of solid nutrient media by
water, air, soil and the human body and reemphasised that, overall,
Robert Koch and his co-workers. To isolate the growth to single points
cultivable microorganisms represent less than 1% of the micro -
in the medium, the mycologist Julius Brefeld in 1872 added gelatin to
organisms observed by direct microscopic examination. This
liquid media to cultivate moulds from single spores.
cultivability could range from 0.1 to 0.01% in oceanic microbiota to
To obtain a pure culture Klebs used fractional culture two years
around 80% for the microbiota on the human forearm. Questions that
later, but this was selective for the microorganism most suitable for
can be asked include: what does this understanding mean to the
growth in the chosen liquid medium. Bacterial enumeration was first
pharmaceutical microbiologist responsible for testing and releasing
achieved in 1878 when the British surgeon Joseph Lister combined serial
drug products? How does this apply to the challenge of evaluating,
dilution of soured milk with inoculating media under a coverslip with a
validating and implementing alternative methods to the compendial
dispensing syringe to enumerate lactobacillus.
microbiological test methods? As a background to a general discussion
Working as a country physician, Koch inoculated barnyard mice with
of these questions amongst microbiologists, it is useful to review the
diseased tissue from farm animals that died of anthrax, reproducing
history of the development of microbiological testing methods.
the disease in these improvised laboratory animals. He cultured the

spore-forming bacterium responsible for anthrax in ox aqueous
From Pasteurs discoveries to the Petri dish
humour, and fixed and stained bacteria with methyl violet on a coverslip
The development of classical microbial methods, broth culture, plate
for microscopic examination. Later he sealed the preparation in Canada
count, serial dilution, enrichment and microbial identification over time
balsam and progressed from illuminating it with direct sunlight to using
has been described in the literature
. The method of growing
Abbe condenser and immersion oil, which gave better definition owing
bacteria in a transparent liquid nutrient medium and inoculating fresh
to it having the same refractive index as glass. In the summer of 1877,
medium from turbid medium to isolate a pure culture of the bacterium
Koch demonstrated to the preeminent German microbiologist
was commonly employed by the French pioneering microbiologist
Ferdinand Cohn, a Professor of Botany at the University of Breslau, the
Louis Pasteur, beginning in 1861. Pasteur used a medium consisting of
proof that the specific bacterium Bacillus anthracis caused a specific
100 parts water, 10 parts dextrose, one part ammonium tartrate and one
disease: anthrax. Cohn recognised the importance of the work, helped
part the ash of yeast cells to grow bacteria. However, before isolation on
publish the findings to clearly establish the scientific basis of the germ
solid media became a standard practice, it can be said that Pasteur did
theory of disease, and helped Koch find positions suitable for a scientist
not truly obtain pure cultures of microorganisms.
of his enormous talent.
1,2,3,4,5,6
The transitional step between liquid and solid culture was the work
The period that followed the appointment of Koch as the Head of
of Schroeter in 1872, who isolated pigmented bacterial colonies of the
the German Imperial Institute for Infectious Diseases was the most
now-described red Serratia marcescens and violet Chromobacterium
significant five years in the development of microbiological techniques.
violaceum on thin slices of heat-treated potato. This pioneering work
For the plate technique, Koch essentially took nutrient broth, which
suggested that different bacteria were unique species, since the colour
supported the growth of pathogens, and added gelatin to make it solid
11
MICROBIOLOGY SERIES
and isolate pure cultures as distinctive colonies on the plate. Two
widely used, but unfortunately many bacteria do not grow on the culture
additional steps were needed to give us the familiar solid agar
media commonly used, while those that grow often occur in large clumps
microbiological medium in a Petri dish. Firstly, gelatin, which had the
that do not break up when plated both of which in fact cause the plate
disadvantages of liquefying at above-ambient temperature incubation
count to be considerably below the actual number of bacteria present in
required by many pathogenic bacteria and was not inert (many bacteria
the material4. Eighty years later, in their influential review article, Staley
hydrolysed it), was replaced by the gelling agent agar, used in Asian
and Konopka11 coined the catchy term the great plate count anomaly to
cooking. Secondly, the flat, indented glass plates that supported the
describe this phenomenon that was known to microbiologists for many
solid media were replaced by flat, double-sided 25 x 100mm glass plates
generations but conveniently often discounted.
that could be sterilised by dry heat, hold the molten nutrient agar
poured into it, and be inoculated and stacked during incubation .
2
As pointed out by Brown and Gilbert in 199512, in most compendial

microbial tests, the inoculum are passaged several times on solid media
The progress that Koch and his co-workers had made in the develop-
to ensure purity, and harvested by washing the colonies off the plate
ment of microbiological techniques is apparent by the inventory of
with suitable diluents. Colony density affects colony size with reduced
equipment, including culture vessels, media, disinfectants, water
nutrient availability and the production of inhibitory substances by
baths, Bunsen burners, inoculating needles, stains, microscopes, and
individual colonies shrinking their overall dimensions. Within a colony,
sterilising apparatus that the 1883-4 German Cholera Expedition took to
oxygen and nutrient depletion occurs in the core of the colony so that
Egypt and India2.
the growth status of individual cells will differ, with the youngest cell at
In his book Uncultivated Microorganisms, Microbiologist Salva
the margins and the oldest at the centre of the colonies. Using bacterial
Epstein from Northeastern University reported that Winterburg (1898)
lawns for inoculum preparation or growing cells in liquid culture can
recognised the limitations of the CFU early on, observing that the
largely overcome this heterogeneity.
number of cells in his samples did not match the number of colonies
growing on a solid microbiological plate. This discrepancy was
A genetic revolution
quantified in 1911 by Amann who found that the non-growing numbers
The field of microbial testing progressed still further following Watson
of waterborne bacteria were around 150 times those growing as colonies
and Cricks discovery of the chemical structure of the DNA molecule in
on an agar plate. Conn8 reported that the total microscopic count of soil
1955, their Nobel Prize winning publication in Nature marking the
bacteria was at least 10 to 100 times greater than plate counts from the
beginning of the new field of molecular biology, which would
same soil samples. He believed that this discrepancy was not due to
revolutionise both biology and medicine. After their discovery, in 1977,
poor dispersion or the presence of dead cells in the samples but down
Woese and Fox13 introduced ribosomal RNA base sequence analysis as a
to the majority of cells not growing on plates.
measure of microbial phylogenetic diversity and reclassified living

organisms into three Kingdoms Bacteria, Achaea and Eucarya.
Plate limitations
Thirty years ago, rRNA analysis was first used to analyse environ-
More recent studies using fluorescent stains have shown that the total
mental samples from less complex ecosystems like hot springs, copper
microscopic counts of soil were up to 1000 times that of plate counts9.
leaching ponds and deep-sea thermal vents with fewer microorganisms.
It has been suggested that plate counts select for rapid growing
As reviewed by Amman et al14 in 1995, 5S rRNA molecules were extracted,
bacteria and do not favour slow growing bacteria but factors including
electrophoretically separated, and sequenced to provide phylogenetic
media selection, gelling agents, elevated temperature during pouring
information about the ecosystem. Today, with the introduction of the
molten agar, incubation conditions and incubation time will influence
polymerase chain reaction (PCR), the larger 16S rRNA gene fragment
the recovery on solid media. Similar patterns were observed in marine
extractions can be rapidly amplified, sequenced and analysed against
microbiology. For example, Jannasch and Jones in 1958 employed five
16S rRNA databases to identify bacterial species. In 1986, Pace and his co-
different cultures and two direct microscopic methods for enumerating
workers stated that The simple morphology of most microbes provide
bacteria in sea water. Direct microscopic methods on membrane filters
few clues for their identification; physiological traits are often
showed 10 to 10,000 times as many bacteria than cultural methods
ambiguous. The microbial ecologist is particularly impeded by these
including macro-colony counts on nutrient agar, silica gel and
constraints, since so many microorganisms resist cultivation, which is an
membrane filters and micro-colony counts on membrane filters.
essential prelude to characterisation in the laboratory15. The Woesian
10
The limitations of microbiological culture methods was described by
revolution was established.
the pioneering American microbiologists, father and son H. W. Conn and

H.J. Conn in their text book Bacteriology A Study of Microorganisms
Current perspectives
and Their Relation to Human Welfare. In it, they stated: Another
Despite the concerns of clinical microbiologists Fredericks and
common application of Kochs technique is the counting of bacteria. If in
Relman16, Kochs postulates may not be applicable to non-cultivable
the material that is mixed with gelatin or agar every microorganism is
microorganisms associated with human infection there is a paucity of
separate from every other one and grows as a colony, it is obvious that
novel human pathogens isolated only by culture-independent
the number of colonies represents the number of microorganisms in the
methods. In contrast to the situation in water and soil microbiology,
material plated. This method is commonly used in estimating the number
culture methods have proved more than adequate for clinical
of bacteria in water, milk, soil, or other materials, although to get a small
microbiology, presumably because pathogens colonise nutrient-rich
enough number of colonies on the plates to be counted, it is often
locations in the body in near pure culture and readily make the
necessary to dilute the materials. This method is so convenient that it is
transition to laboratory culture. Exceptions may be pathogens that
12
MICROBIOLOGY SERIES
enter a non-cultivable state when exposed to salt water, freshwater or
media, and may not be directly statistically related. However, I believe
a low temperature outside the human body .
that they should move directionally with CFU so as to detect adverse
17
Limitations of the microbial methods used for water analysis are
bioburden trends. Cumulative sum (CUM-SUM) charting of the
emphasised in the AWWA/APHA18. As explained in the text, in water
alternative signal will reveal whether changes are occurring over time20.
microbiology, samples are examined to determine their sanitary quality
The CUM-SUM score will increase when there is an adverse trend in
and the plate count and multiple-tube fermentation methods for the
microbial quality and decrease when there is a favourable trend
detection and enumeration of indicator organisms are intended to
in the alternative signal that will reflect the microbial population in the
determine the degree of contamination with faecal waste, using
samples taken during routine monitoring.
coliforms as the indicator. The so-called heterotrophic plate count is
It should be kept in mind that materials with an established fitness
determined by pour plate, spread plate or membrane filtration
for use using CFU will retain their quality level despite the measurement
methods with the understanding that it is only an approximation of the
of an alternate signal. The reduced time to a result is most useful with
number of viable microorganisms in the sample and is useful
in-process bioburden monitoring where manufacturing can respond to
information about the water quality, judging the efficiency of water
an adverse trend in a timely manner and sterility testing of short-lived
treatment processes and the quality of water in a distribution system.
products like cell therapies, compounded sterile preparations and
Efforts to improve the recovery of microorganisms using modified

culture methods are reported in the literature. New cultivation
radiopharmaceuticals. To take advantage of emerging technologies, we

may need to cut the ties to the CFU as a so-called gold standard.
techniques to improve recovery include altering the medium

composition, extending the incubation time, lowering the incubation
temperature, and increasing the cell density on solid media to gain an
increased diversity of colonies. In the belief that standard microbiological media does not meet the metabolic needs of so-called
syntrophic organisms, researchers have added siderphores, vitamins,
other carbon sources or essential nutrients. However, as pointed out by
Nichols et al19 in 2008, these efforts have not significantly increased the
overall microbial recovery rate.
With the adoption of alternative microbiological methods, the
development of specifications independent of the CFU may be
Dr. Tony Cundell works as a Consulting Microbiologist,

supporting the pharmaceutical industry. Prior to November
2013 he worked for Merck Research Laboratories in
Summit, New Jersey, as the Senior Principal Scientist
in early-phase drug development. Earlier in his career,
Dr. Cundell worked at a director level in quality control and
product development organisations at the New York Blood
Center, Lederle Laboratories, Wyeth Pharmaceuticals and Schering-Plough.
He is a member of the 2010-2015 USP Microbiology Committee of
Experts. In June 2009, he co-edited with Anthony Fontana a book entitled
Water Activity Applications in the Pharmaceutical Industry and contributed
two chapters to the book. Tony Cundell has a PhD in Microbiology from the
Lincoln University, New Zealand.
necessary. With a qualitative limit test such as a sterility test, or in the

absence of a specified microorganism test, a statement that
References
the product meets the requirement of the test would be sufficient.
1.
2.
For a microbial enumeration test, the measurement would be defined

in terms other than CFU, e.g., ATP bioluminescence relative light units,
number of genomic units per mL, etc. If these units are not truly
equivalent to the CFU, then new specifications based on the alternative
3.
4.
5.
signal must be established.
6.
USP <1223> perspective on CFUs and method validation
7.
8.
As alternative microbiological methods move further away from
9.
classical methods based on the CFU, the USP needs to respond to this
rapid microbiological method validation challenge. For example, the
current compendial sterility tests are unsuitable for the emerging short-
10.
11.
lived cell-derived drug products, sterile compounded preparations and

radiopharmaceuticals, since these methods are growth-based.
Signals other than CFUs that may be used for microbial enumeration and detection include:
Autofluorescent cells detected by spectrophotometric methods;
Vital stained cells detected by solid-phase and fluid fluorescent cytometry;
12.
13.
14.
15.
16.
PCR amplified nucleic acid targeted sequences;
Number or weight of genomic units;
17.
ATP levels measured by bioluminescence; and
18.
Headspace analysis.
19.
These signals may be numerically higher than CFU, since they are not
limited by the ability of microorganisms to grow in microbiological
20.
Beck RW. (2000) A chronology of microbiology in historic context. ASM Press

Block TD. (1988) Robert Koch A life in medicine and bacteriology. SpringerVerlabg, New York
Bulloch W. (1938) The history of bacteriology. Oxford University Press, New York
Conn HW and Conn HJ, (1923) Bacteriology A study of microorganisms and their
relation to human welfare. Williams & Wilkins, Baltimore pp39-54
Cundell AM. (2003) Historic perspective on methods development In Rapid
microbiological methods in the pharmaceutical industry Martin C. Easter, Editor
Interpharm/CRC pp9-18
Handelsman J. Metagenomics: Application of genomics to uncultured microorganisms.
Microbiol. Mol. Biol. Rev. 2004; 68(4): 669-685
Epstein SS. Uncultivated Microorganisms. Springer, New York
Conn HJ. (1918) The microscopic study of bacteria and fungi in soil. N.Y. Agr. Exp. Sta.
Tech. Bull. 2009; 64: 3-10
Olsen RH and LR Bakken. Viability of Soil Bacteria: Optimization of Plate-Counting
Technique and Comparison between Total Counts and Plate Counts within Different Size
Groups. Microb. Ecol. 1987;13:59-74
Jannasch HW and GE Jones. Bacterial populations in seawater as determined by different
methods of enumeration. Limnol. Ocanogr. 1959; 4: 128-139
Staley JT and A Konopka. Measurement of in situ activities of non-photosynthetic
microorganisms in aquatic and terrestrial habits. Ann. Rev. Microbiol. 1985; 39: 321-346
Brown MRW and P Gilbert (1995) Influence of the environment on the properties of
vegetative microorganisms: an overview. In Microbiological Quality Assurance A guide
towards relevance and reproducibility of inocula CRC, Boca Raton
Woese C and G Fox. Phylogenetic structure of the prokaryotic domain: the primary
kingdoms. Proc. Natl. Acad. Sci. 1977; 74: 5088-5090
Amman RI, W Ludwig and K-H Schleifer. Phylogenetic identification and in situ
detection of individual microbial cells without cultivation. Microbiol. Rev. 1995; 59
(1): 143-169
Pace NR, DA Stahl, DJ Lane and GJ Olson. The analysis of natural microbial populations
by ribosomal RNA sequences. Adv. Microbiol. Ecol. 1986; 9:1-55
Fredericks DN and DA Relman. Sequence-based identification of microbial pathogens:
a reconsideration of Kochs postulates Clin, Microbiol. Rev. 1996; 9 (1): 18-33
Roszak DB and RR Colwell. Survival strategies of bacteria in the natural environment.
Microbiol. Rev. 1987; 51(3): 365-379
AWWA/APHA Standard Methods for the Examination of Water and Wastewater,
20th Edition Section 9010
Nichols DK, Lewis J, Orjala S, Mo R, Ortenberg P, OConnor C, Zhao P, Vouros T,
Kaeberlein and SS Epstein. Short peptides induces uncultivatable microorganisms to
grow in vito. Appl. Environ. Microbiol. 2008; 74(15): 4889-4897
Bandurek GR. Cumulative sum charts for problem solving. BioPharm Intern. 2008;
21 (5): 1-5
13
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16 High-content screening
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Angela Panarella & Jeremy C. Simpson, UCD Cell
Screening Laboratory, University College Dublin
21 Phenotypic screening
using 3D tissue culture
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Horst Flotow, Hit Discovery Constance
25 Screening Roundtable
Moderated by Jeremy C. Simpson, UCD Cell
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High-content screening
accelerates discovery
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Angela Panarella and Jeremy C. Simpson
UCD Cell Screening Laboratory, University College Dublin
In the past decade, the increasing power of informatics combined with the application of automation has facilitated
big data handling and the development of large-scale approaches to address key issues in the life sciences. The
omics disciplines such as genomics, proteomics and metabolomics became established during this time, allowing
researchers to expand their level of investigation to the scale of complex multicellular organisms. In this wider
context, imaging techniques allowing qualitative spatial and temporal subcellular definition of molecules and
organelles have been combined with improved molecular and cell biology tools, automation and image analysis,
giving rise to the field of high-content screening (HCS) microscopy.
HCS represents a step forward from the low sample-throughput and
for biochemical analysis. At the core of HCS is the automated
time-consuming limitations of conventional fluorescence light
quantitative analysis of the image data, always coming from individual
microscopy methods, and at the same time provides the high-
cells in the population, thus increasing confidence and statistical
throughput power associated with biochemical methods such as
significance. HCS is now widely applied in both academia (for
proteomics and mass spectrometry. The bonus of course is that imaging
fundamental systems biology and biological pathway studies) and
techniques retain the detailed spatial (and potentially temporal)
industry (to advance drug discovery and development). Although
information found in cells; information that is lost when cells are lysed
infrastructure costs and levels of expertise required for large-scale
16

screen development and implementation
are still relatively high, the speed of data
acquisition, as well as depth and significance of
information obtained, is unparalleled.
The revealing nature of HCS

In recent years almost all fundamental cellular
processes (for example cell division, membrane
trafficking, lipid droplet formation, autophagy
and neuronal development) have been quantitatively investigated by HCS in conjugation with
protein depletion technologies, particularly
RNA interference (RNAi). An elegant early
example of HCS was carried out by researchers
at the European Molecular Biology Laboratory
(EMBL) in Heidelberg, in which a genome-wide
RNAi screen in a live-cell imaging format for the
detection of regulators of mitosis was applied1.
A human cell line engineered to express a
fluorescently-labelled chromosomally-located
Maeve Long, UCD Cell Screening Laboratory, analyses some cells in the HCS image analysis suite
protein was dispensed into chambered slides containing arrays of
classification of mitosis defect phenotypes into 16 classes. This
specific RNAi reagents targeting the 21,000 human genes.
phenotypic profiling of each individual cell, followed for more than two
The effect on the capacity of these cells to

undergo mitosis (as observed by the distribution of the fluorescent reporter protein in
the nuclear area) following the depletion of the
specific gene products was then evaluated
in a time-resolved fashion. An impressive
days, revealed key genes involved in not only
Imaging techniques retain the

detailed spatial (and potentially
temporal) information found in
cells; information that is lost when
cells are lysed
cell division, but also cell survival and motility,

effectively providing the first systematic
overview of these basic cellular processes.
Other fundamental cell biology questions
into which HCS has provided new insight is with
190,000 time-lapse movies were collected and 200 cell features
regard to how intracellular organelles communicate with one another
were quantified and used to train an algorithm for the automatic
through the processes of membrane traffic. For example, we have used

HCS to map the main regulators of constitutive
secretion in cultured human cells, through the
quantification of more than eight million cells
following RNAi-mediated protein depletion and
phenotypic analysis based on the secretion of a
well characterised secretory cargo2. The several
hundred proteins identified in this study were
then re-tested for their influence on membrane
morphology and intracellular localisation by
overexpressing them as proteins fused with
the green fluorescent protein. This allowed the
spatial mapping of the 15% of genome found
to regulate protein secretion, as well as
suggesting a previously unknown link between
the secretory pathway and the signalling from
cell surface.
Due to the high sample throughput and
small per well volumes required, HCS has also
become appealing to the pharmaceutical
industry in the context of development of novel
therapeutics. One significant challenge in drug
development is the identification of increasingly powerful small molecule drugs, but which
Dr. George Galea, UCD Cell Screening Laboratory, uses a liquid handling robot to prepare a set of plates for
an HCS assay
have minimal side effects and toxicity.

