Biopharmaceuticals 20Jan09 - 2 - Ian Marison DCU

7/28/2019 Biopharmaceuticals 20Jan09 - 2 - Ian Marison DCU

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Overview of Upstream and Downstream Processing ofBiopharmaceuticals

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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: [email protected]


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Outline of presentation

Introduction- what is a bioprocess? Basis of process design

Upstream processing

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Batch, fed-batch, continuous, perfusion Downstream processing

Philosophy

Chromatography Examples

Conclusions


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What is a bioprocess? Application ofnatural or genetically manipulated

(recombinant) whole cells/ tissues/ organs, or parts

thereof, for the production of industrially or medicallyimportant products

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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 bymanipulation of cells and their environment to produce thedesired 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, wholeanimals, 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 othersystems, in solving

environmental,pharmceutical,

industrial and

agricultural problems.

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While some products have entered the marketplace, the difficulties of doingso 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 bioprocessengineering- involves acareful understanding of the conditions mostfavoured for optimal production, and the duplication of these conditionsduring scaled- up production.


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Design criteria

Concentration Productivity (volumetric, specific)

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Quality

Purity

Sequence Glycosylation

Activity (in vitro, in vivo)


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Design criteria forpharmaceutical product

Order of importance

Quality

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oncentrat on Productivity

Yield/ Conversion

High added value products


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Design criteria for bulk product

Order of importance Concentration

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Pro uct v ty Yield/ Conversion

Quality

Low added value products


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Biomass-product

separation

Product purification

Storage properties,

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

Processinte ration

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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


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Choice of production cell line- microbes

Bacterial cells

genetic ease (single molecule DNA, sequenced) high productivity, high

Resistance to shear, osmotic pressure, immortal

-

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,

translational modifications Yeast

High , high cell concentrations, high productivity, goodsecretors, post-translational modifications, glyco-engineeredstrains available

Non-mammalian glycosylation, post-translationalmodifications, complexity of genetic manipulation


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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 ()

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


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Type of bioreactor

Depends on:

Anchorage dependence or suspension adapted,

Mixing- homogeneous conditions, absence of nutrient and

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temperature gra ents 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


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Type of bioreactor

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(STR)

Fluidized-bed reactor

(FBR)

Disposable reactors

Fixed-bed reactor


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Animal cell encapsulationCHO 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


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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

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ta ty o pro uct Productivity

Product concentration

Formation of toxic products

Osmotic stress

Substrate inhibition/ catabolite repression/ diauxic growth

Availability/ Need of PAT (quality by design, consistency)


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Feeding regimes

F S

F S0 F S

V

Continuous

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V

Batch Fed- batch

F S0

F SV

Perfusion


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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?

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,

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!!)


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DSP- the challenge

Proc

ess-relat

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dcontaminants

Product-related contaminants


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Dose-Purity relationship

99.9

99.99

99.997

EPO

SOD

hGH

Purity

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95

99

Diagnostic

In vitro 100 mg 1 g 3 g >10 g

Vaccine

Lifetime doseage

Required Purity as a Function of Dosage


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DSPCell separation

CaptureVolumePurity

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

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Intermediatepurification

Polishing

Fill-Finish


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Purification techniques

Filtration

Precipitation

Liquid-liquid two-phase separation

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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)


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Chromatography

STREAMLINE

CHROMAFLOW

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BPG FineLINEBioProcess Stainless Steel


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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 filtrationCross-flow filtration

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


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Filtration

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Generic monoclonal antibody production scheme

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ceramichydroxyapatite

(flow through mode)


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School of BiotechnologyBioprocess Engineering Group

On- line

monitoring

MolecularBiology

Microbiology

Animal cellCulture

PAT

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n egra e

bioprocessing

Environmentalengineering

Natural andRecombinant

products

Micro- and

Nano-encapsulation

Immunology

Bioinformatics,genomics,proteomics

etc.


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Conclusions

Bioprocesses are, or should be, integratedprocesses designed taking all parts into account

to rovide the uantit and ualit of roduct

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required using the least number of steps, in mostcost-effective manner.

Holistic approach to process design Quality by design


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Thank you for your attention

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Any questions?

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Biopharmaceuticals 20Jan09 - 2 - Ian Marison DCU