Vaccine 31 (2013) 12641267

Contents lists available at SciVerse ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Review

A review on current downstream bio-processing technology of vaccine products

Michael Li, Ye Xian Qiu
School of Chemistry & Biotechnology, Suzhou University of Science and Technology, Gao Xin District, Ke Rui Road, No. 1, Suzhou, China

a r t i c l e

i n f o

Article history:
Received 7 November 2012
Received in revised form
12 December 2012
Accepted 20 December 2012
Available online 9 January 2013
Keywords:
Downstream bio-processing of vaccine
products
Clarication
Chromatography
Process analytical technology
Linear pH gradient

a b s t r a c t
An effective downstream bio-processing of vaccine products requires complete chemical knowledge of
the contaminants that may arise from a given vector expression system. Whether the vaccine is made from
the traditional egg-based or the new cell-cultured process, it is the expression system that determines
the types of impurities that need to be identied and removed from the vaccine product.
There are mechanical and chemical factors that can either reduce the yield or render a vaccine product
to be irreversibly inactive. The choice of equipment and solvents is therefore important in minimizing
product loss, and for maintaining an efcient and optimized manufacturing process.
The frequent out-of-specication, irreproducible data and inefciency in the manufacturing of biologics
were the basis for FDA to propose the cGMP for the 21st Century initiative in the year of 2000. Effective
2004, the concept of quality by design (QbD) has been imposed in the manufacturing of biologics. To
facilitate the implementation of QbD FDA has encouraged the use of process analytical technology (PAT).
Further, FDA believes that an optimized manufacturing scheme requires one to identify and to control
the variables that can negatively affect the yield and quality of the desired product, and PAT can reveal
wrongful data and alert the operator for immediate correction during processing.
2012 Elsevier Ltd. All rights reserved.

Contents
1.
2.
3.
4.
5.
6.
7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264
The chemical variety of potential contaminants in a vaccine product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
Cutting-edge downstream bio-processing technology to improve manufacturing efciency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
Clarication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
Polishing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267

1. Introduction
A vaccine is an antigenic preparation, which induces your
body to produce an active immunity against a pathogen. Vaccines
exist in many different forms. They may contain an inactivated
micro-organism, or live, attenuated virus, or some unique molecular component of the organism that causes the disease. Human
vaccines, for example, for yellow fever and many others are conventionally manufactured in fertilized chicken eggs. However, the
vaccine production scale-up challenges include slow production
time, limited throughput and low yield [1]. The 2009 inuenza

Corresponding author. Tel.: +86 13962103891; fax: +86 512 80912009.

E-mail addresses: mli138@yahoo.com (M. Li), QYX542@mail.USTS.edu.cn
(Y.X. Qiu).
0264-410X/$ see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2012.12.056

A (H1N1) pandemic prompted the World Health Organization to

call for the establishment of an economically sustainable base of
annual u vaccine production around the world. In recent years,
vaccine production platforms, other than egg-based, such as bacteria, micro-organisms, recombinant mammalian cell-culture, and
plant-based cell culture have been examined and signicant progresses have been made.
A major issue in vaccine production is the occurrence of biological contaminants that originate from in vivo production platforms.
Because the vaccine production process is vector dependent, each
production platform poses its own inherent contamination risks.
However, recent improvements have shown promising developments in contaminate-free production methodology. Research
data have proven that a purer vaccine product along with faster
processing time and free of allergenic egg protein can be accomplished using cell culture-based methods [2]. An example is Optau

M. Li, Y.X. Qiu / Vaccine 31 (2013) 12641267

vaccine, which is manufactured by Novartis. In this vaccine, the

virus is grown in a cell-cultured medium instead of the conventional egg-based method [3].
As continual developments in upstream processing have
enhanced the capacity for vaccine production, the main bottleneck
has been in downstream processing. Thus, this review focuses on
the latest development on downstream bio-processing technologies of vaccine products.
2. The chemical variety of potential contaminants in a
vaccine product

1265

eliminate extracellular proteins and other unwanted bio-molecules

so that the purity of the vaccine product satises safety requirements set forth by regulatory agencies [5].
Another concern of vaccine production is product integrity.
Vaccine products can be inadvertently de-natured or rendered
irreversibly inactive due to mechanical damage caused by manufacturing equipment or by deleterious chemical effects from harsh
solvents used during processing. Hence, the choice of equipment
and solvents is important in minimizing product loss, and for maintaining an efcient and optimized manufacturing process.
4. Clarication