Establishing these properties is seen as
17

essential in terms of reducing the amount of animal testing and
improving the success rate of drug candidates reaching clinical
trials stages. HCS presents itself as a fundamental tool to achieve
these aims, since it provides deep and cumulative in vitro singlecell analysis for the determination of acute but also sub-acute
toxicity in various physiologically relevant cell types.
Parameters that HCS can easily and rapidly measure include
changes in nuclear morphology, calcium flux, mitochondrial and
plasma membrane permeability and potential, all of which can be
used as indicators of toxicity. Exploitation of this approach and the
information it provides is now seen as fundamental in helping
predict candidate drug failure at the earliest stages of their
development3. HCS also has roles to play beyond toxicity testing,
for example, in screening large libraries of drugs (biological or
chemical) to determine potential leads suitable for further
optimisation. At later stages of the pipeline, HCS is a fundamental
tool for multiplexing of measurements such as drug uptake,
efficacy and specificity. Again, it is important to emphasise that
high-quality HCS regimes all extract highly parallel information
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from individual cells, and this ensures that precious information is

not lost from specific subpopulations of cells.
Therapeutic approaches are constantly evolving currently
there is much emphasis on the design of improved drug carriers
utilising novel nanomaterials, and we foresee that HCS has a key
role to play here4. In addition, biomedical device-drug delivery
combination products are also emerging as a new approach to
target human disease5. The potential of such devices is truly
exciting, and HCS seems well placed as a technology to enhance
the development pipeline of these products. In such cases it can
provide critical information about how cells physically respond to
the device material, as well as the aforementioned parameters
associated with drug delivery.
The challenges ahead for HCS platforms

Having considered the applications of HCS in both fundamental
and applied life science research, it is worth reflecting on the
technology itself, its direction and challenges faced. In order to
cope with the high processivity of HCS microscopy, the samples
are usually prepared in miniaturised arrays (such as 96-, 384- or
1536-well plates) and processed in parallel by high-throughput
instruments (robotic liquid handlers, cell seeding devices and
multi-well plate washers), which allow both standardisation,
precision and speed at each step of sample processing. Use of
smaller well size formats (3456-wells) would potentially increase
throughput, however these have not proved attractive for HCS
assays, largely because of issues surrounding very low numbers of
cells to quantify from. Indeed, the vast majority of HCS studies
reported do not go beyond the use of the 384-well format.
Assay design remains a critical factor in the success of any HCS
www.perkinelmer.com/YES
campaign, and particular care needs to be given to read-out

(discussed further below) in order to give the greatest chance of
identifying leads. Choices regarding the durability and batch
consistency of materials and reagents (cells, media and buffers,
transfection reagents, fluorescent markers, RNAi reagents or small
molecule libraries), the assay format and plate type (trade-off
Copyright 2015 PerkinElmer, Inc. 400285_14 All rights reserved. PerkinElmer is a registered
trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners.

between glass-bottomed and plastic-bottomed plates in terms of
of negative and positive controls. The definition of the so-
image quality and cell friendliness), controls (strength of phenotype
called primary hits is then followed by appropriate validation
and position in the plate in order to detect artefacts), number of
screens, which are typically more focussed, at a smaller scale and use a
replicates, and assay incubation and acquisition times all need to be
higher number of replicates. Ultimately, in a large-scale screen, only
carefully determined. Such parameters, which may be overlooked when
very few of the images are actually manually inspected, hence
carrying out microscopy experiments involving a small set of samples or
the importance of accurately defining the image analysis routine
targets, become extremely relevant when planning high-throughput
in the first instance.
and automated experiments.

The imaging parameters are also worth consideration. For example,
The road so far and beyond
a typical 20x objective-based acquisition of 8-10 fields of view per well
HCS microscopy and analysis is now established as one of the
with two colours and 200ms acquisition time per channel (plus 1 second
fundamental pillars of data generation for systems biology studies,
dichroic switching time and a 1 second focusing
shedding light on how cells are organised and
time) requires fewer than 20 minutes in a 96-
controlled. It is also embedded in the drug

discovery and development pipeline, providing
well plate, however in a 384-well plate format

the acquisition time becomes more than 2
hours. Such acquisition times, which may not
be critical in fixed cell end-point analyses, do,
Assay design remains

a critical factor in the success of
any HCS campaign
high quality data to inform and accelerate

the identification of novel therapeutics.
This is an exciting time for this technology
the interest in combination medical devices
however, become relevant for live cells assays.
and personalised medicine strategies high-
As increasing numbers of HCS experiments seek

to gather temporal image data, this also presents itself as a limitation.
lights the importance of extracting as much information as possible
Of final note is the choice between wide-field and confocal HCS
from individual cells grown in vitro prior to the deployment of new
systems. Relatively simple assays using conventional 2D cell culture
therapeutics in vivo. As an imaging technology that is founded in
models largely do not require confocality, however the trend in recent
the principle that the data generated must come from individual
years has been to employ 3D cell culture models, for example, cell cysts
cells, it is surely a technology that will continue to evolve at a
or spheroids. While such models may be advantageous in terms of more
tremendous pace.
accurately mimicking the in vivo distribution of cells, they clearly

present a challenge from an imaging perspective. Confocal HCS systems
have the ability to partially optically section such cell models, however
current systems suffer from the limitation of penetration depth, and so
HCS infrastructure developers need to give major consideration to how
the next generation of systems will quantitatively image such models.
HCS is all about accurate image analysis

Arguably, the most critical step in HCS lies in the image analysis routine
deployed. The individual cells present in each image contain rich
information content that needs to be harvested in an appropriate and
quantitative manner. Typical image analysis routines follow four key
steps. Firstly, out-of-focus images are removed and images are preprocessed and enhanced to allow more precise object detection.
Next, informatic algorithms are used to identify objects of interest
such as cell boundaries, nuclei and organelles of interest, as required
for the analysis. Thirdly, the regions of interest in which assay
read-out measurements are to be made are defined. Finally, only after
the software has identified all the appropriate elements of the image
Dr. Angela Panarella has a BSc in Pharmaceutical

Biotechnology (2007) and an MSc in Medical Bio technology (2010) from Universit degli Studi Aldo Moro in
Bari (Italy) working on a project for the design and
optimisation of microfluidic devices for tissue engineering.
In 2015 she was awarded a PhD in Cell Biology from
University College Dublin (UCD) where she carried out a
project under the guidance of Prof. Jeremy Simpson and Prof. Kenneth
Dawson using high-content screening microscopy to investigate the
intracellular trafficking of nanoparticles. She is currently working in UCD as
part of the Cram Centre for Research in Medical Devices.
Prof. Jeremy Simpson carried out his PhD work at the
University of Warwick (UK), and post-doctoral work at
the Scripps Research Institute in San Diego (USA), the
ICRF in London (UK), and the European Molecular
Biology Laboratory (EMBL) in Heidelberg (Germany).
In 2008 he was appointed as Professor of Cell Biology at
University College Dublin, Ireland. His lab currently applies
high-throughput imaging technologies to study membrane trafficking
processes, and how cells interact with nanomaterials. He has authored over 80
peer-reviewed articles and a number of book chapters. He runs the UCD Cell
Screening Laboratory (www.ucd.ie/hcs), and is a principal investigator in the
Cram Centre for Research in Medical Devices (www.curamdevices.ie).
are the in-depth measurements of the parameters (morphology,

intensity, co-occurrence with other markers, distribution profiles
References
etc.) carried out.
1.
Neumann, B et al. Phenotypic profiling of the human genome by time-lapse microscopy

reveals cell division genes. Nature. 2010; 464 (7289), 721-7
2.
Simpson, JC et al. Genome-wide RNAi screening identifies human proteins

with a regulatory function in the early secretory pathway. Nature Cell Biology. 2012;
14 (7), 764-74
3.
OBrien, PJ. High-content analysis in toxicology: screening substances for human

toxicity potential, elucidating subcellular mechanisms and in vivo use as translational
safety biomarkers. Basic & Clinical Pharmacology & Toxicology. 2014; 115 (1), 4-17
4.
Brayden, DJ et al. High-content analysis for drug delivery and nanoparticle applications.
Drug Discovery Today. 2015; 20 (8), 942-57
5.
Couto, DS et al. Lessons from innovation in drug-device combination products.

Advanced Drug Delivery Reviews. 2012; 64, 69-77
The number of matrices generated from this process can be

very complex, but they can also serve as a template for machinelearning linear classifiers to then mine the image data for particular
cell phenotypes. Ultimately, the data produced allow ranking of the
phenotype strengths, and candidate treatments, conditions or
compounds can be selected based on quality control tools such as
Z-scores, strictly standardised mean differences, or arbitrary thresholds
which take into consideration the strength and the robustness
19
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Caenorhabditis elegans
Phenotypic screening
using 3D tissue culture
and whole animal assays
Horst Flotow
Hit Discovery Constance
Phenotypic, or high content, screening (HCS) is an automated microscopy that allows very precise interrogation of
cellular and even whole animal assays. The high throughput is achieved by automating the image analysis and
quantifying the abundance and subcellular distribution of a target such as a protein or organelle using fluorescent
detection reagents including antibodies and specific stains. HCS can measure a compounds effects on a specific
cellular target within individual cells and can be used to screen biologically relevant cell- and whole animal-based
assays (designed as disease models) against large compound libraries. In addition, the technique can multitask
(multiplex) and measure several targets at once, further increasing the information content and utility of these
screens. As a result, a more informed selection of hit compounds that have improved chances of progressing
through the drug discovery pipeline can be made during primary screening.
Phenotypic screening
to target-based high-throughput screening (HTS) assays. The more
Phenotypic assays are actually nothing new; in fact, early drug discovery
laborious and lower throughput phenotypic assays were subsequently
was mostly carried out mostly using simple phenotypic assays such as
employed as secondary assays. However, the increase in the use of
whole cell antibacterial, antifungal and growth-inhibition assays, as well
target-based screens has not been reflected in more productive drug
as whole animal experiments. With the advent of combinatorial
discovery efforts. In the mid 1990s, the first automated microscopes
chemistry and large in-house compound libraries, the limited
were introduced, offering a chance to implement ever more
throughput of these phenotypic assays often meant a shift in emphasis
sophisticated phenotypic assays at vastly improved throughput. HCS,
21
Kazakov Maksim / Shutterstock.com
The embryos of adult zebrafish make the ideal HCS model
as it is now known, offers a chance to use many of these cellular
establish and maintain and have a low throughput, some recent efforts
secondary assays as primary screening assays, thus leveraging on the
are addressing these shortcomings and it is now possible to run
high biological relevance of these assays.
spheroid cultures in high density arrays of up to 384 well microtitre
Identifying compounds with interesting activities without knowing
plate-like devices. Organoid culture models are another kind of 3D
the molecular target for the compounds activity makes it difficult to
model system that may find its way into HCS application. As the name
implies, organoids are mammalian 3D model
initiate medicinal chemistry programmes. Thus,

to provide some clues as to the compounds
target, HCS may be used in combination with
RNAi-mediated gene silencing. siRNA libraries
covering the human genome are now comm-
Zebrafish embryos can be kept in

96 or 384 well plates and compounds
may be administered by simply adding
them to the water in the wells
ercially available1 and can be used to measure
systems, generally derived from stem cells that

display characteristics of organs, albeit in
a smaller footprint and in vitro. Although
organoids are technically challenging, have a
low throughput and lack vascularisation and
phenotypic changes, in response to the gene silencing, which are
higher level (organism) organisation, they are well-characterised,
similar to treatments with the compounds of interest. HCS produces an
sophisticated and biologically relevant model systems with a range of
extensive amount of phenotypic information (features) that may be
potential applications in drug discovery5.
used to classify compounds and may give clues to the mode of action
or target of unknown compounds2-4.
Whole animal HCS assays
With the fast-paced improvements in HCS technology over the past
All early stages of drug discovery can benefit enormously from the use
10 years, sophisticated (complex) assays that were manual, low
of simple and inexpensive animal models that are biologically relevant
throughput assays even a couple of years ago can now be run as HTS
and may offer some additional predictive benefits for compound
campaigns. Two types of phenotypic assays that are of particular
efficacy and toxicity. HCS offers the possibility to bring in animal models
interest for drug discovery using HCS three-dimensional (3D) cell and
earlier and even use them for primary screening. The technological
tissue culture and whole animal assays are highlighted here.
advances in high content imaging instrumentation and the requisite

IT infrastructure have reached a level of sophistication where almost
3D cell and tissue culture
any standard microscopic analysis can now be carried out and analysed
Most cell-based assays, including those currently used for HCS, work
in microtitre plates. Clearly, this kind of HTS can only be carried out with
with cells grown in monolayer (two-dimensional; 2D) cultures on a
animals that are small and can be grown or kept in microtitre plates.
plastic substrate (i.e., tissue culture flasks or microtitre plates).
Zebrafish embryos and the nematode Caenorhabditis elegans are the
This contrasts with the in vivo growth of cells where there is interaction
two kinds of animal models that fit these requirements.
with neighbouring cells and extracellular matrix components. This leads
Zebrafish are a well characterised and established model system.
to the establishment of a unique 3D structure that plays an important
Although adult zebrafish cannot fit into the well of a microtitre plate,
role in normal cell and tissue formation and function. Many of these
their embryos can be kept in 96 or 384 well plates and compounds may
tissue-specific properties appear to be lost when cells are grown in
be administered by simply adding them to the water in the wells.
isolation and in 2D tissue culture conditions.
Another benefit for imaging is that the zebrafish embryos are
Although 3D cell culture models are technically challenging to
transparent, thus the effects of the added compounds can be
23

visualised easily. Although there are still some obvious technical
from imaging and HCS, this provides a powerful proof of concept of
challenges to overcome before zebrafish HCS can be considered
whole animal HTS.
mainstream, a number of groups are addressing the various issues.

In particular, the image analysis will remain a big challenge for a
Conclusions and future directions
vertebrate system like zebrafish embryos and this is leading
HTS appears to be going back to the future, with phenotypic
investigators in the direction of a simpler system, the worm
screening (HCS) becoming widely used for primary screening
(roundworm), Caenorhabditis elegans. This tiny, 1mm-long worm is a
campaigns. Although the majority of HCS campaigns are using standard
genetically well-characterised model system for biological research
fluorescent readouts (albeit increasing on confocal microscopes) and

cell lines grown in standard 2D tissue culture,
in general toxicology in particular. The high

degree of genetic similarity with higher
eukaryotes makes C. elegans a predictive and
therefore highly relevant simple animal model.
In addition, the roundworm is cheap and easy
to maintain in the laboratory and can be grown
There is some movement

towards applying the more biologically
relevant 3D tissue culture technologies
such as spheroids and organoids
for HTS and HCS
in 96 and 384 well microtitre plates for high throughput screening6.
there is some movement towards applying the

more biologically relevant 3D tissue culture
technologies such as spheroids and organoids
for HTS and HCS.
In terms of bottlenecks, it has become
apparent that upgrading the IT infrastructure to cope with the increased
C. elegans are normally grown on solid media with Escherichia coli
volumes of data can be effectively managed by investments in hardware
as a food source. However, the nematodes are killed when grown in
(and software), thus removing this potential obstacle. Instead, the main
liquid media in the presence of a number of human pathogenic bacteria
bottleneck for the effective adoption and use of 3D tissue culture and
and fungi, such as Enterococcus faecalis and Candida albicans. Since
whole animal assays in HTS (HCS) will most likely be the development of
this lethality can be rescued by antibiotics, C. elegans is a very sensitive
novel imaging equipment (for example holographic imaging) and the
and convenient model system for the screening of compound libraries
software, such as tomography, needed to analyse 3D images within a
in antibiotic and antifungal drug discovery . C. elegans grown in the
reasonable time frame.
7,8
presence of Burkholderia thailandensis are rapidly overgrown and killed

by the bacteria.
Based on this simple surrogate animal model for the disease
Horst Flotow is the CEO of the newly formed

Hit Discovery Constance (HDC GmbH), located in
Konstanz in Germany.
Melioidosis, a high-throughput-capable assay in a 384 well microtitre

plate format was developed. Using this assay in an HTS campaign
against a library of 300,000 compounds, those compounds that
might have some clinical applications in the treatment of Melioidosis
were discovered. Additional secondary assays against clinical
isolates of Melioidosis causative organism, Burkholderia pseudomallei,
References
provided a proof of concept of the utility of this kind of whole animal
1.
Kurreck J. RNA Interference: From Basic Research to Therapeutic Applications. Angew

Chem Int Ed. 2009 Feb 9;48(8):137898
HTS approach9.
2.
Lei Z, Tan IB, Das K, Deng N, Zouridis H, Pattison S, et al. Identification of Molecular
Subtypes of Gastric Cancer With Different Responses to PI3-Kinase Inhibitors and 5Fluorouracil. YGAST. Elsevier, Inc; 2013 Sep 1;145(3):55465
3.
Adams C, Kutsyy V, Coleman D, Cong G, Cromton A, Elias K, et al. Compound

Classification Using Image-Based Cellular Phenotypes. Methods in Enzymology.
2006;414:44068
4.
Zhou J, Yong W-P, Yap CS, Vijayaraghavan A, Sinha RA, Singh BK, et al. An integrative
approach identified genes associated with drug response in gastric cancer.
Carcinogenesis. 2015 Apr;36(4):44151
5.
Lancaster MA, Knoblich JA. Organogenesis in a dish: Modeling development and

disease using organoid technologies. Science. 2014 Jul 17;345(6194):12471255
6.
Leung MCK, Williams PL, Benedetto A, Au C, Helmcke KJ, Aschner M, et al.

Caenorhabditis elegans: An Emerging Model in Biomedical and Environmental
Toxicology. Toxicological Sciences. 2008 Aug 18;106(1):528
7.
Giacomotto J, Sgalat L. High-throughput screening and small animal models, where are
we? British Journal of Pharmacology. 2010 Mar 4;160(2):20416
8.
Ewbank JJ, Zugasti O. C. elegans: model host and tool for antimicrobial drug discovery.
Disease Models & Mechanisms. 2011 May 9;4(3):3004
9.
Lakshmanan U, Yap A, Fulwood J, Yichun L, Hoon S, Lim J, et al. Establishment of a

Novel Whole Animal HTS Technology Platform for Melioidosis Drug Discovery. Comb
Chem High Throughput Screen. 2014 Nov 7;17(9):790803
An imaged-based (HCS) approach has also been applied to the

use of C. elegans in anti-microbial drug discovery. A library of
2,200 Pseudomonas aeruginosa mutants was screened for bacterial
genes that attenuate the virulence of the bacteria and enhance the
nematode survival. This revealed a role for several proteins potentially
involved in P. aeruginosa pathogenicity and provided some potential
new targets for antibiotic drug discovery10.
The potential of using C. elegans for the discovery of novel
antibiotics is highlighted in another small-scale screen, using
E. faecalis-infected C. elegans plated out in 384 well microtitre plates.
The automated image analysis makes use of the fact that dead
nematodes take up Sytox Orange and are straight whereas live worms
do not take up Sytox Orange, are S-shaped and move in the well.
The screen revealed several classes of compounds that enhanced the
survival of the nematodes without harming them11. More recently,
a similar HTS-capable microtitre-plate based assay was developed to
screen a small number of peptides. This HCS-capable assay also
included an image-based analysis of the nematode survival12.
Thus, C. elegans offers a simple whole animal screening system
of some utility in drug discovery (target finding and discovery of
novel antibiotics). With the throughput afforded by this simple
screening system and the additional information that can be obtained
24
10. Garvis S, Munder A, Ball G, de Bentzmann S, Wiehlmann L, Ewbank JJ, et al.

Caenorhabditis elegans Semi-Automated Liquid Screen Reveals a Specialized Role for
the Chemotaxis Gene cheB2 in Pseudomonas aeruginosa Virulence Ausubel FM, editor.
PLoS Pathog. 2009 Aug 7;5(8):e1000540
11. Moy TI, Conery AL, Larkins-Ford J, Wu G, Mazitschek R, Casadei G, et al. HighThroughput Screen for Novel Antimicrobials using a Whole Animal Infection Model.
ACS Chem Biol. 2009 Jul 17;4(7):52733
12. Jayamani E, Rajamuthiah R, Larkins-Ford J, Fuchs BB, Conery AL, Vilcinskas A, et al.
Insect-Derived Cecropins Display Activity against Acinetobacter baumannii in a WholeAnimal High-Throughput Caenorhabditis elegans Model. Antimicrobial Agents and
Chemotherapy. 2015 Feb 11;59(3):172837
pedrosala / Shutterstock.com
IN-DEPTH FOCUS: SCREENING ROUNDTABLE
Guillaume Frugier
Jacob Tesdorpf
Joseph M. Zock
Peter Banks
Field Applications Scientist

at Molecular Devices
Director of High Content Imaging

Instruments and Applications,
PerkinElmer
Senior Director,
Product Management,
IntelliCyt Corporation
Scientific Director,
BioTek Instruments Inc
Moderator
Jeremy C. Simpson, UCD Cell Screening Laboratory, University College Dublin
What do you see as being the next exciting technological

developments in the area of screening?
and high speed while simplifying the workflows that involve

complex samples.
Frugier: Increased demand for biologically relevant screening in order

to discover efficacious non-toxic compounds at an earlier stage has
Zock: Utilising the immune system to attack cancer is one of the
driven a move to high-content assays. While assays for plate readers are
hottest areas of research, due in part to the recent clinical
optimised to isolate a particular pathway, high-content screening (HCS)
success reported. Unfortunately, the gold standard tool for measur-
enables visualisation of specific, toxic, and off-target effects in the
ing immune cells, flow cytometry, is a relatively poor screening
same screen or workflow. The growing complexity in screening has
tool due to a lack of throughput, need for expert adjustments,
generated higher demand for high-content, high-throughput screening
large sample/reagent requirements, and lack of plate level analytics.
(HTS) and improvements in acquisition and analysis.
IntelliCyt has overcome these limitations by developing an auto-
To support this demand, we are developing high-content
mated screening platform, using flow cytometry as the detection
technologies for 2D/3D live sample acquisition at high resolution
engine. It offers fast plate processing of small sample volumes,
25

with easy to use assay set up and plate level analysis. True screening
cost per screen, so finding ways to afford those systems while
for immunology!
maintaining the throughput is important. This can be achieved by

automating complex workflows and by further miniaturisation of
Tesdorpf: One of the most exciting advances in screening is the move
cellular assays. Another bottleneck can be in-depth image analysis
towards true multiparametric phenotypic screening of complex cellular
of large screens. Here, we invest heavily into our Columbus platform

to enable, for example, detailed phenotypic
models. Not only does this hold the promise of

higher predictivity, it also provides novel ways
to understand mode of action, as recently
demonstrated by Reisen et al from Novartis
Institutes of BioMedical Research. This paper
The principal bottleneck in primary

screening is the use of more
physiologically relevant models of
disease to screen against
and others show how leveraging a priori
Peter Banks
fingerprinting across large compound libraries.

