Overview of Upstream and Downstream Processing of Biopharmaceuticals

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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

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– Batch, fed-batch, continuous, perfusion

• Downstream processing

– Philosophy

– Chromatography

– Examples

• Conclusions

What is a bioprocess?• Application of natural or genetically manipulated

(recombinant) whole cells/ tissues/ organs, or partsthereof, for the production of industrially or medically important products

• Examples

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• 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

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• 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.

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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’.

Design criteria

• Concentration

• Productivity (volumetric, specific)

• Yield/ conversion

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• Yield/ conversion

• Quality

– Purity

– Sequence

– Glycosylation

– Activity (in vitro, in vivo)

Design criteria for pharmaceutical product

Order of importance

• Quality

• Concentration

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• Concentration

• Productivity

• Yield/ Conversion

High added value products

Design criteria for bulk product

Order of importance

• Concentration

• Productivity

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• Productivity

• Yield/ Conversion

• Quality

Low added value products

Biomass-product

separation

Product purification

Storage properties,

stability

Effluent recycle/disposal

Concentration,

crystallization, drying

Fill-Finish

DSPClear idea of product

Selection of producing

organism

Strain screening

Formulation medium

requirements

Medium optimization

Strain improvement

(molecular biology)

USP

Processintegration

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Field trials

stability

FDA approval

Product licence

Marketting

Sales

Small scale bioreactor

Cultures (batch,

fed- batch, continuous)

Process control

requirements

Scale- up (>100 litre)

Process kinetics

(productivity etc.)

Are yields,

conversion,

productivity

ok?

DSP

integration

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/ post-

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– Negatives: poor secretors, little glycosylation/ post-translational 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

Choice of production cell line- mammalian cells

• CHO/ BHK/HEK/COS…… cells

– Advantages

• Produce ‘human-like’ proteins

• Secrete

• Correctly constructed and biologically very active

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– 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

Type of bioreactor

Depends on:

• Anchorage dependence or suspension adapted,

• Mixing- homogeneous conditions, absence of nutrient and temperature gradients

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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

Type of bioreactor

Stirred tank reactor Membrane reactor

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Stirred tank reactor

(STR)

Fluidized-bed reactor

(FBR)

Membrane reactor

Disposable reactors

Fixed-bed reactor

Animal cell encapsulation

CHO cells secreting human secretory component (hSC)

14PGA, propylene-glycol-alginate

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.

0 days 3 days 12 days

Aim:

to achieve high cell density culturesincrease overall process productivity

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

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• 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)

Feeding regimes

F S

F S0 F S

VContinuous

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V

Batch Fed- batch

F S0

F SV

Perfusion

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,

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– 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!!)

DSP- the challenge

Pro

cess-re

late

d c

onta

min

ants

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rela

ted c

onta

min

ants

Product-related contaminants

Dose-Purity relationship

99.9

99.99

99.997

EPO

SOD

hGH

Purity

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95

99

99.9

Diagnostic

In vitro 100 mg 1 g 3 g >10 g

Vaccine

EPO

Lifetime doseage

Required Purity as a Function of Dosage

DSP

Cell separation

Capture

VolumePurity

USP- Culture harvest(product 10-1000mg/l)

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Intermediate

purification

Polishing

Fill-Finish

Purification techniques

• Filtration

• Precipitation

• Liquid-liquid two-phase separation

• Chromatography

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• 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…)

Chromatography

STREAMLINE™INdEX™

CHROMAFLOW™

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INdEX™

BPG™ FineLINE™BioProcess™ Stainless Steel

Filtration

Ultrafiltration Microfiltration

Reverse Osmosis

Nanofiltration

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0.001 0.01 0.1 1.0pore size (microns)

103

10710

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Approx. molecular weight (globular protein)

Dead end filtration

Cross-flow filtration

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

Filtration

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Generic monoclonal antibody production scheme

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ceramichydroxyapatite

(flow through mode)

School of BiotechnologyBioprocess Engineering Group

Integrated

On- linemonitoring

MolecularBiology

Microbiology

Animal cellCulture

PAT

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Integrated

bioprocessing

Environmentalengineering

Natural andRecombinant

products

Micro- and Nano-

encapsulation

Immunology

Bioinformatics,genomics,proteomics

etc.

Conclusions

• Bioprocesses are, or should be, integrated

processes designed taking all parts into account

to provide the quantity and quality of product

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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

Thank you for your attention

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Any questions…………?