Overview of Upstream and Downstream Processing of Biopharmaceuticals

Ian Marison
Professor of Bioprocess Engineering and Head of School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland E-mail: ian.marison@dcu.ie

Outline of presentation
Introduction- what is a bioprocess? Basis of process design Upstream processing
Batch, fed-batch, continuous, perfusion

Downstream processing
Philosophy Chromatography Examples

Conclusions
2

What is a bioprocess?
Application of natural or genetically manipulated (recombinant) whole cells/ tissues/ organs, or parts thereof, for the production of industrially or medically important products
Examples Agroalimentaire: food/ beverages Organic acids and alcohols Flavours and fragrances DNA for gene therapy and transient infection Antibiotics Proteins (mAbs, tPA, hirudin, Interleukins, Interferons, enzymes etc) Hormones (insulin, hGH,EPO,FSH etc)

Aims of bioprocesses
To apply and optimize natural or artificial biological systems by manipulation of cells and their environment to produce the desired product, of the required quality. Molecular biology (genetic engineering) is a tool to achieve this Systems used include:
Viruses Procaryotes (bacteria, blue- green algae, cyanobateria) Eucaryotes (yeasts, molds, animal cells, plant cells, whole plants, whole animals, transgenics)

Importance of process development

Advances in genetic engineering have, over the past two decades, generated a wealth of novel molecules that have redefined the role of microbes, and other systems, in solving environmental, pharmceutical, industrial and agricultural problems. While some products have entered the marketplace, the difficulties of doing so and of complying with Federal mandates of: safety, purity, potency, efficacy and consistency have shifted the focus from the word genetic to the word engineering. This transition from the laboratory to production- the basis of bioprocess engineering- involves a careful understanding of the conditions most favoured for optimal production, and the duplication of these conditions during scaled- up production.
5

Design criteria
Concentration Productivity (volumetric, specific) Yield/ conversion Quality
Purity Sequence Glycosylation Activity (in vitro, in vivo)
6

Design criteria for pharmaceutical product

Order of importance

Quality Concentration Productivity Yield/ Conversion

High added value products
7

Design criteria for bulk product

Order of importance

Concentration Productivity Yield/ Conversion Quality

Low added value products
8

Clear idea of product Selection of producing organism Strain screening Formulation medium requirements Medium optimization Small scale bioreactor Cultures (batch, fed- batch, continuous) Process control requirements Scale- up (>100 litre) Process kinetics (productivity etc.)

USP

Biomass-product separation Product purification

DSP
Effluent recycle/disposal

Strain improvement (molecular biology)

Concentration, crystallization, drying Fill-Finish

Process integration

Storage properties, stability Field trials FDA approval Product licence Marketting Sales
9

Are yields, conversion, productivity ok?

DSP

Choice of production cell line- microbes

Bacterial cells
genetic ease (single molecule DNA, sequenced) high productivity, high Resistance to shear, osmotic pressure, immortal Negatives: poor secretors, little glycosylation/ posttranslational modifications

Yeast
High , high cell concentrations, high productivity, good secretors, post-translational modifications, glyco-engineered strains available Non-mammalian glycosylation, post-translational modifications, complexity of genetic manipulation
10

Choice of production cell line- mammalian cells

CHO/ BHK/HEK/COS cells Advantages
Produce human-like proteins Secrete Correctly constructed and biologically very active

Disadvantages
Slow growth rate () Low cell densities Low productivity Shear sensitive, osmotic pressure sensitive, substrate/ product toxicity, apoptosis, cell age

Choice of cell line profoundly affects selection of bioreactor, DSP, feeding regime, scale of production 11

Type of bioreactor
Depends on: Anchorage dependence or suspension adapted, Mixing- homogeneous conditions, absence of nutrient and temperature gradients Mass transfer particularly (OTR = kLa (C*-CL) Cell density (qO2.x = OUR)
CHO and BHK qO2 = 0.28-0.32 pmol/cell/h

Shear resistance CIP/SIP Validation issues

12

Type of bioreactor

Stirred tank reactor (STR)

Membrane reactor Fixed-bed reactor

Fluidized-bed reactor (FBR)

Disposable reactors

13

Animal cell encapsulation

CHO cells secreting human secretory component (hSC)

0 days

3 days

12 days

Microscope photographs during the repetitive fed-batch culture. Capsules produced with 1.2% alginate, 1.8% PGA, 4% BSA, 1% PEG, initial cell density 106 cells/ml.

Aim: to achieve high cell density cultures increase overall process productivity
PGA, propylene-glycol-alginate 14

Type of substrate feeding

Depends on anchorage dependence or suspension adapted OTR (poor oxygen solubility; 5-7 mg/L 25 C) Cell density (qO2.x = OUR) Shear resistance Stability of product Productivity Product concentration Formation of toxic products Osmotic stress Substrate inhibition/ catabolite repression/ diauxic growth Availability/ Need of PAT (quality by design, consistency)
15

F S0

FS

Feeding regimes
FS Continuous V

V F S0 Batch Fed- batch V Perfusion FS

16

Questions
Which regime provides for highest product concentration (titre)? Which regime provides for highest productivity? Which regime is used for situations where product is unstable? Which regime is used when substrates are inhibitory, repressive, mass transfer is limiting? Which regime is used to design the smallest installation? Which regime is the easiest to validate? Which USP is easiest to integrate with DSP? etc (think up some of your own questions!!)
17

DSP- the challenge

Process-related related contaminants

Product-related contaminants

18

Dose-Purity relationship
Purity 99.997 hGH 99.99 SOD

99.9

EPO

99

Vaccine

95

Diagnostic

In vitro

100 mg

1g

3g

>10 g

Lifetime doseage

Required Purity as a Function of Dosage

19

DSP
Purity

USP- Culture harvest

(product 10-1000mg/l)

Cell separation

Volume
Capture Intermediate purification Polishing

Fill-Finish

20

Purification techniques
Filtration Precipitation Liquid-liquid two-phase separation Chromatography
Size exclusion (gel filtration) Ion-exchange Hydrophobic interaction Reverse- Phase Hydroxyapatite Affinity (protein A,G etc, dyes, metal chelates, lectins etc) Fusion proteins (tagging, Fc, Intein, streptavidin etc)
21

Chromatography

STREAMLINE

INdEX

CHROMAFLOW

BPG

FineLINE

BioProcess Stainless Steel

22

Filtration
Reverse Osmosis

Nanofiltration

Ultrafiltration
0.001 103 0.01 10
5

Microfiltration
0.1 10 7 1.0

pore size (microns)

Approx. molecular weight (globular protein)

Dead end filtration Cross-flow filtration

Attention: fouling, membrane polarization, cost, protein aggregation/ precipitation, degradation
23

Filtration

24

Generic monoclonal antibody production scheme

ceramic hydroxyapatite (flow through mode)

25

School of Biotechnology
Bioprocess Engineering Group
Molecular Biology On- line monitoring Animal cell Culture Microbiology

PAT

Micro- and Nanoencapsulation

Integrated bioprocessing
Environmental engineering

Immunology Bioinformatics, genomics, proteomics etc.

Natural and Recombinant products

26

Conclusions
Bioprocesses are, or should be, integrated processes designed taking all parts into account to provide the quantity and quality of product required using the least number of steps, in most cost-effective manner. Holistic approach to process design Quality by design
27

Thank you for your attention

Any questions?

28