Banks: The principal bottleneck in primary
screening is the use of more physiologically
relevant models of disease to screen against.
We have transitioned from the use of common
biological knowledge, in-depth image analysis,

and machine learning can provide new insights into compound mode of
immortalised host cells over-expressing putative drug targets to the use
actions. Sequencing technologies such as RNAseq hold additional
of endogenous expression of drug targets in human primary cells or
promise for detailed understanding of cellular response at later stages
even more complex cell models comprised of layered biology using
of the drug discovery workflow.
in vitro methods (3D cell culture) or biopsied tissue. Cost, supply and
difficulty of use limit their potential. Stem cells have the potential to
Banks: The use of the CRISPR-Cas9 system for genome-wide knock-out
alleviate supply issues and in certain cases, such as cardiomyocytes,
screens for identifying potential drug targets is extremely exciting.
have been used in campaigns.
The precision, ease of use and relative freedom of this gene editing tool
from off-target effects will ensure widespread adoption in genome
Frugier:
screening. In HTS, the use of kinetic ligand binding also deserves a nod.
1.
Model choice: Physiological relevance is crucial for meaningful data
Drug residence time, or the reciprocal of its off-rate, is beginning to be
and successful translational research. Selecting and optimising the
understood as an equally important (sometimes more important)
model, choosing the parameters to focus on, and acquiring and
measure of drug potency as compared with equilibrium-based IC50s.
analysing models with systems like the ImageXpress Micro Confocal

imaging system are all important elements needed to simplify
What are the major bottlenecks in screening

for drug discovery?
discovery.
2.
Tesdorpf: Coming off a record year in United States Food and Drug
Administration approvals, the challenge is to further grow productivity
at stagnant budgets. More complex cellular models often equal higher
Data complexity: Acquiring images and generating numerical data is

faster and simpler with HCS systems. However, extracting meaning
from results still remains a challenge to users.
3.
Data management: Terabytes of raw image data places a burden on

users networks and storage capabilities. It is
important to consider proper investment in
information technology.
Zock: Many bottlenecks exist in the drug discovery process, from finding drug-able targets, to
dealing with large data sets, to identifying/fine
tuning a lead with the right activity profile to
move into the clinic. Many of these bottlenecks
suffer from a lack of technologies that can
provide the right amount of actionable data, at
the right level of pharmacological relevance, with
enough capacity, at the right time in the process
to make a decision. High content technologies,
like the iQue Screener, hold the promise to
address this problem for targets in immunooncology, immunotherapy, and immune-toxicity
Andrej Vodolazhskyi / Shutterstock.com
profiling in all therapy areas.
26
What are the challenges associated

with screening using more complex
cell models such as spheroids?
Frugier: Challenges start at the sample
preparation stage with the selection of the
science photo / Shutterstock.com
physiologically relevant model, the microplate, an extracellular matrix
each screen that is why the Opera Phenix High Content Screening
or gel, and the staining protocol needed to generate appropriate dye
System provides fast and gentle live cell imaging for maximum sensitivity
penetration. Historically, using live cells in situations where 3D
with simultaneous multi-channel acquisition to maintain throughput.
acquisition was required forced researchers to compromise between
In many cases, more complex models will also generate significantly
resolution and phototoxicity during z-sectioning. Modern confocal
larger data volumes, especially when working with live cells or 3D models.
systems are more sensitive and have advanced illumination techniques
Careful assay optimisation can help to establish the minimal number of
allowing them to screen high resolution images at the speed of
time-points or z-planes required for the desired assays window.
widefield . Time lapse or 3D data also increases complexity of image

1
Zock: Increasing the physiological relevance of cell models will yield
What is your current perspective on collaboration

between the academic and industrial communities
in screening?
better data to drive drug discovery. The two major challenges I see are
Zock: Academic screening centers, using the breadth of commercially-
a) creating reproducible models with robust responses to screen
available screening technology, continue to increase. However the
analysis and interpretation.
against, and b) how to get the tags to the

biological targets so they can be measured. A
lot of effort is on-going to develop models from
spheroid creation to iPSC of disease states.
However, reproducibly staining targets inside
chasm between screening for knowledge and
When establishing a screen,

greater predictivity of more complex
models must be balanced against
higher cost and effort
these structures to get at their secrets remains

difficult and content limiting. I believe diss-
Jacob Tesdorpf
screening for revenue remains quite large.

Questions of ownership, patent protection, and
profit distribution often make collaboration in
screening difficult. Several pharmaceutical
companies, including my old alma mater Eli
Lilly, have created processes (OIDD) to attract
ociation, staining, then measuring by suspension screening may
new targets and compounds from academia promoting collaboration.
provide a route to success in some situations.
We have also seen a large amount institutional collaboration in the

burgeoning area of immunotherapy where many potential research
Banks: The principal challenges with using more complex cell models in
tracks and combinatorial approaches are being explored to harness the
screening are cost, supply, and difficulty of use. For spheroids, the two
immune system to eradicate cancers.
methods for their production are using hanging drop technology or

round bottom microplates with coatings that reduce cell adherence to
Frugier: The assumption that screening technologies are mainly
the point where cells self-aggregate. The former technology typically
adopted by industry is more removed from the current paradigm, since
requires transfer of the produced spheroid to another microplate to
an increasing number of academic institutions are investing in high-
conduct a screening assay, which can be alleviated through the use of
content technologies. The withdrawal of pharmaceutical companies
the latter technology. Each is somewhat limited in the type of cell used
from particular research areas have paved the way for academia to step
for aggregation, although conditions can be manipulated to encourage
in and fill the gap, or enabled collaborations between these two
spheroid formation.
communities. These ventures are extremely positive for drug discovery

and are driving expansion of assays that can be run on high-content
Tesdorpf: When establishing a screen, greater predictivity of more
systems, such as whole organism imaging, antibody and protein
complex models must be balanced against higher cost and effort.
internalisation, infectious agent (virus and bacteria) internalisation,
The high sample cost mean it is important to maximise the data from
and 3D spheroid assays.
27

Tesdorpf: The current trend is very promising
since it provides the academic scientist with
access to large compound libraries and stateof-the-art screening infrastructure, while drug
discovery companies benefit from access to
new targets, model systems and analysis
strategies. In addition, the knowledge transfer
strengthens academic drug discovery efforts for
neglected diseases. PerkinElmer tries to
support such approaches, for example by
ensuring that both the Opera Phenix HCS
system and Operetta system are run by the
same Harmony software for image acquisition
and analysis, making it easy to transfer assays
between labs.
infrastructure required for practical screening

campaigns. This includes the automated
instrumentation and the content to be
screened. Yet academia does have the patience
and depth of knowledge to develop appropriate
Alex_Traksel / Shutterstock.com
Banks: Academia typically does not have the
disease models that can be utilised in

screening programmes. This includes identifying and validating putative
Content Screening: Science, Techniques, and Applications edited by
drug targets and developing complex cell models that can effectively
Steven Haney.
mimic the disease state in vitro. This type of collaboration forms the
Tesdorpf: Whole well read-outs provide fast and robust read-outs of the
basis of most academia/industry relationships.
effects under investigation. They tend to be cost effective and produce
What do you see as the relative advantages and

disadvantages of whole well-based readouts versus highcontent individual cell-based readouts?
minimal amounts of data. This allows for very high throughput screens
Banks: Whole well-based readouts provide cell population-averaged
amounts of data, but generate a more complete picture of the cellular
data. Assay performance can be improved by the use of more cells,
response down to the organelle level and allow discrimination between,
although there is a cost downside to this. Also, there are many off the
for example, responding and non-responding subpopulations or
shelf detection technologies that can be used and screened rapidly.
between different modes of action. The PerkinElmer EnSight
with streamlined experimental and analytical workflows. In contrast,

high content readouts take considerably longer and produce large
High content individual cell-based readouts

are typically performed by microscopy and
allows for cell sub-population analysis. Cell
segmentation algorithms from associated
image analysis software also provide spatial
Multimode Plate Reader combines typical well-
The advantages of whole

well are isolation of a particular
biological readout, speed, and
simplicity of analysis
information important for phenotypic assays,
Guillaume Frugier
such as drug target translocation. Microscopy is
based readouts with orthogonal fast imaging to

enable normalisation of cell populations.
Frugier: The advantages of whole well are
isolation of a particular biological readout,
speed, and simplicity of analysis. However,
also required for live cell, kinetic spheroid-based assays to obtain
whole well assays limit the ability to analyse the complexity and
suitable signal to background ratios.
multi-parametric nature of biological processes. New sensitive, large

field-of-view high-content widefield and confocal systems capture
Zock: Although whole well-based readouts from various plate
large cell populations at higher magnifications. Increasing flexibility of
readers offer throughput, simplicity of assay creation/analysis, and
HCS instruments, such as our ImageXpress Micro Confocal, allow whole
almost universal access, they suffer from limitations in providing
well and cellular resolutions to be easily run on an all-in-one machine.
cellular context and/or multiplexing endpoints per cell and well.
High-content throughput is now capable of running >200,000 wells/day;
High-content technologies, like IntelliCyts iQue Screener, allow a
we have driven approximately a 50-fold increase in the last 15+ years
deeper interrogation of each well by isolating cellular subpopula-
from 4000 wells/day.
tions, accessing multiple cell health/functional endpoints per

cell, and correlating the interplay when exposed to test compounds,
proteins, cellular constructs, or environments. A more detailed
answer from me can be found in chapter one of the book High
28
Reference
1.
Sirenko et al., Assay and Drug Development Technologies, 13: 402, 2015
WEBINAR REVIEW GE HEALTHCARE
Development and applications of

biosensor assays for fragment
screening of wild-type GPCRs
European Pharmaceutical Review is proud to present this GE Healthcare-sponsored webinar, which was broadcast
live on the 5th November 2015. The topic was the use of G-protein coupled receptors (GPCRs) as drug targets, and
the webinar included details of how assays are being improved for fragment screening of wild-type GPCRs.
Our keynote speakers were Dr. Iva Navratilova,
Screen campaigns involving >2700 fragments from
Chief Scientific Officer at Kinetic Discovery,
the Maybridge Ro3 library. By screening for
University of Dundee, and Dr. Tim Fagge, Applica-
undesirable binding behaviour to the sensor
tion Specialist at GE Healthcare.
surface matrix, a library of fragments that is better
GPCRs are the primary target class of currently

marketed drugs, accounting for around a third of
Dr. Iva Navratilova
prepared for FBDD campaigns using Biacore

Systems has been established.
all drug targets of approved medicines. Fragment
Since fragments inherently exhibit low
screening of wild type GPCRs using surface
affinities, high concentrations are typically
Dr. Tim Fagge
plasmon resonance (SPR) which can be used to measure
required in screening assays; many fragment libraries are there-
biomolecular interactions in real time without the need for labelling
fore designed for solubility at high concentrations (mM).
is an alternative ligand discovery strategy. It has proved more
However, this optimisation does not remove the possibility of sticky
beneficial than current screening efforts since it directly measures
substances that can lower the data quality, so in order to minimise
GPCR-ligand interactions, independently of the binding site.
the need for repeat experiments it is important to identify and
Dr. Iva Navratilova works with SPR, and GPCRs are one of her
subsequently remove these compounds prior to binding and/or
companys main focuses. In her presentation, Dr. Navratilova
affinity analysis. Tim touched on the dedicated Clean Screen tool
discussed the applications of this technique, as well as future
which Biacore systems have; this performs a pre-analysis elimination
developments (for example the use of SPR assays for a large variety of
of undesirable sticky compounds so that efficient identification can
membrane proteins).
take place.
Following Dr. Navratilovas interesting presentation, Dr. Tim Fagge
To watch the webinar on demand and find out more about
introduced GE Healthcares new SPR-based sensor, the Biacore S200.
screening of wild-type GPCRs, SPR and the Biacore S200, feel free to
This was previously used in collaboration with Maybridge to run Clean
visit our website.
This webinar was sponsored by GE Healthcare
www.gehealthcare.com
The webinar is available on-demand via the

European Pharmaceutical Review website
VIEW IT NOW AT:
http://www.europeanpharmaceuticalreview.com/webinar24
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29
PRODUCT HUB
Dr. Michael Schneider, Sr VP BU Life Science

for Chemspeed Technologies AG, discusses
Chemspeeds SWILE Automated Workstation
(first true gravimetric one-to-one pick and
dispense of compounds)
The compound library is at the heart of every pharmaceutical company,
Chemspeed Technologies believes. Built from years of synthetic work,
most potential blockbusters start and are stored here, since it is the
source of all screening activities. These compounds are extremely
precious: often difficult to synthesise or extract, and sometimes the
worlds entire resource is in that little vial. Compound management
groups from pharmaceutical companies typically handle more than
tens to thousands of thousands of compounds in their libraries, and
have to deliver small fractions (in milligram scale) for biological tests.
The conventional way to automate these one-to-one dispenses
with an extruder has hit its limit, Dr. Michael Schneider, Senior Vice
President of Business Development for Chemspeed Technologies AG,
tells European Pharmaceutical Review. It requires higher amounts of
flowable crystalline material, is prone to cross-contamination or
plugs into the overall workflow and handles standard source vials as
compound loss, and simply doesnt meet the required accuracy. More
well as virtually any recipient, including Matrix TM tubes. The modular
and more compounds are not available in the required amounts, are too
robotic platform additionally handles supporting tasks like
sticky or oily, and the required manual dispensing creates a bottle-neck.
capping/uncapping of either crimped or screw-capped vials, barcode
This is where the SWILE solution comes in. The SWILE Automated
reading and tracking, dissolution, agitation, and there are a vast
Workstation was introduced into Chemspeeds portfolio of
number of further options.
pharmaceutical R&D workflow solutions in 2014, driven, Dr. Schneider
So how can SWILE be applied to the next generation of compound
explains, by an industry need for automated smallest amount solid
logistics? SWILE easily plugs into any existing compound
handling for, for example, compound management and catalyst
management/logistics solution rack from storage; one-to-one solid
screening in conjunction with the companys proprietary portfolio of
reformatting directly from source vials into a vast range of destination
overhead gravimetric dispensing tools for solids, liquids, viscous
vials/tubes/plates; and rack back to storage, Dr. Schneider explains,
liquids, pastes and waxes.
adding: It can be part of an entire Chemspeed compound
So how can the advantages of using SWILE over existing
management solution.
conventional methods of compound management be summed up?
When asked to summarise some of the companys other workflow
It is the first true one-to-one gravimetric automated pick and dispense
solutions, Dr. Schneider is happy to elaborate: Chemspeed
solution for solids, Dr. Schneider points out. He adds that the
Technologies provides a vast suite of automated pharmaceutical
Workstation gives pick and dispense capability from almost any source
R&D solutions [including] powder/solid dispensing, compound
to any destination, and allows a vast range of solid compounds, from
management, excipient compatibility screening, content uniformity
< 0.1mg to 30mg, to be dispensed. Then there is the use of economical,
screening, polymorph screening, library synthesis in discovery
disposable positive displacement tips, which enables an unparalleled
chemistry, catalyst screening, route scouting, process research
variety of physical consistencies to be handled, eliminating any cross
and optimisation, solid formulation, and liquid formulation.
contamination at the same time. A further benefit of SWILE is the

complete lack of dead volume.
Further advantages include a recovery rate equal or higher than
that in manual procedures, and the ability to operate the Workstation
unattended (with an optional inert atmosphere). The technology
www.chemspeed.com
31
Jens Goepfert / Shutterstock.com
PCR
MIQE compliance in
expression profiling and
clinical biomarker discovery
Irmgard Riedmaier, Melanie Spornraft, Benedikt Kirchner and Michael W. Pfaffl
Technical University of Munich
Molecular diagnostics and biomarker discovery are gaining increasing attraction in clinical research. This includes
all fields of diagnostics, such as risk assessment, disease prognosis, treatment prediction and drug application
success control1,2. The detection of molecular clinical biomarkers is very widespread and can be developed on
various molecular levels, like the genome, the epi-genome, the transcriptome, the proteome or the metabolome.
Today, numerous high-throughput laboratory methods allow rapid and holistic screening for such marker
candidates. Regardless of which molecular level is analysed, in order to detect biomarker candidates, high
sample quality and a standardised and highly reproducible quantification workflow are prerequisites. This article
describes an optimal and approved development strategy to discover and validate transcriptional biomarkers in
clinical diagnostics, which are in compliance with the recently developed MIQE guidelines3. We focus on the
importance of sample quality, RNA integrity, available screening and quantification methods, and biostatistical
tools for data interpretation.
The application of molecular biomarkers is a common research field in
profiling of specific gene transcripts (transcriptomics), to the screening
many different areas, including clinical diagnostics, therapeutic
of functional proteins (proteomics) and the deposition of degradation
prediction, risk assessment and food safety. By applying molecular
products (metabolomics)2,4,5.
biomarkers, different physiological or pathophysiological conditions
The primary essential information for each protein is encoded in
can be identified in patients, or stages of disease progress can be
the genome, where epigenetic modifications have a great influence
distinguished. There are numerous molecular levels on which such
on the expression profile and expression rate of specific genes. This first
biomarkers can be determined: from the detection of DNA mutations or
step of building a functional protein is the transcription of the specific
methylation patterns (genomics and epi-genomics), over-expression
gene into the coding messenger RNA (mRNA). Beside mRNA, the
32
PCR
transcriptome also includes a huge variety of non-coding RNAs which
been shown multiple times that the integrity of all RNA transcripts has
have regulating functions on protein formation, like tRNAs, rRNAs,
a tremendous effect on RT-qPCR performance, either for mRNA or
miRNAs, piRNAs and long non-coding RNAs. All these expressed
microRNA quantification7,8. Obtaining high integer RNA starts with the
transcripts are summarised in the transcriptome .
quality of sampling, which needs to be fast and clean to avoid
Compared to the technically very complex analysis of post-
contamination with RNases. Storing tissue samples in formalin-fixed,
translational modified proteins or chemically closely related
paraffin-embedded, as it is mostly performed in clinical routine
metabolites, today the analysis of the

transcriptome is relatively fast and easy. Hence,
the development and application of transcriptional biomarkers for different purposes has
risen tremendously in clinical diagnostics
during the recent years4,5.
diagnostics, leads to a crosslinking and high
In order to detect biomarker

candidates, high sample quality
and a standardised and highly
reproducible quantification
workflow are prerequisites
The gold standard method for the reliable
degradation rate of RNA and therefore delivers

limited or misleading biomarker information9.
Storing samples in stabilising solution, e.g.,
PAXgene blood or tissue (PreAnalytix),
LeucoLock (Life Technologies), or RNAlater
(Ambion), or snap freezing in liquid nitrogen
and quantitative analysis of single gene transcripts is still reverse
would be the preferred sampling and storage strategy to obtain high
transcription polymerase chain reaction (RT-qPCR). However, if
quality RNA. It has been shown that the RNA integrity number, which is
someone wants to perform a holistic screening for a high number of
determined by Agilent TechnologiesAgilent 2100 Bioanalyzer, directly
transcripts or even for all transcripts in a biological sample, recently
correlates with the resulting Cq value and hence with the quantification
developed next-generation sequencing (RNA-Seq) will be the method of
result. The better the RNA integrity and the higher the RIN value, the
choice. Since RNA-Seq is still relatively expensive to perform on any
more specific the RNA or microRNA molecules that can be quantified in
large sample set, preliminary screening on a subset of representative
the respective sample7,8.
samples is conducted most of the time. To validate these candidates in

all available biological samples, those transcripts can then be
Strategy for transcriptomic biomarker discovery
quantified and confirmed using RT-qPCR, and if such transcriptomic

biomarkers form a valid and stable biomarker signature, they are
Screening by next generation sequencing and

validation by RT-qPCR
suitable for the implementation in RT-qPCR routine analysis in clinical
There are two main strategies for biomarker detection for a specific
molecular diagnostic laboratories. It is also an advantage to apply RT-
physiological status, disease or treatment. On the one hand, there is
qPCR for molecular diagnostics, since it is fast to perform, relatively
the so called targeted approach, whereby a limited number of well-
cheap, already established in almost all clinical laboratories and if
known and established biomarker candidates that are potentially
applied according to the MIQE guidelines, valid and highly reproducible.
influenced by the examined condition are quantified. Regarding the
This article describes the technical require ments for the
analysis of the transcriptome, RT-qPCR will be the first method of
development of high quality transcriptomic biomarkers according to
choice. Depending on how many biomarker candidates shall be
the MIQE guidelines3, using RNA Seq and RT-qPCR.
analysed, single gene assays or qPCR arrays can be applied5.