To meet the specic purity goals of regulatory agencies, a feasible and effective purication scheme requires complete chemical
knowledge of the contaminants that may arise from a given vector
expression system. In the case of in ovo production of inactivated
virus, live viruses as well as uid-containing antibiotic is purposely
injected into fertilized eggs during the incubation cycle. The live
viruses and antibiotics are subsequently removed from cell lysates
via ltration or centrifugation. The puried viral product is then
inactivated with a disinfectant followed by treatment with detergent to break up the viral oligonucleotides into particles. Hence,
the eventual removal of additives is necessary and important, and
should be considered as part of the total purication program. Some
live vaccines, in addition to non related viruses, may also contain
bacteria, host cell proteins, DNA, RNA, and endotoxins. If the vaccine is made from an alternative platform such as cell culture-based
methodology, host cell proteins should be considered as potential
contaminants for removal. Therefore, it is the expression system
that determines the types of impurities that need to be identied
and removed from the vaccine product [4].
Contaminants are broadly divided into two categories, namely
gross contaminants and micro-heterogeneity in the structure of the
nal product. Gross contaminants are bacteria, viruses, proteins,
nucleic acids, lipids, polysaccharides and low molecular weight
substances commonly found in the host. Micro-heterogeneity
refers to the presence of trace amounts of modied versions of the
product itself due to truncated fragments, incomplete transcription, degradation, oxidation, or reduction processes. Regardless of
whether one works with egg-based, cell-based or other types of
production process, the eventual removal of all contaminants and
non-essential excipients in the vaccine product is a crucial step
that requires the implementation of the appropriate purication
technology.
3. Cutting-edge downstream bio-processing technology to
improve manufacturing efciency
Regardless of which vector expression system is chosen to manufacture a vaccine product, whether it may be in ovo or ex ovo,
the general categories of downstream bio-processing unit operations do not vary. A brief outline of the general sequence of
unit-operations in process ow will be given here. Afterward, each
step will then be detailed further with an eye toward equipment
considerations.
Generally, the rst step in downstream vaccine production is
known as clarication, and is used to concentrate the vaccine product obtained from the chosen expression system. Because most
viral production occurs intracellularly, the rst step in clarication is cell lysis. The liberated intracellular uid is then either
centrifuged at low speed or ltered to remove any undesirable
precipitants and cell debris. Depending on the chemistry of the
potential contaminants, either afnity or ion exchange chromatography may be employed to further purify the vaccine product.
Tangential ow ltration or size exclusion may be the nal step to

Clarication is the rst step to remove cell debris, particulates

and insoluble matter after cell lysis. When transporting the uid
containing the product to either a centrifuge or through a ltration
device, careful consideration must be given to the class of pump
that is used. Classes of pumps to be avoided are centrifugal, lobe,
peristaltic, and piston pumps. Single diaphragm, but not multidiaphragm pumps, can also pose issues. High shear forces exist in
centrifugal and lobe pumps that can permanently reduce product
integrity or irreversibly de-nature the biological properties of protein and nucleic acid-based vaccine products [6,7]. On the other
hand, peristaltic and single diaphragm pumps have limited utility
due to high pulsation and inconsistent ow that may cause adverse
effects on the stability and reproducibility of unit operations. The
frequent breakdown of piston pumps affects their reliability, which
poses an unnecessary risk for the transfer of high value product in
a uid delivery regime.
The use of a multi-diaphragm pump has several advantages over
other pump classes. Diaphragm pumps, whether single or multiunit are the gentlest class of pumps to bio-molecules. Recently,
an alternating quarternary diaphragm pump, which can provide a
smooth, nearly pulsation-free ow, was introduced by the Quattroow Fluid Systems headquartered in Germany [8]. The low
internal velocity and specially designed Duckbill valves make Quattroow suitable for a shear-sensitive product. The gentle nature
of the pump was instantly recognized by many multi-national
companies with business interests in downstream bio-processing.
RJ Bio Process in Suzhou, China has advanced this technology
further by integrating a computer and a programmable logic controller (PLC) along with a pressure regulator into the Quattroow
pump technology (Photo 1, [9]). This modication has afforded
uniform and consistent uid control that is resistant to sudden
pressure changes due to clogged lters or uid viscosity differentials. Furthermore, the sanitary design, clean-in-place feature,
on-line process monitoring and electronic signature capability can
satisfy cGMP requirements.
5. Chromatography
After clarication, further removal of molecular impurities from
the vaccine product is necessary. With the vaccine product contained in typically large volumes of claried liquid, only liquid
chromatography can perform such a purication step economically
and efciently. Generally, an ion exchange step is often employed to
concentrate and capture the viral product of interest. Ion exchange
resins, either positively or negatively charged, have the ability to
interact with the opposite charges of any bio-molecules through
electrostatic forces [10]. In essence, the separation relies on the differences in the electrostatic charges between the viral product and
its impurities. Depending on the nature of these electrostatic differences, either a positive or negative ion exchange strategy can be
utilized. Negative ion exchange employs the use of anion exchange
resins to retain viral products which have negative charges on their

