Overview of Upstream and Downstream Processing of ...

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: [email protected]

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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)

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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)

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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

Biomass-product separation

USP

Selection of producing organism Strain screening

Product purification Strain improvement (molecular biology)

Formulation medium requirements Medium optimization

Fill-Finish Process integration

Storage properties, stability Field trials

Process control requirements

FDA approval Product licence

Process kinetics (productivity etc.)

Effluent recycle/disposal

Concentration, crystallization, drying

Small scale bioreactor Cultures (batch, fed- batch, continuous)

Scale- up (>100 litre)

DSP

Are yields, conversion, productivity ok?

DSP

Marketting Sales 9

Choice of production cell line- microbes • Bacterial cells – – – –

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

• Yeast – High µ, 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 (µ) 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

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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

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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 Continuous

FS

V

V F S0 Batch

Perfusion

Fed- batch V

FS

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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

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Dose-Purity relationship Purity

99.997

hGH

99.99

SOD

99.9

EPO

99

95

Vaccine

Diagnostic

In vitro

100 mg

1g

3g

>10 g

Lifetime doseage

Required Purity as a Function of Dosage 19

USP- Culture harvest

DSP

(product 10-1000mg/l)

Cell separation

Purity

Volume Capture Intermediate purification Polishing

Fill-Finish

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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™

BPG™

INdEX™

FineLINE™

CHROMAFLOW™

BioProcess™ Stainless Steel 22

Filtration Reverse Osmosis

Nanofiltration

Microfiltration

Ultrafiltration 0.001

0.01

103

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pore size (microns)

5

0.1

1.0

10 7

Approx. molecular weight (globular protein)

Dead end filtration Cross-flow filtration Attention: fouling, membrane polarization, cost, protein aggregation/ precipitation, degradation 23

Filtration

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Generic monoclonal antibody production scheme

ceramic hydroxyapatite (flow through mode)

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School of Biotechnology Bioprocess Engineering Group Molecular Biology

PAT

Microbiology

On- line monitoring Animal cell Culture

Micro- and Nanoencapsulation

Integrated bioprocessing Environmental engineering

Immunology Bioinformatics, genomics, proteomics etc.

Natural and Recombinant products

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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…………?

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