On the other hand, there is the so called untargeted approach,
Sampling and RNA quality assessment
whereby all transcript sequences of a sample will be screened and
An important consideration in biomarker research is the quality of the
therefore quantified. This allows for the detection of new and unknown
analysed samples and the nucleic acids contained within. It has already
biomarker candidates that would not be recognised by the researcher

using the targeted approach. Some years ago, microarray
technology was the gold standard for the broad screening of
differentially expressed genes. Since next-generation
sequencing (NGS) technology, with its array of applications
like RNA-Seq or small RNA-Seq, has become more and more
popular, it has displaced microarray analysis10. Applying NGS,
a holistic and very sensitive quantification of all different RNA
species is possible. Compared to microarray analysis, it has
no upper limit of quantification, nearly no background signal
and a higher dynamic range in the expression levels11.
If a stable and treatment-specific biomarker signature
should be established, a high number of biological samples is
required to exclude markers that are also dependent on
generic factors, like age, gender, stress and nutrition. As
screening methods like RNA Seq are still very expensive, the
untargeted approach is usually applied only in a small subset
of samples. After analysing the data for potential biomarker
Figure 1: Correlation analysis of miR-3431 expression between results obtained from small
RNA-Seq analysis and single miRNA target RT-qPCR analysis
34
candidates, they can be validated via single RT-qPCR assays in

all available samples11,12. Validating small RNA-Seq data using
PCR
single target RT-qPCR assays showed that the
obtained read count correlated well with the Cq
values from qPCR (Figure 1; page 34).
Biostatistical tools for

multivariate data analysis
The physiological answer to a treatment, drug
application or disease is usually a complex regulation
cascade, whereby the expression of multiple mRNAs
and their connected small RNAs are impacted. For the
most part, a meaningful expression pattern of
significantly regulated gene transcripts is the outcome of transcriptomic biomarker research13,14. But to
obtain the intended unique biomarker signature,
highly advanced bioinformatical tools for data visualisation, data comparison, data grouping or treatment
cohort separation must be applied. The goal is the
visual and statistical separation of the treated
abnormal, or patho-physiological, from the normal
physiological status. So-called multivariate data
analysis tools like hierarchical cluster analysis (HCA),
heatmaps or principal components analysis (PCA) can
be ideally used for this purpose13.
HCA is a frequently-used tool for the twodimensional illustration of multiple parameters
available for a sample. Within HCA analysis, the
expression profile of different samples is divided into
subgroups, with the goal to create subsets that share
as many common characteristics (transcriptional
biomarkers) as possible. The more common the
expression profile of two biological samples appears,
Figure 2: HCA (A) and heatmap (B) analysis of a biomarker research trial with 21 controls and 14 treatment
samples. Herein 11 independent gene transcripts were integrated in the analysis applying Genex software15
(MultiD, Sweden)
the nearer they are positioned in the created cluster.

Clustering is performed in many repeating cycles within the probands
distance between the samples based on their individual expression
and the best biomarker similarities are combined in one cluster. Finally,
profile of the applied parameters13,15.
this leads to a tree-shaped dendrogram (Figure 2A) that displays the
A special application of HCA is the creation of a two-dimensional

heatmap (Figure 2B). Clustering can be performed in parallel for the
measured parameters, e.g., quantified transcripts and investigated
samples/individuals. Using a heatmap, those two parameters can be
combined in one plot, resulting in a colour-coded presentation of the
complete experimental matrix (applied parameters vs. samples).
PCA is a further biostatistical method for visualising the affiliation
of a sample to a group based on the similarity of specific parameters.
PCA is also based on a statistical procedure, able to convert big
multi-dimensional data sets into two-, or three-dimensional variables
called principal components 13,16,17. Using PCA in transcriptomic
biomarker discovery, the classification of samples is mostly based on
the expression values obtained from NGS or high-throughput RT-qPCR
expression profiling experiments, whereat each sample/individual is
represented by one data point on the PCA graph (Figure 3).
Summary and conclusion

The use of transcriptomic biomarkers has already entered different
Figure 3: PCA analysis of a biomarker research trial with 21 untreated controls
(blue circles) and 14 treated probands (red triangles). Herein, 11 independent
gene transcripts were integrated in the analysis applying Genex software 15
(MultiD, Sweden)
fields of clinical research. Obtaining reliable and reproducible results

is most important in developing valid biomarker signatures 2,5.
High sample quality and high RNA integrity is a first essential step to
35
PCR
reach this goal. For the detection and expression profiling of
transcriptomic biomarkers, different general quantification strategies are available. Either the expression of a set of predefined
genes is quantified using RT-qPCR or a holistic approach is taken to
monitor the whole transcriptome of a biological sample, applying
RNA-Seq10. Regardless of which of those strategies is followed, the
result is ideally a set of candidate genes, whose expression is changed.
To get the intended information out of such a biomarker set,
multivariate data analysis tools, like HCA, PCA or heatmaps are
very helpful. In the research field of establishing new sensitive detection methods for drug abuse in veterinary molecular
diagnostics, those biostatistical methods have already been
successfully employed18.
Michael W. Pfaffl studied Agriculture with a focus on

Animal Science in 1986 at the Technical University of
Munich (TUM). His second TUM university degree, in
Biotechnology, was performed in parallel with his PhD.
In 1997, he obtained his PhD in Molecular Physiology, in
the field of molecular muscle and growth physiology at
TUM, at the Chair of Physiology. In June 2003, he
completed his Habilitation (Dr. habil.) at Center of Life and Food Sciences
Weihenstephan with the title Livestock transcriptomics: Quantitative
mRNA analytics in molecular endocrinology and mammary gland
physiology. In early 2010 he became Professor of Molecular Physiology at
the TUM in Freising. Today, he has reached the Principal Investigator status
at the Institute of Physiology and is one of the leading scientists concerning
RT-qPCR technology and its data analysis in mRNA and small-RNA
expression profiling. In 2004 he founded, together with Sylvia Pfaffl
(MBA), the Biotec Marketing, Communication and Consulting company
bioMCC. Contact Michael at: Michael.Pfaffl@wzw.tum.de.
References
References
1. Hulka BS (1990) Overview of biological markers. In: Biological markers in epidemiology
(Hulka BS, Griffith JD, Wilcosky TC, eds), pp 315. New York: Oxford University Press
2. Atkinson AJ (2001) NCI-FDA Biomarkers Definitions Working Group; Biomarkers and surrogate
endpoints: preferred definitions and conceptual framework; Clin. Pharmaco. Ther. 69: 8995
3. Bustin, SA, Benes, V, Garson, JA, Hellemans, J, Huggett, J, Kubista, M, Mueller, R, Nolan,
T, Pfaffl, MW, Shipley, GL, Vandesompele, J, Wittwer, CT. The MIQE guidelines: minimum
information for publication of quantitative real-time PCR experiments. Clin. Chem., 2009, 55
4. Sewall CH, Bell DA, Clark GC, Tritscher AM, Tully DB, Vanden Heuvel J, Lucier GW
(1995) Induced gene transcription: implications for biomarkers. Clin Chem. 12(2):
1829-1834
5. Riedmaier, I. and Pfaffl, MW. Transcriptional biomarkers high throughput screening,
quantitative verification, and bioinformatical validation methods. Methods, 2013, 59
6. Kiss T.: Small nucleolar RNA-guided post-transcriptional modification of cellular RNAs.
EMBO J. 2001, 20(14): 3617-3622
7. Becker, C, Hammerle-Fickinger, A, Riedmaier, I, Pfaffl, MW. mRNA and microRNA quality
control for RT-qPCR analysis. Methods, 2010, 50
8. Fleige, S and Pfaffl, MW. RNA integrity and the effect on the real-time qRT-PCR
performance. Mol. Aspects Med., 2006, 27
9. Kalmar, A, Wichmann, B, Galamb, O, Spisak, S, Toth, K, Leiszter, K, Tulassay, Z, Molnar, B.
Gene expression analysis of normal and colorectal cancer tissue samples from fresh frozen
and matched formalin-fixed, paraffin-embedded (FFPE) specimens after manual and
automated RNA isolation. Methods, 2013, 59
10. Wang, Z, Gerstein, M, Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nat.
Rev. Genet., 2009, 10
11. Riedmaier, I, Benes, V, Blake, J, Bretschneider, N, Zinser, C, Becker, C, Meyer, HH, Pfaffl,
MW. RNA-sequencing as useful screening tool in the combat against the misuse of anabolic
agents. Anal. Chem., 2012, 84
12. Riedmaier, I, Becker, C, Pfaffl, MW, Meyer, HH. The use of omic technologies for biomarker
development to trace functions of anabolic agents. J. Chromatogr. A, 2009, 1216
13. Bergkvist, A, Rusnakova, V, Sindelka, R, Garda, JM, Sjogreen, B, Lindh, D, Forootan, A,
Kubista, M. Gene expression profiling Clusters of possibilities. Methods, 2010, 50
14. Kubista, M, Andrade, JM, Bengtsson, M, Forootan, A, Jonak, J, Lind, K, Sindelka, R,
Sjoback, R, Sjogreen, B, Strombom, L, Stahlberg, A, Zoric, N. The real-time polymerase
chain reaction. Mol. Aspects Med., 2006, 27
15. GenEx qPCR data analysis software version 5.0 (MultiD, Gothenburg, Sweden)
16. Lee, G, Rodriguez, C, Madabhushi, A. Investigating the efficacy of nonlinear dimensionality
reduction schemes in classifying gene and protein expression studies. IEEE/ACM. Trans.
Comput. Biol. Bioinform., 2008, 5
17. Beyene, J, Tritchler, D, Bull, SB, Cartier, KC, Jonasdottir, G, Kraja, AT, Li, N, Nock, NL,
Parkhomenko, E, Rao, JS, Stein, CM, Sutradhar, R, Waaijenborg, S, Wang, KS, Wang, Y,
Wolkow, P. Multivariate analysis of complex gene expression and clinical phenotypes with
genetic marker data. Genet. Epidemiol., 2007, 31 Suppl 1
18. Riedmaier, I, Pfaffl, MW, Meyer, HH. The physiological way: monitoring RNA expression
changes as new approach to combat illegal growth promoter application. Drug Test. Anal.,
2012, 4 Suppl 1
piotr_pabijan / Shutterstock.com
CONTINUOUS MANUFACTURING
Designing a novel
continuous manufacturing
plant with superior
monitoring and control
Ravendra Singh, Jun Zhang, Marianthi Ierapetritou and Rohit Ramachandran
The State University of New Jersey
There is a growing interest in manufacturing the pharmaceutical product continuously1. Along with other
advantages2, it provides an appropriate platform to implement suitable monitoring and control architecture, to
improve the product quality and minimise product rejection and operating expenses. Continuous pharmaceutical
manufacturing can be also considered as a data rich operation since data is continuously generated from the
substantial experiments employed in the process development activities or process operation. Therefore, a
systematic framework is needed through which suitable sensing and control architectures can be designed,
developed, evaluated and implemented into a continuous pharmaceutical manufacturing plant. In addition, an
efficient data management system is required for data storage, organisation and subsequent applications.
The objective of this article is to introduce a continuous pharmaceutical manufacturing process and pilot plant,
and highlight the process analytical technology (PAT), control and data management system integrated with it.
The pharmaceutical industry is a global business sector with around
inherent limitations that can be overcome through continuous
$1 trillion annual sales focused on performance-based products
manufacturing2, which itself offers the advantages of high efficiency,
designed to address the healthcare needs of the worlds population.
ability to enhance product quality, better controllability and reduced
Traditionally, pharmaceutical products involving solid dosage forms
need for space, labour and resources.
were produced using batch processes. One major advantage of batch
Driven by these benefits and as a result of initiatives taken by
processing is flexibility of using the same equipment for multiple
United States National Science Foundation Engineering Research
purposes. However, batch manufacturing processes have some
Center on Structured Organic Particulate Systems (C-SOPS), the
37
pharmaceutical
industry
is
currently
undergoing a paradigm shift from batch to

continuous manufacturing. The US Food and
Drugs Administration (FDA) has also been very
encouraging and supportive of the changes1.
Consequently, most of the large pharma ceutical companies are in process of either
building continuous pharmaceutical manufacturing plants or filing for approval to
regulatory agencies such as the FDA. One
continuous pharmaceutical manufacturing
plant, described in this article, recently
received regulatory approval, which indeed
serves as a motivating example for this
paradigm shift.
The benefits of harnessing the rich data
accumulated in continuous pharmaceutical
manufacturing is that a clear landscape about
target product and process is generated, and
the operational space of pharmaceutical
processes optimised3. However, less attention
has been given to data utilisation, as indicated
by a survey which demonstrated less than 10%
Figure 1: Continuous direct compaction tablet manufacturing pilot-plant (adapted from Singh et al.6): (1) feeders,
(2) co-mill and blender, and (3) tablet press
of data use by pharmaceutical companies. This can be traced to a
A continuous direct compaction tablet manufacturing pilot plant
lack of systematic organisation of the data. Furthermore, few
has been installed and situated at C-SOPS, Rutgers University.
approaches have been developed to extract information from such data
A snapshot of the pilot-plant is shown in Figure 1. The pilot plant is built
sets automatically. To address this challenge, a general computational
on three levels at different heights to take advantage of gravitational
framework has been developed that allows the process data to be
material flow. The top level is used for feeder placement and powder
harnessed for the development of a continuous manufacturing process.
storage, the middle level is used for delumping and blending, and the
An integrated process flowsheet model

of continuous pharmaceutical tablet
manufacturing has also been developed and it
has several applications such as virtual
experimentations, process design, process
optimisation, risk assessment, sensitivity
bottom level is used for compaction. There are
The benefits of harnessing the rich

data accumulated in continuous
pharmaceutical manufacturing is that
a clear landscape about target product
and process is generated
analysis, feasibility analysis, flexibility analysis,
three gravimetric feeders with the capability

of adding more that feed the various
formulation components (active pharma ceutical ingredient [API], excipient, etc.).
The feeders consist of a hopper, a load cell that
is integrated with a gravimetric controller, and a
design space generation, sensing and control architecture design4,5.
conveying screw. A co-mill is also integrated after the feeder hopper,
The current focus is to further modernise continuous pharmaceutical
primarily to enable the powders to be de-lumped and to create contact
manufacturing through integration of efficient in-line/on-line real-time
between components.
monitoring tools, advanced feed-forward/feed-back control and

systematic data management systems.
Conical screen mills each consist of a cone-shaped screen

with an impeller inserted into the center. The impeller rotates and
material is ground between the impeller and the screen until it is small
Flexible continuous tablet manufacturing

process and pilot-plant
enough to pass through the holes in the screen and leave the mill.
A closed-loop flexible multipurpose continuous tablet manufacturing
and the lubricant feeder is added after the mill to prevent over-
The co-mill eliminates any large, soft lumps within the powder,
process has been developed . In this process, a typical pharmaceutical
lubrication of the formulation in the mill. These feed streams are then
tablet can be produced via the different paths of direct compaction
connected to a continuous blender within which a homogeneous
(route one), dry granulation using roller compaction (route two) or wet
powder mixture of all the ingredients is generated. Subsequently,
granulation (route three), and the most appropriate route is decided on
the outlet from the blender is fed to the tablet press via a rotary feed
based on the properties of the raw materials and the desired
frame. The powder blend fills a dye and is subsequently compressed to
characteristics of the final product. Route one is the simplest process
create a tablet. The NIR sensor for inline monitoring of blend uniformity,
while routes two and three provides enhanced flowability characteristics
blend composition and blend density has been integrated through a
of the powder blends; route two is particularly suitable for cases where
chute placed in between tablet press and blender. The chute is
powder feeds are moisture-sensitive. The process is described in detail
specifically designed to provide an interface within which the
in Singh et al.6 and has been previously extensively studied2,4,5.
new powder layer can form by displacing the old powder layers.
38
The integration of the chute with the blender
and sensor is reported in Singh et al.
Integration of PAT
Along with other monitoring tools, PAT
has been integrated with the continuous
pharmaceutical manufacturing plant described
to enable real-time monitoring of critical
Common data file

used for data storage,
Data representation:
e.g., particle size
Data will be saved in
a defined format (xml)
process parameters. An advanced control

system has then been added to control the
critical process parameters to achieve desired critical quality attributes. The local level
controllers are inbuilt in each feeder in order to
control the powder flow rate. A ratio controller
User defines query, e.g.,

which API, which excipient,
and search function returns
a set of relevant data (xml)
has been added that provides the flow rate set

points of API, excipient and lubricant feeders
for a given total flow rate and API composition7.
Six supervisory control loops are added.
The first loop is for the PAT-based feed-forward
control which takes a corrective action for
variations in the powder bulk density8. An NIR
Predictive model
clustering results
A set of relevant data
will be used by
multivariate data analysis
algorithm
sensor together with real-time prediction tool

has been used for inline monitoring of the
powder blend density9. The signal of powder
blend density has then been sent to the feedforward controller that manipulates the fill cam
Figure 2: Flowchart depicting use of accumulated data for process design
depth of the tablet press proactively. The second loop is added to
method is used for online monitoring of the powder level and the drug
control the main compression force of the tablet press. This control
concentration is controlled through the PAT based on the sixth control
loop is in a cascade arrangement with a master controller (loop three)
loop7. The NIR sensor has been used for inline real-time monitoring
specifically designed to control the tablet weight. The input of this
of drug concentration and the powder bulk density and drug
master controller is weight and it generates the set point of the main
concentration is monitored using a single NIR probe9. Two partial least
compression force.
square (PLS) models are used to predict the powder bulk density and
Loop four is designed to control the tablet hardness by
drug concentration separately.
manipulating the punch displacement . Checkmaster (Fette) is used for
In order to implement the control system into the pilot plant, the
monitoring the tablet weight and hardness. Loop five is added to
NIR spectra are sent to a real-time prediction engine that utilises
control the powder level in the instrumented hopper. A webcam
the NIR calibration models for blend density and drug concentration
and a real-time prediction tool (OLUPX)

generates the signals for the control variables
in real time7. The generated signals are then
sent to a commercially-available control
platform via an OPC where the combined FF/FB
control loop has been implemented. The blend
density is the input for the feed-forward
controller while the blend composition
together with powder level, tablet weight and
hardness are the inputs of the feed-back
control system9.
Efficient data management system

This framework includes data and ontology
representation, search function and a
multivariate data analysis (MVDA) algorithm
(see Figure 2). The data representation can
Figure 3: Process dynamic analysis: (a) production rate and turret speed, (b) fill depth, (c) main compression force,
and (d) pre-compression force
systematically represent process data, which is

generated by different characterisation devices
39
or operation units, as a set of human-readable
specifications. These specifications can be
organised as an XML file, which is a standard
data vehicle for none-gap communication
between various database/systems. Each data
point is an XML file with unique filename as the
ID, and in each XML file, the specifications are
organised as a hierarchical structure whereby
each node represents a specification, i.e., a
specific measurements name with associated
value. Ontology representation describes
the relationship of terminologies referred in the
data, e.g., paracetamol is a type of API. These
terminologies and relationships are usually
visualised as a hierarchical structure that has
been discussed in detail10,11.
Ontology establishes logic interactions
between different data to facilitate data search
and selection.
The search function is developed to
retrieve desired process data, and it consists of
a user interface and search algorithm. The user
Figure 4: Use of PLS algorithm to predict powder density after co-mill in gPROMs platform
interface allows the user to define which data should be studied and
to 10mm. As shown in the figure, after initial lag time the fill depth
retrieved, e.g., API name, as well as the numerical criterion to be used
approached the set point.
for data searching, e.g., 10% of defined viscosity. The ontology can also
To study the effect of fill depth on critical process parameters such
be used to structure the data. For example, Zhang (2011) described an
as main and pre-compression forces, the fill depth has been changed
example of harnessing strain ontology to search Chinese hamster ovary
from 8mm to 10 mm and back to 7mm. The achieved main compression
cell data, which could expand the search space for further data
and pre-compression forces are shown in Figures 3c-d (page 39).
understanding. A search algorithm compares the users defined
The achieved fill depth is also shown in the figure. As shown, the main
specifications with data points organised as individual XML files to
and pre-compression forces follow the profile of fill depth with some
confirm the data satisfies the users definitions, which would be used
delay times. The response of main compression force is relatively
for information extraction. An example of the search algorithm and how
more delayed than pre-compression force response and it is essentially
it works for data searching is provided by Zhang and Hunter et al.11
because the distance between filling station and main compression
12
Based on the returned data, MVDA algorithms serve to extract
station is more in comparison to that of pre-compression station and
information. For example, clustering algorithms such as hierarchical
filling station. Similarly, the dynamics of other process variables have
clustering and K-mean clustering can quickly sort data into groups that
been analysed. Based on the experimental data, a process model can
allows users to understand commonalities/differences embedded in
be developed that can be used to analyse the process and design the
the data; regression algorithms, e.g., PLS, can harness returned data to
control system.
generate the prediction model for estimated results. Such a prediction
The control hardware and software integration13 and closed-loop
model could ensure the completeness of process data, e.g., the missing
performance of the continuous tablet manufacturing plant has been
data can be predicted to facilitate process simulation and
previously described 7, and the performance of combined feed-
understanding.
forward/feed-back control system has been evaluated by Singh et al8.