1266

M. Li, Y.X. Qiu / Vaccine 31 (2013) 12641267

Fig. 1. PAT-based in-line buffer dilution NaOAC 0.01 M.

Photo 1. A four-diaphragm pump integrating with a computer and PLC for data
processing, courtesy of RJ Bio Process, Suzhou, China.

surfaces. As sample is loaded into the column, any uncharged or

positively charged molecules will not be retained by the positively
charged resins and will be the rst to elute from the column. The
negatively charged viral product will be retained by the stationary phase, and is thus separated from the rest of the contaminants,
which migrate through the column under the inuence of the ionic
character of the mobile phase. If positive ion exchange is deemed
more suitable, then cation exchange resins may be used to effect
the necessary separation in a manner opposite to anion exchange
chromatography.
In recent years, an alternative to traditional column chromatography has emerged. The labor-intensive and time involvement to
pack and to unpack a column with resins has evolved a new mode
of process scale separation referred to as monolithic sheet. Its mode
of throughput is based on convective ow, which offers a type of
separation that falls somewhere between traditional column chromatography and membrane-based ltration [11].
To effect a good ion exchange based separation, either pH or
ionic strength or both should be precisely controlled at any given
time as a critical process parameter. Accordingly, accurate mobile
phase preparation and delivery will ensure good chromatographic
efciency and reproducible manufacturing.
In 2000, the U.S. Food and Drug Administration (FDA) was very
dissatised with the frequent occurrence of out-of-specication
product, the lack of reproducible process data, and the overall
inefciency in manufacturing operations. Therefore, a new cGMP
for the 21st Century initiative was proposed [12]. In 2004, the
FDA imposed new guidelines that required any new pharmaceutical drug applications had to incorporate the concept of quality by
design (QbD) as part of the chemistry, manufacturing and control
(CMC) program. Furthermore, the FDA strongly recommended the
use of process analytical technology (PAT) to facilitate the implementation of QbD [13].
What exactly is PAT? According to FDA, the working denition
of PAT is a set of tools or a system for designing, analyzing, and controlling manufacturing through timely measurements

