overview of upstream and downstream processing of...
TRANSCRIPT
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: [email protected]
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…………?