The real-time monitoring of powder bulk density for FF/FB control has
Results and discussions
also been previously demonstrated9.
Continuous plant operation and process dynamic analysis

The dynamics of important continuous tablet manufacturing operating
Efficient data management and applications
parameters along with PAT and control have been studied. Tablet
To illustrate how this framework works, a demonstration has been
press unit operation has been considered here as a demonstrative
developed based on gPROMs, which is a dynamic process simulation
example. The production rate changed from 7500 tablets/h to 30,000
platform. The flowchart of demonstration is given in Figure 4, where the
tablets/h. The set point, along with achieved production rate, is shown
raw material properties data were stored in excel files that can be
in Figure 3a (page 39). As shown in the figure, the achieved production
represented as XML files. Search function is developed to return
rate is delayed in comparison to actual set point. The turret speed is
relevant data satisfying users interests. This returned data is harnessed
also shown in Figure 3a (page 39). As expected, the production rate
by PLS algorithm to generate predicted models for prediction of
is proportional to the turret speed. The dynamics of fill depth is shown
concerned parameters requested by flowsheet modelling in gPROMs.
in Figure 3b (page 39). The fill depth has been changed from 8mm
The function referred in this framework can be realised by the general
40
programming languages, Python (i.e., data representation and search
function) and C++ (i.e., PLS algorithm), which are selected due to
conformability of gPROMs platform.
For a case study, the flowsheet model of continuous direct
compaction tablet manufacturing process was used. The bulk density of
powder coming out from the co-mill was considered for prediction
using the PLS method because it is the key attribute and currently there
is no other model available to predict it. Sixteen data points of powder
properties were stored in the database, and the search function
returned eight data points consisting of six PLS input variables: namely
API, excipient, API concentration, excipient concentration, feeder
throughput and co-mill rotation speed, and one PLS output variable
namely powder density. These returned data points were harnessed by
PLS algorithm to establish a prediction model to predict powder density
for the corresponding information defined in the simulation. The whole
process and results are given in Figure 4 (page 41). The predicted density
was calculated by experimental facts rather than theoretical
assumption/calculation which makes the simulation results closer to
the real situation.
Conclusions
A novel continuous pharmaceutical tablet manufacturing process
integrated with real-time monitoring tools, advanced control and a
systematic data management system has been developed. The process
operation dynamics have been analysed. The data management
framework described in this paper shows that reusing the process data
for process development is a promising approach and a flexible one,
which can easily be implemented and integrated with pharmaceutical
companies current data management systems. It provides a costeffective big data strategy to accelerate a products commercialisation.
Future work will include the implementation of a combined feedforward/feed-back control system into the pilot plant.
Acknowledgements
This work is supported by the National Science Foundation Engineering
Research Center on Structured Organic Particulate Systems, through
Grant NSF-ECC 0540855.
Dr. Ravendra Singh is Assistant Research Professor at the

Department of Chemical and Biochemical Engineering,
Rutgers University, US. He is also serving as a manager of
multi million dollars, NSF ERC-SOPS projects. He
obtained his MS from IIT Roorkee India, and his PhD from
Technical University of Denmark. He is the recipient of a
prestigious EFCE Excellence Award given in Recognition
of an Outstanding PhD Thesis, from European Federation of Chemical
Engineering. He has published more than 43 research papers, written three
book chapters, and presented at over 72 international conferences. He is lead
guest editor of Journal of Chemistry special issue for PAT. He can be
contacted at: ravendra.singh@rutgers.edu (phone: [001] 8484454944).
Dr. Jun Zhang obtained his BE from Tianjin University,
China, his MS from Peking University, China and his PhD
from University College London (UCL), UK. He completed
his post-doctoral research at Rutgers University, New
Jersey, US. He is currently employed at GlaxoSmithKline,
UK. Major awards he won in 2010 and 2011 include
Overseas Research Students Awards (from UCL),
Bioprocess UK 2010 Conference Award for Poster Competition and UCL
Graduation School Conference Award. He has published several papers and
delivered presentations to national and international conferences. He can be
contacted at: Jonison.zhang@gmail.com.
Dr. Marianthi Ierapetritou is a Professor and Chair of
Chemical and Biochemical Engineering at Rutgers
University. She obtained her BS from National Technical University in Athens, Greece (magma cum laude), her
PhD from Imperial College London in 1995 and
subsequently completed post-doctoral research at Princeton
University (New Jersey) before joining Rutgers University
in 1998. Among her accomplishments are the Outstanding Faculty Award in
2012, the Rutgers Board of Trustees Research Fellowship in 2004, and the
prestigious NSF CAREER award in 2000. She has published over 190
papers and delivered over 200 presentations to national and international
conferences. She can be contacted at: marianth@soemail.rutgers.edu
(phone: [848] 445-2971).
Dr. Rohit Ramachandran is currently an Assistant
Professor at the Department of Chemical & Biochemical
Engineering, Rutgers University, in New Jersey (US). He
completed his bachelors and masters degrees in Chemical
Engineering at the National University of Singapore. This
was followed by his PhD in Chemical Engineering at the
Centre of Process Systems Engineering, Imperial College
London. He then undertook postdoctoral work at MIT in the Process
Systems Engineering Laboratory and the Novartis-MIT Center for
Continuous Manufacturing. His research interests include modelling, control
and optimisation of pharmaceutical and chemical processes. He can be
contacted at: rohit.r@rutgers.edu.
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41
PAT
44 Process analytical control:
An overview
David P. Elder, GlaxoSmithKline and JPAG
48 Expert View
Kaiser Optical Systems
50 Monitoring, understanding
and assessing
pharmaceutical process
and product quality
Huiquan Wu, Bogdan Kurtyka, Geoffrey Wu,
Jennifer A. Maguire, Rapti Madurawe, Christine
M.V. Moore, Robert Iser, Mansoor A. Khan,
Robbe C. Lyon, Patrick Faustino, Lucinda Buhse,
Sau Lee, Lawrence Yu, United States Food and
Drug Administration
57 PAT Roundtable
Moderated by David P. Elder,
unpict / Shutterstock.com
SPONSORS
43
akank / Shutterstock.com
IN-DEPTH FOCUS: PAT
An overview of process
analytical control
David P. Elder
In a quality by design (QbD) paradigm (ICH Q81, Q92 and Q103) it is envisioned that a full understanding of critical
quality attributes (CQAs) and critical process parameters (CPPs) will facilitate better product understanding and
thereby improved process control. However, critical material attributes (CMAs), CQAs and CPPs are intrinsically
complex and exhibit multi-factorial inter-relationships, which in turn necessitate multi-variate control strategies,
such as multivariate data analysis (MVDA) and statistically-based design of experiments (DOE). The United States
Food and Drug Administration foresees that an integrated approach to product and process understanding based
on these methods4, together with real-time process analytical technology (PAT5), should facilitate better
understanding, monitoring and process control6.
Facilitating enhanced process understanding
methodologies should be based on direct measurements of process
The FDA has defined PAT as a system for designing, analysing, and
variables, rather than pre-defined process parameters, such
controlling manufacturing through timely measurements (i.e., during
as processing times (e.g., addition times, granulation times, etc.), or
processing) of critical quality and performance attributes of raw and
predefined consumption of materials (e.g., during film coating).
in-process materials and processes, with the goal of ensuring final
When adequate levels of PAT data have been generated, on-line/
product quality . Real-time PAT monitoring has been extensively
at-line PAT process monitoring can evolve into real-time release testing
applied to all stages of pharmaceutical processing. The PAT
(RTRT), whereby at least some of the analytical testing is no longer
44
IN-DEPTH FOCUS: PAT

required or necessary to demonstrate product quality, since the real-
(RSD) as a function of time have also been developed. This approach
time information that has been generated provides an assurance that
assesses the convergence of the expected cumulative NIR spectra,
product quality is acceptable1,7,8-10. This is sometimes termed parametric
based on a predefined, statically-based endpoint, i.e., 1% RSD, to
release. Indeed, in an ideal world, RTRT could potentially allow process
establish blend homogeneity28,29. The major advantage of this approach
intervention to address real-time issues and realign the system to the
is that there is no requirement for pre-existing data. In contrast, the
desired state. The key concepts of PAT are multivariate data acquisition
major disadvantage is that although differences between spectra from
using process sensors and analysers, process monitoring and
a single blend are measured, allowing qualitative decisions to be made
subsequent control and supporting information management tools.
with respect to the homogeneity of the blend, quantitative
PAT can either monitor one unit operation, two interconnecting unit
measurements will still be required using off-line testing30. This can
operations or, ideally, the entire process. This article will provide a brief
be addressed by using a limited number of off-line samples to
overview of some of the applications of PAT and RTRT.
develop a model that can accurately predict content, in addition

to homogeneity30,31.
Active pharmaceutical ingredient applications
Another downside is that NIR produces a weak signal (except with
The last stage of API manufacturing is typically crystallisation. However,
water), and consequently there are inherent limitations to the
this stage can often be challenging since there are numerous variables
sensitivity of these methods for high potency drug formulations, where
to be optimised, i.e., yield, control of impurities, residual solvents
drug loading may be 1% in the blend. To address these sensitivity
arising from the crystallisation process, crystallinity, version (salt,
concerns, other analytical approaches have been investigated.
co-crystal, etc.), form (polymorph, solvate,
Lai et al.27 developed a laser-induced fluor-
hydrate, etc.), habit, particle size, shape and
escence (LIF) method to monitor blend homo-
distribution11. In turn these input variables can
Real-time PAT monitoring has been

extensively applied to all stages of
pharmaceutical processing
affect filtration rates, drying rates (particularly

in agitated drying systems), particle fragility,
particle surface roughness and overall API
geneity. LIF involves irradiating blend samples

at a suitable wavelength for excitation and
assessing the emission at a second wavelength.
The method is rapid, sensitive (API loadings as
processing times12. Some of the factors can
low as 0.05%w/w have been measured),
significantly impact on secondary processing, particularly particle size
accurate and homogeneity is established when the LIF response at
and shape, powder flow and powder compaction13. PAT is commonly
steady state is the same between two successive sampling points.
used in the monitoring and controlling of crystallisation processes due

to its ability to provide real-time data14 leading to real-time control13.
Tabletting
The FDA has advocated the usage of PAT during crystallisation and
Blend homogeneity is intrinsically interrelated with content
identified some in-line and on-line monitoring tools . The habit
uniformity of the resultant drug product and the latter is defined by all
and growth of crystals during crystallisation can be monitored in real
of the major pharmacopoeias as well as being a required attribute for
time within the reactor using in-process visualisation techniques, such
release testing of all drug products 23,24,25. Both reflectance and
as particle vision monitoring (PVM)16 and other related in situ imaging
transmittance NIR have been used in content uniformity testing with
approaches
. In addition, the developing crystal size and related
encouraging results30. NIR has been used for real-time monitoring of
crystal size distribution can be monitored using focussed beam
content uniformity of a tablet product in a production environment
reflectance measurements (FBRM)19, as well as in-line spectroscopic
using partial least squares (PLS) modelling 32 and the real-time
tools such as attenuated total reflectance ultraviolet/visible (ATR-UVV)20
monitoring of both API and excipient content uniformity of a
and ATR Fourier transformation infrared (ATR-FTIR) for real-time solute
tablet product, again using PLS33. In addition, Raman, terahertz and
concentration measurements. In parallel, polymorph form control can
chemical imaging analysis (CIS) have been applied34,35, but the quantities
also be monitored using FBRM and PVM
of data can be enormous and are often difficult to interpret,
15
17,18
21
16,22
necessitating multivariate chemometric approaches35. Interestingly,
Drug product applications
by adopting multivariate methodologies, the user can monitor and
Blending
control both API and excipients33. Indeed, for certain dosage forms,
The preparation of a homogenous blend of API and excipients is an
the role of these key excipients can often be as important as the
important step in the manufacturing process of solid oral dosage forms,
API itself in controlling the performance of the product, e.g., controlled-
such as tablets or capsules. This is particularly true for low-strength and
release products.
highly potent drugs23,24,25. However, sampling using classical approaches

allied with off-line analysis of the sample blend is inefficient, can
Tablet film coating
introduce sampling errors and can necessitate long processing times26.
Film coating is extensively utilised within the pharmaceutical ind-
For these reasons, there are opportunities for PAT methods to
ustry. Film coats can be applied as non-functional, immediate-release
provide a more rapid and consistent approach towards the assessment
coatings, i.e., to enhance stability, to improve palatability or to provide
of blend homogeneity27.
a distinctive commercial image. Alternatively, they can be applied as
Non-invasive monitoring of powder blends using near-infrared (NIR)
functional, modified-release coatings, to address poor chemical
spectroscopy has been extensively reported. Calibration-free
stability in gastric media (delayed release via enteric coating) or to
approaches based on the mean spectral residual standard deviation
modify the in vitro release profile (controlled release via osmotic
45
IN-DEPTH FOCUS: PAT

controlled release oral delivery system; OROS). Traditionally, the
diprophylline43,45. Real-time monitoring using in-line and off-line Raman
film coat thickness is monitored and controlled by the average
spectroscopy was applied to the active coating process of an OROS
weight gain of the tablets, e.g., ca. 3% w/w target for non-functional
dosage form. Wirges et al.46 were able to demonstrate that their in-line
film coats. This requires sampling and off-line or at-line analysis
calibration model gave better accuracy than the off-line calibration
which is inherently inefficient.
model and the former correlated well with the off-line reference HPLC
NIR in the reflectance mode can be used as an off-line , on-line ,
assay. The authors indicated that this was the first time in-line Raman
or in-line 38,39 PAT tool by monitoring the linear reduction in the
calibration models had been applied to complex dosage forms or high
absorbance signal of the tablet core, whilst in parallel monitoring
coating levels, i.e., coat thickness of up to 400m.
36
37
the linear increase in the absorbance signal of the coating materials.

In-line monitoring has been applied to both fluid bed38 and pan
Real-time release testing
coating39 operations. NIR models can determine film coat thicknesses
The principles of RTRT may be applied during any stages of the
much more cheaply and rapidly than either

scanning electron microscopy (SEM) or
imaging and the method gives good com40
parability with the classical weight gain

methodology . NIR has been used in con 30
junction with terahertz pulsed imaging (TPI)
manufacture of synthetic or biological products
There are opportunities for PAT

methods to provide a more rapid and
consistent approach towards the
assessment of blend homogeneity
and multi-variate modelling to monitor tablet
resulting in the exclusion some, or all, specified

tests on the specifications of the finished API or
the finished drug product47. However, the most
obvious and well documented example of RTRT
is terminal heat sterilisation of parenteral
products. Here a review of the documentation
coating41,42. TPI was also utilised to assess the coating requirements of a
obtained during process monitoring, e.g., pressure, temperature and
push-pull OROS system to provide off-line PAT control and correlate the
time for the autoclave cycle by the quality assurance group, together
effect of coating thickness with in vitro release performance43.
with ongoing good manufacturing practice (GMP) compliance, will
Raman has also been used as a PAT tool for film coating, offering
provide the necessary assurance of the quality of the process and
advantages in terms of enhanced selectivity, good robustness and
thereby the product47. It is worth noting that although QbD is not
reduced interference from water signals (a significant deficiency for
directly linked with RTRT, it would be unlikely to envisage RTRT without
NIR), allowing calibration models to be developed based on limited
both a science-1 and risk-based2 approach. Additionally, not all PAT
batch data. A multi-variate model was developed to assess both
approaches lead to RTRT and a design space is not needed for RTRT.
the amount and the uniformity of an active coat of the drug
However, a product specification is still required48.
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item_files/ed21/attachments/31745/original/304.pdf. Accessed on 24th September, 2015
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IN-DEPTH FOCUS: PAT

Conclusion
treatment between various instrument vendors;
PAT plays a fundamental role within the QbD paradigm, but it is not a
new approach, since process analysis and control have been used in API
Standardisation of analytical data formats allowing unified data
reactors for several decades49. PAT needs to be part of an integrated
Enhanced robustness of in-line sensors aligned with

continuous monitoring;
control system. There are deemed to be three levels of process
Rapid maintenance and repair of in-line monitors;
control , one or all of which can be used inter-changeably within a
In situ cleaning of sensors (this is particularly relevant for solution-
50
based processes, such as crystallisation);
designated process:

Real-time, automated testing and flexible normal operating process
Costs of sensor and monitoring devices;
ranges, with the ability of the control system to respond
Enhanced analytical sensitivity for RTRT; and
interactively to input variability;
Enhanced quality and ease of data interpretation by non-experts.
Reduced end-product specification testing, flexible CMAs and CPPs

within a designated design space; and
In summary, there is a requirement to provide confidence that the
End product testing together with constrained material attributes
proposed PAT is robust and will operate consistently, that there is an
and process parameters.
adequate return on investment and that regulators will accept

the proposal51.
It is also important to differentiate between those PAT tools required

to develop in-depth process understanding and those systems
used for day-to-day production/process monitoring, which can be very
simple analytical tools for measuring fundamental variables, such as
weight, pressure or temperature51. There is also a balance between
on- and off-line process monitoring. In addition, current regulatory
interpretation necessitates retention of all historical spectral
data; which for a fully PAT-aligned, data-rich system could be a
significant undertaking. This is a technical burden that other industries
do not share. A recent equipment, analytical and academic symposium
on continuous manufacturing 51 highlighted some of the major
PAT challenges:
Dr. Elder obtained BSc and MSc degrees in Chemistry from