(i.e. during processing) of critical quality and performance

attributes of raw and in-process materials and processes with the
goal of ensuring nal product quality. This illustrates how difcult
it is to improve a process without knowing a process. In non-PAT
based production-scale bio-processing, mobile phase preparation
and delivery, to effect afnity or ion exchange separation, is often
inaccurate and irreproducible, and therefore can adversely affect
the chromatographic performance and outcome.
In recent years technological advancements have improved
upstream yields tremendously. As upstream yields continue to
increase, downstream purication involving process solution
preparation and delivery must increase in proportion to keep pace
with demand. Thus, productivity and efciency of downstream unit
operations have become the bottleneck of manufacturing. Recently,
the concept of in-line buffer dilution and on-demand buffer preparation was found to be useful and practical in manufacturing by
bio-processing scientists [14,15]. To facilitate QbD requirements, a
PAT-based process liquid chromatography system should have inline pH and conductivity sensors coupled to PLCs to measure and
adaptively correct the variability of the incoming buffer strength
and pH through process control feedback effects before allowing the mobile phase to enter the chromatographic column. This
technology can mitigate the uctuation of unpredictable buffer
concentration and pH as critical quality attributes, and thus create
compliance with the FDAs PAT initiative.
A PAT-based in-line buffer preparation skid offers several
important benets for vaccine production. The rst is buffer
preparation on-demand. This feature allows the manufacturer to
ramp-up or scale-back production capacity as product demand
uctuates, without any advanced buffer preparation necessary.
Secondly, it provides the ability to use buffer concentrates that
can be mixed (binary or ternary blends) and diluted in-line from
10 or more to a 1 buffer that can be pumped directly onto the
chromatographic column. This greatly reduces the large footprint
caused by buffer tank farms. A third benet is the ability to ensure
that only in-specication buffer is directed to the column, since
real-time monitor allows for the diversion of out-of-specication
buffer to be diverted from the column to avoid potential catastrophic product loss. A fourth benet to PAT-based in-line buffer
dilution is real-time quality control and ongoing process validation
that comes from the real-time monitoring and recording of critical
quality attributes [14].
Shown in Fig. 1 is an example of in-line buffer dilution
preparation obtained from concentrate. Sodium acetate (0.1 M)
concentrate was prepared and diluted 10 to 0.01 M. The pH and
conductivity set point of 0.01 M sodium acetate was determined
to be 6.3 and 0.85 mS/cm, respectively, and programmed in the

M. Li, Y.X. Qiu / Vaccine 31 (2013) 12641267

process control software. Further, QbD concept was applied. The

upper limit of conductivity was chosen to be 1.0 and the lower
limit to be 0.7 mS/cm. At the same time, the upper pH limit was
chosen to be 6.5, and the lower 6.1. In the 72 min run, either pH
or conductivity value was well within the specication limit. The
experiment has demonstrated that the ability to monitor and control the process values of conductivity and pH in real time using PAT
could provide for process robustness and could adaptively adjust
to variability in raw material buffer feed stocks.
Traditionally, ion exchange resins aforementioned have been
used to separate impurities from the product. One has to seek an
alternative method if ion exchange resins fail to provide the separation and desired product purity. Another important development
of PAT is to afford a linear pH gradient which otherwise is not possible. A group of process scientists was successful to separate two
proteins, Trypsinogen and Ribonuclease A, based on a linear pH
gradient methodology [16].
6. Polishing
The nal step in the downstream bio-processing of vaccine
products is either size exclusion or tangential ow ltration. The
main purpose of this step is to remove trace amounts of bacteria, viruses, and any other small molecules that are required to be
absent from the nal product. The size of the contaminants relative to the size of the vaccine product would dictate the pore
size of the lters. There are many commercially available nano
or ultra-ltration devices, such as those produced by Millipore
(Massachusetts, USA), Sartorius Stedim (Goettingen, Germany), Pall
(New York, USA) and GE Healthcare (Pittsburgh, USA). End users
should consult with membrane manufacturers to determine which
chemical and physical properties of the membrane lter will be
best suited for their processes.
7. Discussion
To develop a strategy for downstream bio-processing of vaccine products one should rst understand the physico-chemical
properties of potential contaminant and by-products of the host
expression system that has been chosen. This knowledge will

1267

direct the engineering of an effective and efcient downstream

bio-processing operation. Care must be taken in choosing the
appropriate equipment and technologies to ensure product purity,
integrity and safety, while creating an economic and efcient
process based on sound manufacturing science. Hence, the employment of PAT is necessary as a standard process monitoring and
controlling feature in downstream bio-processing equipment.
PAT not only can provide buffer dilution on-demand and in-line
process validation, but also produce a linear pH gradient methodology as an alternative to the traditional ion exchange process. The
inclusion of PAT will ensure compliance with the cGMP for the 21st
Century Initiative promulgated by the FDA, which has required that
QbD be an integral part of the CMC le for any new marketable
pharmaceutical products effective as of 2004.
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[3] Optau vaccines, http://www.novartis.com/products/vaccines; November 1,
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[12] Pharmaceutical cGMP for the 21st Century a risk-based approach. U.S.FDA;
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[13] FDA, PAT guidance for industry a framework for innovative pharmaceutical development, manufacturing and quality assurance, September 2004,
http://www.fda.gov; October 22, 2012, pp. 155.
[14] Malone T, Li M. Bioprocess Int 2010:408. January.
[15] Matthews T. Pharm Manuf 2009;8(4):3641.
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2010:404. October.