Newcastle upon Tyne, before he moved to Edinburgh to
study for a PhD in Crystallography. He is a visiting professor
at Kings College, London. Dr. Elder has 37 years
experience at a variety of different pharmaceutical
companies (Sterling, Syntex and GSK). He is currently a
director within the product development group in GSK
R&D. Dr. Elder is a member of the British Pharmacapoeia (Expert Advisory
Group PCY: Pharmacy), a council member of the Analytical Division, Royal
Society of Chemistry (RSC), UK and a council member of the Joint
Pharmaceutical Analysis Group, UK. He is a fellow of the RSC (FRSC) and
a member of the Royal Pharmaceutical Society (SRPharmS). He has coedited one book on the Analytical Characterisation and Separation of
Oligonucleotides and their Impurities.
27. Lai C-K, Holt D, Leung JC, Cooney CL, Raju GK, Hansen H. Real time and noninvasive
monitoring of dry powder blend homogeneity. AIChE J. 2001; 47, 2618-2622
coating thickness and surface area of pharmaceutical pellets using fluorescence microscopy
and image analysis. J Pharm Biomed Anal. 2000; 20, 325-339
28. Hailey PA, Doherty P, Tapsell P, Oliver T, Aldridge PK. Automated system for the on-line
monitoring of a powder blending process using near infra-red spectroscopy. Part I. system
development and control. J Pharm Biomed Anal. 1996; 14, 551-559
41. Gendre C, Genty M, Boiret M, Julien M, Menier L, Lecoq O, Baron M, Chamiade P, Pean
JM. Development of a process analytical technology (PAT) for in-line monitoring of film
thickness and mass of coating materials during a pan coating operation. Eur J Pharm Sci.
2011; 43, 244-250
29. Blanco M, Gozlez Ba R, Bertran E. Monitoring powder blending in pharmaceutical

processes by use of near infrared spectroscopy. Talanta. 2002; 56, 203-212
30. Moes JJ, Ruijken MM, Gout E, Frijlink HW, Ugwoke MI. Application of process analytical
technology in tablet process development using NIR spectroscopy: Blend uniformity, content
uniformity and coating thickness measurements. Int J Pharm. 2008; 357, 108-118
31. Bertsson O, Danielsson L-G, Lagerholm B, Folestad S. Quantitative in-line monitoring of
powder blending by near-infrared reflection spectroscopy. Powder Technol. 2002; 123, 185-193
32. Skibsted ETS, Westerhuis JA, Smilde AK, Witte DT. Examples of NIR based real time release
in tablet manufacturing, J Pharm Biomed Anal. 2007; 43, 1297-1305
33. Karande AD, Heng PWS, Liew CV. In-line quantification of micronized drug and excipients
in tablets by near infrared (NIR) spectroscopy: Real time monitoring of tableting process. Int
J Pharm. 2010; 396, 63-74
34. Ravn C, Skibsted ETS, Bro, R. Near infrared chemical imaging (NIR-CI) on pharmaceutical
solid dosage forms comparing common calibration approaches, J.Pharm Biomed Anal.
2008, 48, 554-561
35. Gordon, KC, McGoverin, CM. Raman mapping of pharmaceuticals. Int J Pharm. 2011;
417, 151-162
36. Kirsch JD, Drennan JK. Determination of the film-coated tablet parameters by near-infrared
spectroscopy. J Pharm Biomed Anal. 1995; 13, 1273-1281
37. Kirsch JD, Drennan JK. Near-infrared spectroscopy monitoring of the film coating process.
Pharm Res. 1996; 13, 234-237
38. Andersson M, Folestad S, Gottfies J, Johansson MO, Josefson M, Wahlund KG. Quantitative
analysis of film coating in a fluidized bed process by in-line NIR spectrometry and
multivariate batch calibration. Anal Chem. 2000; 72, 2099-2108
39. Prez-Ramos, JD, Findlay, WP, Peck, G, Morris, KR. Quantitative analysis of film coating in
a pan coater based on in-line sensor measurements. AAPS PharmSciTech. 2005; 6,
E127-E136
40. Andersson, M, Holmquist, B, Lindquist, JO, Nilsson, O, Wahlund, KG. Analysis of film
42. Gendre C, Genty M, Chamiade P, Pean JM. Real-time predictions of drug release and end
point detection of a coating operation by in-line near infrared measurements, Int J Pharm.
2011; 421, 237-243
43. Malaterre V, Pederson M, Ogorka J, Gurny R, Loggia N, Taday, PF. Terahertz pulsed imaging,
a novel process analytical tool to investigate the coating requirements of push-pull osmotic
systems. Eur J Pharm Biopharm. 2008; 74, 21-25
44. Mller J, Knop K, Wirges M, Kleinebudde P. Validation of raman spectroscopic procedure in
agreement with ICH Q2 with considering the transfer to real time monitoring of an active
coating process. J Pharm Biomed Anal. 2010; 53, 884-894
45. Mller J, Knop K, Thies J, Uerpmann C, Kleinebudde P. Feasibility of Raman spectroscopy
as a PAT tool in active coating. Drug Dev Ind Pharm. 2010; 36, 234-243
46. Wirges M, Funke A, Serno P, Knop K, Kleinebudde P. Monitoring of an active coating
process for two layer tablets-model dependent strategies. J Pharm Sci. 2013; 102, 556-564
47. EMA. 2012. Guideline on real time release testing (formerly guideline on parametric release),
EMA/CHMP/QWP/811210/2009-Rev1, 29th March 2012
48. Moore CMV. 2011. Regulatory perspective on real time release testing (RTRT). AAPS
Annual Meeting, Washington DC, 27 October 2011 http://www.fda.gov/downloads/
AboutFDA/CentersOffices/OfficeofMedicalProductsandTobacco/CDER/UCM301055.pdf.
Accessed on 28th September, 2015
49. Rantanen J, Khinast J. The future of pharmaceutical manufacturing sciences. J Pharm Sci.
2015; DOI: 10.1002/jps.24594
50. Yu L, Amidon G, Khan M, Hoag S, Polli J, Raju GK, Woodcock J. Understanding
pharmaceutical quality by design, AAP J. 2014; 16, 771-783
51. Page T, Dubina H, Fillipi G, Guidat R, Patnaik S, Poechlauer P, Shering P,
Guinn, M, McDonnell, P, Johnston C. Equipment and analytical companies meeting
continuous challenges, May 20-21, 2014 Continuous Manufacturing symposium, J Pharm
Sci, 104, 821-831
47
Dima Sobko / Shutterstock.com
EXPERT OPINION: KAISER OPTICAL SYSTEMS
Beyond API monitoring:

in-line Raman spectroscopy
for process control
The FDAs Quality by Design (QbD) initiative brought a paradigm shift to
which could be used to identify process deviations and improve the
pharmaceutical manufacturing, and leading manufacturers have
tablet compression operation in real time.
realised improved processes after adopting QbD. Raman spectroscopy

and continuous approaches to pharmaceutical manufacturing 1.
In-line Raman spectroscopy to

increase process robustness
Since the 1980s, Raman spectroscopy has been used to examine
A multi-part customer webinar shows practical examples of integrating
active pharmaceutical ingredients (APIs)2. As a tool for API analysis,
Raman spectroscopy into a process environment, including demon-
Raman has been described for many applications including polymorph
stration of coupling in-line Raman spectroscopy with a QbD approach to
identification, quantitative analysis, in situ crystallisation monitoring,
improve a process reaction11. Raman spectroscopy was proposed as an
real-time release testing, pharmaceutical unit operations and process-
in-line PAT tool to control post-reaction age because the reaction
induced transformations 37. In addition to identifying isolated
conditions required in-line monitoring. The Raman method resulted in
polymorphic forms, mixtures of forms can be analysed and quantified8,9.
an increased understanding of the reaction and expanded the design
The diverse structures that have been quantified by Raman, from the
space, resulting in a more robust process.
is an established process analytical technology (PAT) tool, enabling QbD
discovery laboratory to the manufacturing environment, show that

Raman methods can reliably provide quantitative data. In-line Raman
Conclusions
spectroscopy can control critical process parameters, enables real-
Raman is a proven technique in the world of PAT. Kaiser is a leader in
time process corrections, and ensures consistent production of the
this technology area, supporting their customer applications and
correct API form .
process analysis with their phase-optimised Raman solutions, imple-
Scientifically and financially, successful Raman applications have
mentable across all scales, with demonstrated transferability.
been demonstrated at all scales, from at-line in-process development

to in-line within PAC and PAT manufacturing environments. Customer
References
examples shown in this article illustrate the advantages of Raman
1.
F Adar, in Handbook of Raman Spectroscopy: From the Research Laboratory to the

Process Line, eds. I. R. Lewis and H. G. M. Edwards, Marcel-Dekker, New York, NY
USA, 2001
2.
BA Bolton and PN Prasad, J. Pharm. Sci. 1981; 70, 789793
3.
CJ Strachan, T Rades, KC Gordon and J Rantanen, J. Pharm. Pharmacol. 2007; 59, 179192
4.
A Hdoux, Y Guinet and M Descamps, Int. J. Pharm. 2011; 417, 1731
5.
T De Beer, A Burggraeve, M Fonteyne, L Saerens, JP Remon and C Vervaet, Int. J. Pharm.

2011; 417, 3247
6.
J Mller, K Knop, M Wirges and P Kleinebudde, J Pharm. Biomed. Anal. 2010; 53,
884894
7.
H Wikstrm, PJ Marsac and LS Taylor, J. Pharm. Sci. 2005; 94, 209219
8.
KL Davis, MS Kemper and IR Lewis, in Pharmaceutical Applications of Raman

Spectroscopy, ed. S. Sasic, John Wiley & Sons, 2008.
9.
F Wang, JA Wachter, FJ Antosz and KA Berglund, Org. Process Res. Dev. 2000; 4,
391395
spectroscopy in quantifying API in a blended granule and in expanding

the design space of a process-scale reaction.
Blend uniformity
Jayawickrama et al used Raman spectroscopy to predict API content in
granules at the feed frame of the tablet press, which is the last step
before tablet compression 10. Raman-calculated blended granule
uniformity and HPLC-calculated content uniformity of the final tablets
were found to be in good agreement. Predicted API of the granules as
they passed from the hopper into the feed during tablet compression
correlated well with the nominal API concentration of the blend. Raman
10. D Jayawickrama, Am. Pharm. Rev., November/December 2006, 1017
data were used to isolate the compression start time and end time as
11. J Wasylyk and R Wethman, From Development to Plant Implementation of Raman

Methods: Strategy, Challenges, and Solutions, http://bit.ly/1OdRjIh
well as continuous and non-continuous compression time periods,
48
SHOW PREVIEW
30th International
forum and exhibition
IFPAC is the essential meeting place for the latest developments in process analytical technology (PAT) and quality by
design (QbD) within the pharmaceutical, biotechnology and related industries. For over 25 years, IFPAC has brought
together leading experts in industry, academia, research institutions, manufacturing and supplying, as well as
international and United States regulatory agencies to discuss the latest trends in technologies, standards and controls.
High-profile speakers at IFPAC will include those from the FDA, EMA,
Technical Sessions
NIST, CPAC, MIT, Duquesne, Rutgers, and global representatives from
Tuesday 26th January 2016
numerous pharmaceutical and biotechnology corporations. The
Morning sessions include: clinical relevanceQbD; quality risk
extensive range of topics provides critical tools and solutions for
managementII; process monitoring and control-II: PAT implementation
continuous improvement, innovative thinking and overall process
for drug product development and manufacturing; lifecycle
understanding and control.
management of analyser and method robustness; MSAT in biologics

manufacturing; and high resolution imaging.
Benefits of IFPAC 2016
Afternoon sessions include: innovative/emerging technologies for
Share in pro-active discussions with the FDA, EMA, industry leaders,
manufacturing; quality risk managementI; process monitoring and
and academia from across the globe;
controlI: automation/intelligence-based manufacturing; enhanced
Develop new skills and applications to boost productivity,
approaches to analytical methods; quality metrics; and NIR
streamline implementation processes, enhance operational
implementation/QbD experience.
efficiency, and maximise resources;

Become a part of positively impacting the future of your industry!
Also in the afternoon: RtRT/new technologies; fundamental

University research and its industrial applications; product and process
robustness; from data to decisions: leveraging analytics to drive
Comprehensive tracks will cover important research, trends,
process decisions: international consortium for innovation and quality
technological advances, case studies and the latest in regulatory
in pharmaceutical development; advanced manufacturing/bio -
guidance. It will be held in conjunction with the Food Quality, Safety and
pharmaceuticals (bio-manufacturing consortium); and particle
Analysis Symposium.
characterisation/engineering.
IFPAC 2016 themes
Wednesday 27th January 2016
Innovative technologies for agile manufacturing;
Morning sessions include: advancing pharmaceutical science in the
Process predictability through controls and modelling;
generic industry; knowledge and control of materials during continuous
Quality risk management;
pharmaceutical manufacturing of solid doses; product lifecycle and QA;
Regulatory advancements and emerging initiatives.
excipients; real-time monitoring of bio-reactors and bio-processing;

and portable and handheld Instrumentation.
A comprehensive exhibition of PAT equipment and services, poster
Afternoon sessions include: continuous process verification the
sessions and social events will be available throughout the conference
ultimate PV frontier and continual improvement enabler: practices,
to provide numerous networking opportunities. IFPAC provides a
challenges and opportunities; continuous manufacturing; emerging
unique opportunity to interact with other industries that have been
technology and integrated control strategy for modern pharma-
using PAT for nearly 60 years!
ceutical manufacturing; 1-D process sampling in batch and continuous

manufacturing; process control for biotechnology/pharmaceutical;
IFPAC 2016: Programme overview

Sunday 24th January 2016
In the morning, there will be a pre-conference short course: The Critical
PAT implementation; and PAT for packaging, screening/surveillance.

See our website for additional details, continuous updates, and
information on how to register. Posters are still being accepted.
need of the Theory of Sampling (TOS) for PAC, PAT, Process Monitoring
QC and Product QA, by Kim H. Esbensen, PhD, Research Professor,
Join your fellow exhibitors at IFPAC-2016
Geological Survey of Denmark and Greenland.
Full exhibition services will be provided during the three day exhibit.
In the afternoon, a pre-conference regulatory workshop, Attribute

Based Process Control: Pros and Cons, will take place.
Companies interested in exhibiting are invited to contact exhibition

management as soon as possible.
Monday 25th January 2016

In the morning, there will be a Plenary on: Embracing the Future of
Manufacturing: Quality, Agility, Predictability, chaired by: Sau (Larry)
Lee, FDA, Jose Cardoso de Menezes, Sonja S. Sekulic and Ernie Hillier.
Date: 24-27 January 2016

Location: Virginia (Washington, D.C.) U.S.A
Website: www.IFPACglobal.org
49
0 piotr_pabijan / Shutterstock.com
IN-DEPTH FOCUS: PAT
Monitoring, understanding
and assessing pharmaceutical
process and product quality
1,2
2,4
Huiquan Wu , Bogdan Kurtyka , Geoffrey Wu , Jennifer A. Maguire , Rapti Madurawe , Christine M.V. Moore , Robert Iser ,
1,a
1,b
1
1
5
5
Mansoor A. Khan , Robbe C. Lyon , Patrick Faustino , Lucinda Buhse , Sau Lee and Lawrence Yu
Process analytical technology (PAT) is a groundbreaking approach for pharmaceutical innovation in the 21st century,
representing a new dimension of pharmaceutical manufacturing and quality regulation. It provides unprecedented
opportunities for the pharmaceutical sector to monitor, understand and assess pharmaceutical manufacturing
processes and ensure product quality. In this article, we briefly discuss the history of PAT. A critical scientific review
and technological analysis is conducted in three areas: scientific excellence achieved by the global PAT community;
enabling technology available for PAT development; and current industry trends in utilising PAT for materials
characterisation, process/product development and understanding, and manufacturing. It is hoped that these three
areas together provide the necessary science and technology to support future PAT development. Finally, some of
the future challenges and opportunities for PAT progress are discussed in the spirit of further stimulating the
development and adaption of PAT in the pharmaceutical and academic communities from both pharmaceutical
engineering and public health perspectives.
Brief history of PAT
fundamental-level process understanding 2,8, continuous process
As a major quality initiative and an essential component of the United
monitoring, active process control, real-time release testing (RTRT)9,
States Food and Drug Administrations (FDAs) 21st Century Initiatives ,
and science- and risk-based approaches and continuous
the PAT Initiative2 celebrated its 13th anniversary this year. Two broad
manufacturing10-11. Collectively, the scope and depth of pharmaceutical
pharmaceutical quality programs: FDAs Critical Path Initiative and
innovation has been substantially enriched, as reflected by the
quality by design (QbD)4-5, were launched after the PAT Initiative. During
significant progress made in the vital aspects of PAT development.
this evolution in the FDAs approach to enhancing pharmaceutical
Advances have been seen in innovative PAT research, approved
quality and manufacturing6-7, several concepts have emerged, such as
regulatory submissions for both innovator and generic drugs
50
IN-DEPTH FOCUS: PAT

incorporating PAT into the control strategy, and increasing
popularity of continuous manufacturing and state-of-the-art
manufacturing strategies12 utilising PAT.
The current status of PAT implementation

For either batch or continuous manufacturing, the worst-case
scenario is a poorly understood process that leads to high
variability that, in the end, impacts manufacturing efficiency
and product quality. Ideally, one should have a fundamental
process understanding that is coupled with a technically
advanced control strategy to effectively accommodate input
variability in both raw materials and process parameters
to ensure consistent end product quality. PAT is one
groundbreaking approach to mitigate the potential risk to
pharmaceutical quality2. It has afforded opportunities for
gaining in-depth process understanding, as well as
elaborating complicated process phenomena and dynamics
via in situ real-time measurements at much higher
Figure 1: Applying PAT to the drug substance crystallisation process14,19,28-30,32-33
frequencies. Most importantly, PAT has allowed for the implementation
anticipated in the dynamic process environment; (ii) implementing a
of advanced process control strategies for pharmaceutical
rationale process window-based endpoint determination; (iii) allowing
manufacturing and facilitated the implementation of product life-
active feeder control based on composition in the blender; and (iv)
cycle management strategies, as highlighted in the recent ICH Q12
linking incoming material attributes with critical quality attributes of
concept paper13.
final drug product or establishment of a process design space.
The global scientific community has invested substantial effort into
Additionally, utilising PAT for blend monitoring during continuous
embracing PAT, as evidenced by hundreds of publications summarised
manufacturing can aid in maintaining this critical step in a state
in recent reviews
of control.
. As a science-based regulatory agency, the FDA has
14-18
also conducted both independent and collaborative PAT research

(2) technical feasibility for real-time monitoring of many pharmaceutical
Case study 3: Integrated top-down PAT approach with

a bioreactor cell culture process
unit operations; (3) process dynamics and process design space
The technical feasibility of using FTIR Spectroscopy to track and monitor
mapping via real-time monitoring and multivariate analysis (MVA);
four key cell culture metabolites (including glucose, glutamine, lactate
(4) improved process control; and (5) evaluation of emerging
and ammonia) and the monoclonal antibody yield of a bioreactor model
technologies. To highlight these efforts, three representative case
IgG3 cell culture process in real time was demonstrated via a top-down
studies employing integrated PAT approaches are shown schematically
rational stepwise approach. The level of complexity of the experiments
in Figure 1, Figure 2 (page 53) and Figure 3 (page 55) with the main
was increased incrementally. The important bioprocess implications
advantages of each approach highlighted afterwards. For in-depth tech-
include early indicators to enable a go or no-go process decision,
nical details and discussion, please refer to the original literature19-36.
accelerated understanding of cell culture process dynamics, and
studies in the following areas19-35: (1) in-depth process understanding;
provision of vital information for enhanced process control.
Case study 1: Integrated PAT process engineering

approach for crystallisation
The enabling technology has arrived
The advantages of the PAT approach in this case study lies in gaining
The PAT initiative has fostered the development and commercialisation
an in-depth understanding of the pharmaceutical crystallisation
of at- in-, and on-line methods and technologies. In turn, those
process, including: (i) the process trajectory such as when significant
technological advancements have greatly facilitated the development
events such as nucleation and crystal growth occur; (ii) the derived
and implementation of PAT in the pharmaceutical sector. A PAT book37
kinetics of the crystallisation process, enabling better design of
discussed some of the spectroscopic tools and implementation
vessels and control of crystal size; and (iii) the process design space,
strategies for the chemical and pharmaceutical industries as of 2009.
resulting in a drug substance with desirable quality attributes for
Based on the variety of material properties being measured or
downstream processing.
monitored, a non-exhaustive list of existing or potential technologies

are summarised in Table 1 (page 53). A recent review40 highlights the
Case study 2: Integrated PAT and QbD process engineering

approach for powder blending
most important recent developments in infrared (IR), near-infrared
The main advantages of applying PAT to a pharmaceutical powder
laser-induced breakdown spectroscopy (LIBS)41-42 and X-ray fluorescence
blending process are: (i) gaining an in-depth understanding of
(XRF) spectroscopy.
(NIR), Raman, inductively coupled plasma-mass spectrometry (IC-MS),
the blending process thermodynamics and kinetics conceptually, as
For IR spectroscopy, the single most important development
well as the dynamics due to perturbation, and measurement of errors
has been the expansion of instrumental designs and capabilities.
51
IN-DEPTH FOCUS: PAT

New radiation sources and improved computing, enhanced detector
as a valuable alternative to the traditional means for the data
technology, and advances in theoretical understanding have led to
acquisition of critical process variables, process monitoring, and other
novel designs and expanded choices such as nano-spectrometers,
tasks related to process control.
quantum cascade lasers, and coherent 2D IR spectroscopy.
However, it is imperative to understand the relation of these soft
Incorporating attenuated total reflection (ATR) crystals into a variety of
sensors to drug product critical quality attributes, before they are
sampling devices has made FT-IR ATR ubiquitous in both laboratory and
implemented widely. Regardless of which tool is selected for PAT
process environments36. For NIR spectroscopy, the key developments
application, users should address key quality issues from a measure-
have mainly been associated with new imaging systems43, reduced
ment perspective, such as measurement error34, signal/noise ratio,
instrument size, and reduced cost. For Raman spectroscopy,
averaged value and estimated value of the property measured, data
technological advancements in stimulated Raman scattering (SRS),
quality, etc. For particulate matter measurement and monitoring, it is
coherent anti-Stokes Raman scattering (CARS), tip-enhanced Raman
important that consideration is given to the fundamental chemical and
scattering (TERS) and surface-enhanced Raman scattering (SERS) have
physical mechanisms responsible for the particulate process and how
made Raman imaging more practical regarding speed, applicability, and
these processes affect the subsequent details of the sampling and
spatial resolution.
analysis procedure48, such that a mechanistic understanding of the
Such improvements have effectively established it as a useful

technique for pharmaceutical PAT and have allowed Raman
particulate process itself and its measurement can be established and

will facilitate the interpretation of the particle measurement results.
spectroscopy to further advance into life science and biomedicine.

These spectroscopic advances, in conjunction with the develop-
Current industry trends
ment of innovative process chemometrics, will push the boundary of
The industrial application of PAT in several vital technical areas is
qualitative and quantitative analysis beyond what was previously
described below.
perceived to be possible. Enhanced state-of-the-art analytical

capabilities will fuel future PAT development in the pharmaceutical
Materials characterisation and process development
science and manufacturing arenas.
Technologies employed include NIR, Raman, laser diffraction, and
Furthermore, there are several physical/biological/microbiological
focused beam reflectance measurement (FBRM). The development of
measurement tools that could be potentially adopted as PAT tools.
Raman spectroscopy, and in particular the availability of reliable hand-
In the past two decades, soft sensors or advanced control algo-
held analysers, allows the use of this technology for raw materials
rithms44-47 have been reported in certain advanced manufacturing
screening/identification based on the establishment of spectral
process control applications. Soft sensors have established themselves
libraries capable of identifying multiple products. Drug product analysis
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Operations Production
Tablet Coating
Polymorphism / Crystallization
Process-induced Transformations
Extrusion Monitoring
IN-DEPTH FOCUS: PAT

by PAT may involve assays, uniformity of dosage units, and
moisture content analysis by NIR, and identitification by NIR
and/or Raman. Often these tests are performed as part of
RTRT, which has the potential to provide a better, more
statistically meaningful measure of product quality than final
release testing alone, due to increased sample size and
significantly higher method throughput.
Establishment of process control strategies

It is well recognised that in-process measurements take
full advantage of PAT. The testing speed often allows use
of PAT results in real-time process control. Sometimes
this great advantage may be offset by time-consuming
PAT model development, which requires both engineering
and statistical expertise. However, once the model has
been developed and validated, the value of PAT implementa-
Figure 2: Applying PAT to a pharmaceutical powder blending process23-24,34
tion can be effectively realised on a daily basis. Additionally, PAT results
The design of many continuous manufacturing systems without
are sometimes used as input variables of prediction models for
PAT tools would be extremely difficult.
RTRT. For example, tablet dissolution surrogate models include factors
Given the accelerated development pathway for generic drug
such as blend potency measured by NIR and granules particle
products, the generics industry in general has taken a unique approach
size distribution measured by laser diffraction. PAT measurements are
to actively exploring broader applications of PAT for improving the
also frequently used as model elements of multivariate statistical
efficiency of batch manufacturing and enabling QbD and continuous
process control.
manufacturing. A gradual increase in the application of PAT, mostly

proposed as prior approval supplements to abbreviated new drug
An enabling tool for continuous manufacturing
applications, has been observed over the past ten years. Such a
PAT technologies with their analysis and predictive characteristics
post-approval strategy minimises interruption to the market, whilst
fit ideally into continuous manufacturing control strategies.
allowing manufacturers to continuously improve their product and/or
PAT analysers provide timely results that can be used for feedback or
process using PAT tools during the product lifecycle10. Specifically, the
feed-forward control to achieve and maintain the process under
following NIR spectroscopy applications have been observed in generic
control. They also deliver in-process analytical results for RTRT, either
drug manufacturing: (i) at-line content uniformity (CU) measurement of
directly or as input for models. One PAT analyser often has multiple
the finished drug product; (ii) in-line blend uniformity (BU) monitoring
functions, providing data for both control and RTRT at the same time.
to determine blending end-point; and (iii) as an alternate in-line
Table 1: List of spectroscopic techniques that have been, or potentially, are to be adopted as PAT tools (adopted from Reference38)
Techniques
Quantitative Data
Qualitative Data
Raw material ID
Physical Parameters On-Line Capability
Mid-Infrared
Yes, with attenuated

total reflectance
(ATR) and probes;
usually only
liquid samples
Yes, with ATR and

probes; powder
and liquid samples
Yes, not easy

but similar to
qualitative data
Yes, but not as clean

as NIR
Yes, but more difficult Yes, samples may

than NIR/Raman;
be taken in near
fibers expensive
real time
and brittle
Near-Infrared
Yes, any physical

form; transmission
or reflection
Yes, large numbers

of chemometric
algorithms
Yes, all forms;

ID and quality
Yes, hydrogen
bonding makes it
sensitive to
all changes
Yes, either wireless

units or by fiber
probes from
hardened units
Yes, ruggedised units

are standard
Fourier-Transform
Infrared (FTIR)
Yes
Yes
Yes
Yes
Yes. Promising
in bioreactor cell
culture monitoring
Yes
Chemical Imaging
(NIR and Raman)
Yes, possible with

chemometric
algorithms
Yes
Yes
Yes, for example,

hardness, domain
size statistics, etc.
Not yet. But possible.

One system was
tested in a pilot plant
via a FDA-Pfizer
CRADA39
Not yet. Excellent

for characterising
samples off-line
(intact) quickly
Far-Infrared
or Terahertz
Possible with
chemometric
algorithms
Yes, sensitive to
crystalline materials
Yes, but mostly for

crystalline materials
Yes, intermolecular
THz absorption
makes it easy to
detect interfaces or
layer boundaries
Yes
Yes. Also has

excellent off-line
characterisation
capability (both intact
or destructive mode)
Raman
Yes, as quantitative
as NIR
Yes, insensitive
to water
Yes, portable units

are available
Yes, hydrogen
bonding seen
Yes, mainly liquids
Yes, both portable and

traditional units
Near-Line Capability
53
IN-DEPTH FOCUS: PAT

moisture measurement throughout the granule drying process to
work collaboratively to overcome the challenges and embrace
determine end-point.
the opportunities.
Through procedures and guidance, the FDA strongly encourages the

pharmaceutical development and manufacturing. For instance, FDA
Establishing integrated PAT approach

for critical process variables
CDER Manual of Policies and Procedures (MAPP) 5015.1049 on Question-
Most small-molecule drug PAT applications to date have focused on
based Review includes questions related to on-line/in-line/at-line
real-time process monitoring, rather than active control. Feed-forward
monitoring technologies for routine commercial production and for
or feedback control in a control engineering sense has been rarely
real-time process monitoring and control with the intention of
pursued. Thus, the process control concept as originally proposed in
encouraging drug manufacturers to implement PAT. In the recent FDA
the FDAs PAT Guidance has been adopted in a broad sense, i.e., using
Draft NIR guidance50, the fundamental concepts of development and
real-time process monitoring as part of the control strategy for
validation can be applied to other PAT technologies such as Raman,
endpoint determination, for achieving the targeted value of a certain
FBRM, particle imaging and X-ray, among many other potential
critical material attribute and intermediate, or for critical quality
technologies. With this and other supportive regulatory guidance
attribute of the final product.
adaptation of emerging technologies, including various PAT tools for
documents that have been released or are under development, it is
PAT approaches have been used for manufacturing processes
anticipated that more NIR and other PAT tools will find increased use in
involving solid and liquid oral dosage forms, transdermals and
pharmaceutical development and manufacturing.
parenterals. From a risk-based perspective, once unit operations

deemed to be critical to the process performance and to achieving the
Future challenges and opportunities
clinically relevant CQAs of the final drug product are identified, it may be
Based on the discussion so far, significant and very positive progress
desirable to develop and adopt an integrated PAT approach to monitor
has been made in advancing the scientific and technological aspects
and control critical processes parameters. Such approaches can help to
of PAT. As such, it has catalysed pharmaceutical innovation and
minimise the impact of common cause variability due to raw materials,
helped to advance pharmaceutical manufacturing. To stimulate
equipment capability and operators. PAT can also be used to detect and
more interest in the adaptation of PAT and ensure continued
quickly diagnose special cause variation in less critical unit operations.
success of pharmaceutical innovation, several key challenges and

opportunities are discussed below. It is key to recognise the
Building close collaborations
multidisciplinary nature of proper development and adoption
Given the multidisciplinary nature of PAT, close relationships are
of PAT, necessitating that industry, academia and regulators
essential. Technology vendors, often in collaboration with academia and
IN-DEPTH FOCUS: PAT

industry, have spearheaded the design and development of
technologies potentially suitable for pharmaceutical adoption.
The pharmaceutical industry, including both innovator and
generic companies, has been on the frontier, first pushing
and then growing those prototype technologies into matured
and commercialised manufacturing technologies. Meanwhile,
the FDA and other regulatory authorities encourage innovative
manufacturing technologies for the benefits of public health.
Several successful industrial manufacturing technology
examples, which have PAT control characteristics, include laser
drilling technology for osmotic products (precision
manufacturing and control), 3D printing technology for solid
oral products (algorithm control), bi-layer technologies for
controlled-release products (closed-loop thickness control),
and continuous manufacturing for breakthrough therapeutic
products (closed-loop steady state control). On the analytical
side, wide arrays of advanced analytical technologies have the
potential to be adopted for PAT implementation.
PAT tools, whether quantitative or qualitative, should
Figure 3: Applying FTIR-oriented PAT to bioreactor in a cell culture process36
provide adequate assurance of the measurement results, such as
coating, compression or encapsulation; (iii) improved analytical
through fulfilling relevant analytical method validation requirements51-52
sensitivity to safeguard against drug contamination, especially from
or by following first principle approaches . For a successful PAT
highly toxic impurities and to enable early detection of potentially
application, additional unique aspects need to be addressed
carcinogenic substances; (iv) novel manufacturing processes or drug
adequately. When implementing probe-based PAT process monitoring
delivery system manufacturing; and (v) facilitating robust product
for quantitative purposes during the manufacture of solid oral dosage
lifecycle management. The upfront capital investments for PAT could be
forms, it is important to address the scale of scrutiny
21
, a key issue
more readily justified with the realisation of the benefits afforded by
regarding how the probe-based measurement relates to the population
these PAT advancements and the positive impact on product quality
value of the entire body of materials in the process equipment. This is
and, ultimately, the patient.
23,34
especially important for a batch process and an unsteady process

where material property heterogeneity is expected prior to reaching the
Concluding remarks
true process endpoint.
The first decade of the PAT journey has been characterised by global
In addition, when using statistical design of experiments
scientific excellence and innovative research, the significant
approaches to study, involving large-scale manufacturing, it can be
advancement of PAT-enabling technologies, and successful regulatory
beneficial to balance the data representativeness with the cost of
submissions for PAT being applied to material characterisation, drug
experiments, especially at commercial-scale manufacturing. One factor
substance/drug product development and manufacturing monitoring
that has greatly accelerated the successful implementation of PAT in
and control. These chemistry, manufacturing, and control activities,
pharmaceutical manufacturing is the availability of experienced
together with other positive changes, have collectively stimulated
professionals in the areas of process engineering, control engineering,
pharmaceutical innovation, in-depth process understanding, and
formulation science, analytical chemistry, chemometrics and applied
enhanced pharmaceutical control. Although significant challenges
statistics. Multiple academic centres offer training on these subjects,
remain, exciting collaboration opportunities exist for advancing PAT
which allow pharmaceutical scientists and engineers to develop the
development, such as developing integrated PAT approaches to
complex predictive models required for PAT applications. Applying
monitor and control critical unit operations and developing advanced
statistical methods to sampling plans assures quality of the entire
PAT systems with sufficient sensitivity and accuracy to address unmet
batch from discrete PAT measurements. However, development of
public health needs or concerns. Continued close collaboration among
advanced process chemometrics to handle advanced process control
industry, academia and regulators will be instrumental to expand the
algorithms for complex manufacturing processes, such as fully
technology frontiers beyond the domain of todays possibility and
integrated continuous manufacturing lines, has been challenging.
realise the full public health benefits of PAT.
Addressing unmet public health need
Acknowledgements
Opportunities exist for future PAT advancements to address public
We are very grateful to Dr. Janet Woodcock, Director of the Center for Drug
health needs and/or prevent potential safety issues due to
Evaluation and Research (CDER) at the FDA, for leading the FDAs
compromised manufacturing and quality. Such opportunities include:
Pharmaceutical Quality Initiatives in the 21st Century. Special acknowl-
(i) process monitoring and controlling narrow therapeutic index drug
edgements are extended to: Dr. Ajaz S. Hussain who championed the PAT
manufacturing; (ii) monitoring and controlling polymorphic form
Initiative; to the original FDA PAT Teams (including the PAT Steering
transformation or other phase transition during wet granulation, drying,
Committee, PAT Policy-Review-Inspection-Compliance-Research-
55
IN-DEPTH FOCUS: PAT

Education Teams, and Chemometrics Working Group) for their foundational PAT work about a decade ago; and to the FDA-Pfizer Chemical
Author affiliations
Imaging PAT Cooperative Research and Development Agreement (CRADA)
1.
Office of Testing and Research (OTR), Office of Pharmaceutical

Quality (OPQ), Center for Drug Evaluation and Research (CDER),
Food and Drug Administration (FDA), Silver Spring, MD 20993, USA
2.
Office of Process and Facilities (OPF), OPQ, CDER, FDA, Silver

Spring, MD 20993, USA
3.
Office of Lifecycle Drug Products (OLDP), OPQ, CDER, FDA,

Silver Spring, MD 20993, USA
4.
Office of Regulatory Affairs (ORA), FDA, Silver Spring,

MD 20993, USA
5.
OPQ Immediate Office, CDER, FDA, Silver Spring, MD 20993, USA
a.
Current address: Texas A&M Health Science Center, Rangel College of

Pharmacy, College Station, TX-77843, USA
b.
Current address: 12013 217th PL NE, Arlington, WA 98223, USA
Team and the FDA-Novartis PAT Design Space CRADA Team for their multiyear innovative works. Huiquan kindly acknowledges Drs. A. M. DSa, S.
Chatterjee, F. Weichold, V. Vilker (retired), Mr. F. Friedman, the AIChE PD2M
Forum leadership team, a team of subject matter experts at Actavis Florida
for technical discussions on innovative manufacturing technologies, and
colleagues and fellows at the Division of Product Quality Research (DPQR,
OTR, OPQ, CDER, FDA) for their outstanding support. All of the PAT
practitioners in the global community across the pharmaceutical industry,
developers of technologies and providers of PAT instrumentation,
academia, and governmental agencies, including those original works not
being cited here due to space constraints are appreciated for their creative
works over the past decade.
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IN-DEPTH FOCUS: PAT ROUNDTABLE
Dr. Marek Hoehse
Albert Tulke
Karen Esmonde-White
PAT Department,
Sartorius Stedim Biotech GmbH
Head of PAT Solutions at

Bayer Technology Services
PhD, Senior Marcom Specialist,

Kaiser Optical Systems, Inc.
Moderator
David P. Elder, GlaxoSmithKline and JPAG
Current pharmaceutical regulatory interpretation

(21 CFR Part 11) requires the retention of all historical
spectral data; which for any fully PAT-aligned,
data rich system could be a significant undertaking.
This is a technical burden that other industries
do not share. How does the panel think this
requirement can be addressed?
users, since a number of PAT instruments do not possess the technical
Hoehse: One solution could be to divide the spectral data into two
itself or by an external service provider.
prerequisites to comply with pharmaceutical regulations. It is most

desirable that in future, software of PAT devices will be in accordance
with 21 CFR Part 11 requirements. Furthermore, data should be transferred online, be traceable and safely routed to data management, data
storage or MES systems, ideally also in a non-wired environment,
e.g., GMP-conform clouds. Data storage may be done by the company
branches any spectral data that lead to any action on the product or
process will have to be stored completely, and all other data do not
Esmonde-White: High volumes of data produced by in-line PAT systems
have to be stored since they have no influence on process or product.
pose challenges in data analysis and data storage. Backwards
Additionally, one could reduce the data load by only storing the
compatibility of data readers is an important consideration in order to
first few scores of a principal component analysis instead of
pull meaningful information from archived data. The first step towards
full spectra, since the first scores likely explain more than 95% of the
addressing how to store large volumes of data and their associated
overall variations.
metadata is to define the volume. An open dialog with analytical

instrument vendors, process engineers, data management system
Tulke: Currently, there are technical solutions existing to fulfil these
vendors and regulatory agencies may provide innovation solutions such
requirements. These solutions are often tailor made and put in place by
as stratifying storage approaches based on the data criticality.
57

There is currently a lack of analytical data format
standardisation for example, object linking and
embedding for process control (OPC), and allotrope
which would allow unified data treatment between
various instrument vendors. What efforts are
analytical instrument vendors putting in place
to address this shortfall?
Standardised solutions as field device integration (FDI ADI) or software
Hoehse: There are some attempts to establish a standard for device
Hoehse: A standard interface as discussed in the last question would be
communication and thus data structure and transfer, e.g., OPC-UA.
helpful for PAT integration. Additionally, intelligent data collection
However, a standard cannot be forced to the market; only a broad
software allows the coupling of dozens of different interfaces and
customer acceptance will lead to a standard. So far, customers demand
combines data from different origins into one database with a standard
various technical solutions depending on their individual process
data format, independent of individual process or IT infrastructure.
for unified data treatment are part of these efforts.
Similarly, for better feed-forward or feedback control

there is a need for better integration of PAT into the
control systems. How does the panel think this can
best be implemented?
control infrastructure. Furthermore, interfaces

like OPC-UA require substantial license payments and thus are only affordable for complex
sophisticated analytical techniques and
less attractive for simple analytical sensors.
A standard platform will have to be attractive
In combination with multivariate control tools
Integration of PAT into a control

system is a personalised process that
requires a customer to work closely with
the analytical instrument vendor and a
data management system
for all analytical devices and sensors.
this will be a powerful solution as correlations

between data is revealed and used for
improved predictive process control.
Karen Esmonde-White
Tulke: OPC UA allows bidirectional connectivity. Challenging is rather registration of
production processes and qualification of such autonomous systems.

Esmonde-White: Analytical spectroscopy instrument vendors already
Using one common communication standard would simplify the
have industry guidelines regarding standard data formats, including
integration of PAT into control systems like DCS. In this respect,
JCAMP-DX and ASTM specifications. And most spectroscopy instrument
the United States Food and Drug Administration and the User
vendors enable options to save data in standardised formats.
Association of Automation Technology in Process Industries, NAMUR,

gave a recommendation for OPC UA. The next step should be the overall
Tulke: Bayer is not only an instrument user but also a supplier.
agreement of the suppliers on a common standard.
Therefore, we are very aware of these topics and Bayer is a partner in

the Allotrope Foundation. Analytical instrument vendors offer a broad
Esmonde-White: Integration of PAT into a control system is a
range of interfaces, which start at 4-20mAMP and extend via Profi- or
personalised process that requires a customer to work closely
Modbus connections up to OPC or OPC UA. Reduction of this variety
with the analytical instrument vendor and a data management
of interfaces would decrease the effort for analytical data collection.
system, whilst considering the end user and plant environment.

Kaiser has several features in its analyser software
to enable its integration with third party control
systems and PAT Knowledge Management
Solutions. As a result, the end user can run and
control the Kaiser analyser using familiar control
system tools without needing to learn instrumentspecific software.
There may be evolving requirements for

enhanced analytical sensitivity for realtime release testing, that is, <0.2% levels
for related substances (in line with
ICH Q3A/Q3B). How do you think this
can be accomplished?
Esmonde-White: Analytical technologies and
methods are an exciting field of research growth,
with new methods always in development. It is
possible that when the regulatory guideline, as
Tonhom1009 / Shutterstock.com
stated in ICH Q3A/Q3B, becomes a requirement,
58
there will be analytical methods that can deliver

high-speed trace measurements in a real-time
release testing context. Existing spectroscopy,
chromatography, mass spectrometry and nuclear
magnetic resonance methods are continually
Gayvoronskaya_Yana / Shutterstock.com
being evaluated for their suitability in a real-time release
in situ cleaning by rinsing the sensors surface in-line or at-line, or
testing context. I expect that, as part of that evaluation, the method
alternatively, a single use concept for sensors. By strengthening their
sensitivity and limit of detection will be balanced with speed and
expertise regarding PAT applications the appliers are able to develop
method transferability.
and optimise together with the vendors cleaning solutions and

maintenance concepts. Furthermore, vendors may offer material that is
Tulke: Highly specialised expert groups are required for the
less susceptible for adherence of particles.
development and application of these sensitive PAT solutions.
Most desirable is the possibility of in situ cleaning of the
In the past decade the pharmaceutical industry has been working to
sensors surfaces without disturbing or contaminating the process.
build up such groups. In other industries, this

development is already much more advanced.
In addition, this topic is addressed by the
implementation of research projects in order to
develop new selective and sensitive PAT
In addition, real-time monitoring should be
Highly specialised expert

groups are required for the
development and application of these
sensitive PAT solutions
methods by the vendors refer to NAMUR

sensor roadmap like it is funded by the EU
Albert Tulke
implemented in order to track the success of

cleaning steps.
Hoehse: Sensor surfaces like sapphire
have a lower surface roughness compared to the steel of the reaction vessel.
project Horizon 2020. Furthermore, it is also necessary that universities
Thus, if sensor clogging is a specific problem, the complete
provide enough qualified graduates with a sound PAT know-how.
process might lack homogeneous distribution of all ingredients.

The sensor is just the only part of the process where clogging is
Hoehse: There will not be a single standard technique that will meet all
detected! In some cases surface treatments of sensor interfaces,
requirements for all applications. The combination of different
e.g., with layers of nanomaterials, might be one solution to improve
orthogonal analytical techniques and their combined data evaluation
surface quality and to reduce particle adhesion. On the other side,
might result in reduced LODs. Optical spectroscopy is already proven to
the overall trend towards single-use technologies, e.g., single-use
have limits of detection below 0.2%, however the limits are not several
reaction vessels for bioprocess applications, makes in situ cleaning
degrees below. Therefore, mass spectrometry might be the candidate of
obsolete in the first place.
the future, since it combines fast measurement with extremely low

detection limits. However, this technique will likely only be suitable for
Esmonde-White: Phase-specific probes for in-line measurements
bulk material and not for analytics on single dosages.
address challenges of measurement, cleaning and recalibration. Kaiser

has phase-optimised probes for gases, solids and liquids. For
There is a clear need for better in situ cleaning of

sensors and/or selection of improved construction
materials and this is particularly applicable for solutionbased processes, such as crystallisation, where particles
can adhere to sensor surfaces. What future improvements
do the panel envisage to overcome this issue?
applications in liquid-based processes, Kaisers immersion probe
Tulke: On the market, different ways to overcome this are available:
coatings and probe designs.
features a bevelled tip to avoid particle aggregation at the probe tip. We

have also addressed process compatibility through customised
solutions for customer plants, and we are developing probes that can
be removed, cleaned and reinserted without affecting the process.
Looking ahead, we anticipate technologies in disposables, probe
59
SHOW PREVIEW
Michael M. Gottesman (left), M.D.,

National Cancer Institute and
Adam Diedrich Steltzner (above), NASA
Jet Propulsion Laboratory, will be hosting
keynote presentations at SLAS 2016
SLAS is proud to present the Societys Fifth Annual Conference and Exhibition. Bringing together the best and
brightest minds vested in developing and using technology for life science R&D, SLAS2016 will serve as the epicenter
of science and technology for five days this January.
Information
Nowhere else will you have such convenient access to a wealth of new
Podium presentations, case studies, posters, tutorials, exhibits, short
technology, product expertise and user experiences.
courses, user perspectives, career services, panel discussions and off-
Visit SLAS2016.org for the latest list of exhibiting companies,
site facility tours make SLAS2016 five days of high-volume, high-impact
product descriptions, an exhibition floorplan and a schedule of events
information.
in the exhibition.
Innovation
Networking
Whats new in scientific innovation is at SLAS2016; from breakthrough
A hallmark of the SLAS experience is intelligent network building. Attend
products to breakthrough applications of established technology,
SLAS2016 and reap the benefits of being a part of the SLAS community,
SLAS2016 will showcase what is coming and what has arrived and
not only during the conference, but for the other 360 days of the year.
how that innovation can drive value.
Networking activities at SLAS2016 include daily meals and receptions in

the exhibition, evening functions and programming, a fun run with
Inspiration
fellow attendees, Special Interest Groups (SIGs), special programming
Be it in a hallway conversation with a fellow attendee or hearing a case
for students, early career professionals and international guests, and
study from a podium, SLAS2016 will be serving up a full dose of aha
much more.
moments. As a community united by pioneering new scientific horizons

using technology, and with a programme curated by a committee of
SLAS2016 last night celebration
practicing researchers, SLAS2016 will leave attendees inspired.
The final evening of SLAS2016 will be one to remember as conversations

continue aboard the USS Midway one of Americas longest-serving and
Scientific programme
most impressive aircraft carriers. From stem to stern, the entire ship will
The scientific programme is the cornerstone of SLAS2016. Compiled by a
belong exclusively to the SLAS community. Attendees will explore the
team of practicing scientists and scientific technology users, the
20 stories high, 1,000 ft. long, 64,000 ton, 212,000 horsepower aircraft
programme represents original research, case studies and innovative
carrier while meeting old friends and making new professional contacts.
technology advancement. In-depth tracks include:

Advances in Bioanalytics, Biomarkers and Diagnostics;
Career connections
Assay Development and Screening;
SLAS2016 provides a host of unique resources to help you accelerate
Automation and High Throughput Technologies;
your career and distinguish yourself in a competitive job market. Career
Cellular Technologies;
coaching, career workshops, one-on-one mentoring and resume
Drug Target Strategies;
reviews are among the complimentary services provided.
Informatics; and
Micro/Nano Technologies.
Registration
SLAS2016 is pleased to offer significant registration discounts to
See complete details on the scientific programme on the website.
advance registrants, for groups of registrants from the same industry

organisation, for professionals from academic institutions and
The exhibition
government agencies, and for students. Advance discounts are available
The SLAS2016 exhibition is an all-in-one venue allowing you to see the
until the 18th December 2015.
latest technologies and to visit with product experts and developers

from more than 300 leading multinational providers of scientific
Date: 23-27 January 2016 Location: San Diego, California, USA

More information: www.SLAS2016.org
technologies. From robotics to reagents, SLAS2016 is a unique

opportunity to see, hear, touch and feel breakthrough innovation.
60
Visit European Pharmaceutical Review at Booth 337
mindscanner / Shutterstock.com
REGULATORY INSIGHT
To float or not to float?

Sue Staunton
James Cowper Kreston
For many years, global markets had focused their attention on traditional sectors such as natural resources rather
than on life science companies, so initial public offerings (IPOs) for such companies were uncommon. The past few
years have, however, seen a sea-change and life science companies from biotech to medical devices firms have
come very much back into favour as the must-have stocks; with opportunities for significant upsides.
The turnaround started in 2013; the markets began to pick up that year and 66 life science IPOs were completed
globally. By 2014, there were an extraordinary 133 life sciences companies completing IPOs, raising some $11 billion.
The first quarter of 2015 seemed to show a dramatic downturn in life
price of $22 per share, its valuation almost immediately jumped to
science stock market launches, however, with a number of analysts
$37 per share. Business was booming.
suggesting that this presaged the closure of the biotech IPO window.
However, the market for biotechs is particularly vulnerable to
This was not to be the case, and the second quarter saw a resurgence;
changes in public policy. On 21 September the US democratic party
June 2015 was the busiest month on the market for the sector in
presidential hopeful, Hilary Clinton, tweeted that she would release a
15 years. During this month alone there were 35 companies that went
plan to combat the high price of prescription drugs. She was apparently
through United States IPOs, raking in some $5.9 billion. When, in early
responding to the news that Turing had acquired an older antibiotic and
September this year, gene therapy company RegenxBio floated at a
increased its price by more than 5,000%. Immediately following this
61
REGULATORY INSIGHT
tweet there was a dramatic drop on the Nasdaq Biotechnology Index. It
announced plans to list on AIM in May 2015, and in September 2015
is clear that Clintons tweet hit a nerve since the sector had already
Finnish company, Faron Pharmaceuticals joined Puretech in
been concerned about drug pricing, but there appeared to have been a
announcing its plans to float in the UK.
knock-on impact on the markets, with a number of US floats, including

Cytomax, Aclaris Therapeutics, Nabriva, Edge and Mirna, not reaching
Turning tides
expected valuations. At the beginning of October, UK company
So has the tide turned for the UK markets in the life science sector? It
Shield Therapeutics postponed its IPO on the London Stock Exchange,
does stand head and shoulders above its European neighbours,
noting that current market conditions did not make it a conducive
however it still trails way behind the US in terms of life science
time to raise cash.
presence. Within the three top US biotech clusters Boston, San
This all serves to indicate the inherent volatility of the public
Francisco and San Diego, for example, there are more than five times as
markets and the consequential vulnerability of life sciences companies
many drugs in development than in the UK. But does that matter? Is
with a presence there. It makes more complex the decision by such a
access to funding not rather more of an issue?

There has been speculation that one
company as to whether or not to float. And

such a decision is never taken lightly. For these
companies, however, that choice is further
complicated by the issue of where to list.
The US has dominated the recent boom in
The market for biotechs is

particularly vulnerable to changes
in public policy
reason UK companies, such as Oxford Immunotech, Lombard and GW Pharmaceuticals, float

in the US is due to the funding valley of
death in the UK at the post seed stage. This
year has seen significant activity to try and fill
life-science IPOs with 106 of the 133 IPOs during

2014 being on US-exchanges and raising some $7 billion, but there has
the gap, with London Mayor Boris Johnson launching a 10 billion
been activity in the UK market and also on other exchanges including,
biotech fund for London in June and Neil Woodfords new 700 million
notably, Australia.
fund Woodford Patient Capital Trust similarly being set up.
Where to float
very exciting and dynamic life sciences businesses, well supported by
So should UK life science companies stay in the UK or take their comp-
academia. We have the science, the clinical development and
any overseas? According to the Financial Times, UK life sciences
infrastructure in key centres such as Cambridge, London and Oxford,
companies hit a seven-year high in raising finance in 2014. Big sums
with a number of other vibrant hot spots. We also have well proven,
have been achieved; take for example, Circassia, the anti-allergy
world-class management. All the ingredients are there, and whilst the
specialist that raised 200m through its IPO. Other significant 2014 floats
long-term capital might not yet support it, there is a strong investor
included those of Abzena and Horizon Discovery. More recently, UK
community. With the establishment of significant funds such as
company Acacia announced plans to raise funds through a UK IPO.
Johnson and Woodford, the relative attraction of the US markets for UK
But will these make a difference? The UK is certainly home to some
Moreover, Boston healthcare company, Puretech, announced plans
biotech may diminish.
in May 2015 to raise $160 million on the London Stock Exchange rather
And there is, of course, the advantage of being physically close to
than through US markets; US biotech group, Verseon, similarly
the markets. The investment community expects regular briefings and

updates from the companys senior management team, and this can be
James Cowper Krestons guide

to a successful float
To achieve a successful float, companies
must consider the following:
Raising funds before
IPO it is not something
to do on the cheap
A finance team that is

familiar with market rules
and regulations is on hand
based in the same country. But, and these are the critical questions, will
a biotech business raise as much in the UK as it would if it were to go
public in the US; would the company achieve as high a valuation in the
UK as it would across the pond; and in the long term does a US float
help the business to develop more than a UK float would?
For all of the inroads the UK has made, particularly recently, the
The right advisers

are recruited:
US still remains the preferred global location for life science IPOs.
Accountancy firm,
2014 and overseas companies are continuing to move there. The main
Having a strong
management team,
preferably with a CEO
that has taken a
company public before
Ensuring a capable board
of directors is in place
time consuming. It is clearly much easier to put this into effect if you are
with experience
of the life science
sector and knowledge
of the relevant
market regulations
The number of foreign issuer IPOs climbed from 12 in 2012 to 60 in

attraction of the US markets is always going to be the much larger pool
of potential investors that understand the life science sector with the
risk appetites to match. This generates greater interest leading to
higher amounts raised and an increase in value post-IPO. Until the
recent drop, Nasdaqs biotech index outperformed a lot of other
Law firm
sectors during 2015. And with a significantly larger investor and analyst
Underwriter
community, businesses may find it that much easier to tell their story in
the US. There is a lot less education required of the investor than is the
An experienced CFO
is in place
case here in the UK where life science investment remains a highly

specialist and niche market concentrated in relatively few hands.
However, on the flip side, businesses that struggle or do not
62
Taras Vyshnya / Shutterstock.com
REGULATORY INSIGHT
Worth considering: Australia has experienced a resurgence in liquidity
sufficiently play the game may all too quickly fall out of favour in the
road tours, visiting and pitching the companys offering to future
US. A number of orphan companies listed here are ignored by analysts
investors; they will also have to take significant time away from the
and struggling to progress. The US life science investor community is
front-line of the business in working on the IPO process. This will be
fairly ruthless. The volume of new deals coming to the table means that
the case wherever the float market is, but the impact of having to carry
if sufficient progress is not being made within a company, the investors
this out overseas cannot be under estimated.
will move on to the next opportunity.
Once the company has floated, of course, there is the additional

need to provide the investor community with access to the senior
Further afield
management team. This will usually mean two or three visits a year,
Other markets have their attractions as well; Australia, for example, is
again taking time away from the day-to-day operations. Dealing with
regarded as worth considering following a resurgence in liquidity for
this can necessitate the implementation of a second tier of manage-
the sector there as a result of the collapse of the gold price and the
ment within the company. Yet this is unlikely to deter ambitious and
difficulties in the mining industry during 2013.
forward-thinking life sciences companies
However, as with the other markets the

Australian Exchange is also vulnerable to
volatility brought about by external forces.
Nevertheless, the Australian life science and
looking to go public. On average the businesses
Businesses that struggle or do not

sufficiently play the game may all too
quickly fall out of favour in the US
healthcare sector is becoming much more
that launched onto the US capital markets in

2014 ended the year with 35% rises in market
valuations. And that is a powerful pull.
The decision as to which market to float on
mature, and some reasonable deals have been executed. Adherium
is ultimately and primarily dependent upon the liquidity of that market,
raised Aus $35 million in August in its IPO, backed by its partner,
the volume of biotech-savvy investors located there and the
AstraZeneca. The subscription was reportedly oversubscribed by two
valuations that are being achieved there. But of no less significance are
times. An interesting feature of this deal was the position taken by
company-specific factors and contacts the company may have in any
AstraZeneca which put in $3 million of the fundraising. By the beginning
particular location; the influence of investors; and potentially the
of October, however, things had changed with Australias largest x-ray
presence of a lead investor or pool of investors in that location with a
provider, I-MED Radiology, making its IPO, which had been originally
special interest in the very specific technology area the company is
slated to raise Aus $350 million. Integral Diagnostics did get a deal away
involved in.
but its float was priced very much at the lower end of expectations.
Whilst clearly affected by a wider life science market uncertainty, the
Australian market is significantly influenced by its close trade partner,
China, which is gong through a slow-down at the moment.
Flotation on an overseas market whether in the US or elsewhere
has its own challenges and costs to offset against these benefits. During
Sue Staunton is a Partner at accountants and business

advisers James Cowper Kreston. She can be contacted
by email at sstaunton@jamescowper.co.uk. Visit
www.jamescowperkreston.co.uk
the pre-IPO phase the senior management team will have to carry out
63
UNDER THE MICROSOPE
Caroline Richards, Editor of European

Pharmaceutical Review, asks Dr. Arnaud Carlotti,
PhD, HDR, President of Eurofins IDmyk, to provide
his insights on the microbial testing industry.
Why do customers choose your microorganism

identification and typing services?
allows clear cut identification in the majority of cases. For instance, the
The most common reasons for our customers requiring microorganism
when using long 16S rRNA gene sequence, but not when using partial
identification and typing services are that they want the most
sequencing (Bacillus atrophaeus and Bacillus vallismortis are not
comprehensive, cost-effective services with short turnaround times.
differentiated by partial sequencing). Additionally, long sequencing of
They generally need highly reliable identification at the species level of
16S rRNA genes is required in the description of new species in scientific
either bacteria or fungi. They need definitive identification in cases
literature this is the reference, and partial sequence is not acceptable.
biological indicator Bacillus atrophaeus is identified at the species level
of investigations, such as sterility test failures, media fill test failures,
of isolates of a given species in order to determine if they correspond to a
What are the advantages of characterising isolates

at the level of the strain (beyond the species)
by fingerprinting to determine generic relatedness?
given strain (same clone, same origin) or to a different strain (different
The ability to characterise in this way, using molecular methods, is a
clones, different origin) to trace the route of contamination in order to set
critical improvement for investigations. Here too, we have developed
up effective corrective and preventive actions.
and validated original methods and reagents to be able to generate
product non-compliance, environmental monitoring alert level and

action level investigations. For root cause analysis, they also require typing
reliable fingerprints of microorganism isolates in order to determine
Do laboratories most commonly prefer to have

their samples assessed for phenotype, genotype,
or both together, and why?
their genetic relatedness. This information is useful to determine if
Generally, genotypic identification is preferred since it is globally more
cause of contamination.
isolates encountered in sterility test failure investigations are linked or

not, in order to trace the route of contamination and identify the root
reliable than any traditional phenotypic method for definitive
This is real expertise that can help solve problems, answer
identification at the species level of any microorganism. While they
efficiently regulatory expectations and prevent economic losses for the
performed well for years and still do for some species, phenotypic
industries. Our experience over almost 15 years of operation allows us
methods were overtaken by genotypic methods because the latter are
to determine the most frequently involved species in sterility test
not influenced by environment variations; they are universal, and they
failures, media fill test failures, etc., and we have solutions for all those
permit identification at least at the genus level, and most valuably at
species when it comes to typing analyses.
the species level. Even non-living microorganisms can be identified.
recognised as the most reliable methods for identification of
What is the future for microbial testing methods

and tools do you see the field evolving still
further and will changing customer requirements
shape any such evolution?
microorganisms at the species level.
The future for microbial testing methods and tools relies on future
Moreover, technical and scientific improvements have led to the

availability of genotypic methods for rapid and cost effective analyses.
Genotypic methods have been evaluated extensively and are
technical and scientific improvements. Regarding phenotypic methods,
Your Microbial Identification Database offers long

sequencing of 16S rRNA encoding genes for genotypic
identification. Could you explain why this is better
than other, more conventional, sequencing methods?
matrix assisted laser desorption time of flight (MALDI-TOF) (proteomic
Our long sequencing of 16S rRNA encoding genes (roughly 1,300-1,400
sequences of multiple genes allow simultaneous and highly reliable
base pairs) is more reliable and more powerful for identification than
identification, and even typing of multiple strains, in a very short
partial sequencing (450-500 bp) utilised by other laboratories.
amount of time. We have applications already validated in this field.
analysis) technology is really promising for routine identification of

many bacteria. Regarding genotypic methods, with the development of next-generation sequencing, comparative analysis of
Our extensive database, validated with more than 8,400 bacterial
We can expect the field to evolve still further, as long as the
species reference sequences, covers almost all the recognised species
proposed solutions will be scientifically sound and recognised, rapid,
encountered in the pharma industry. Long sequencing helps
highly reliable and cost effective. Only then will customers expecta-
discriminate species not distinguished by partial sequencing, and it
tions and confidence be fulfilled.
64
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www.europeanpharmaceuticalreview.

You want to be sure

RUSSELL PUBLISHING LTD

VOLUME 20 ISSUE 6 2015

European Pharmaceutical Review

Jumping on the M&A bandwagon

Caroline Richards, Editor,

Designing a novel continuous

Ravendra Singh, Jun Zhang, Marianthi Ierapetritou and

Use of the purge tool in

The limitations of the colonyforming unit in microbiology

43 IN-DEPTH FOCUS: PAT

To float or not to float?

MIQE compliance in expression

Sue Staunton, James Cowper Kreston

64 UNDER THE MICROSCOPE

COMING UP IN THE NEXT ISSUE:

NIR In-Depth Focus

Raman In-Depth Focus

Published February 2016. Dont miss out on

VOLUME 20 ISSUE 6 2015

European Pharmaceutical Review

Innovation with Integrity

Use of the purge

the medium and liquid/liquid extraction. Finally, chromatography is

generating significant amounts of supporting analytical data. This

allocated values of 10-100 based on the efficiency of the final separa-

approach exemplifies a quality by testing (QbT) paradigm it is very

resource-intensive (especially when applied to every PMI/MI that could

a stage purge factor. Then each stage purge factor is multiplied to

occur in a synthetic process), it can be technically challenging, and it

produce the overall purge factor2,3.

runs contrary to the underlying principles of quality by design (QbD).

Lhasa recently initiated a cross-industry collaboration (Mirabilis)

Tellingly, this QbT approach fails to acknowledge that these reactive

to further develop the purge tool to generate a semi-quantitative and

MIs will likely be purged by prevailing downstream chemistry conditions.

reproducible approach to predicting the probable purge factor for these

As a consequence, impurity fate mapping (or purging capability),

impurities at each stage within a synthetic process. One of the key

analytical testing and control strategies for MIs/PMIs in drug sub-

goals of this consortium is to make the process consistent, open and

stances and products have received significant focus during the

transparent, so that regulators can easily view and interrogate the

evolution of this guidance.

findings, increasing the chances of regulatory success and providing

One of the areas of particular interest to Industry is purging of MIs,

regulators with increased assurance of the output. The initial version of

because of the huge analytical resource burden required to support the

the Mirabilis software was released to the existing consortium

current GRA process. Significant reduction in the analytical resource

(comprising Lhasa and eight pharmaceutical companies) in late 20144.

a synthetic process to purge or remove a particular MI. The underlying

principles supporting the purge factor are straightforward and involve

The implementation of the purge factor and the activities of the

identifying those intrinsic physicochemical factors affecting the

Mirabilis consortium have the potential to significantly decrease

processes capability to remove a specified MI. The most important

analytical testing of MI/PMIs without any additional impact on patient

parameters are reactivity, solubility, volatility, ionisability, as well as any

safety. This approach is clearly aligned with the guidance provided in

other physical processes that remove impurities, e.g., preparative

chromatography. These parameters are then allocated a relative weight

(based on a pre-defined scoring system) in the overall purge factor .