Overview of Upstream and Downstream Processing

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
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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’.
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Design criteria
•
•
•
•
Concentration
Productivity (volumetric, specific)
Yield/ conversion
Quality
–
–
–
–
Purity
Sequence
Glycosylation
Activity (in vitro, in vivo)
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Design criteria for pharmaceutical product
Order of importance
•
•
•
•
Quality
Concentration
Productivity
Yield/ Conversion
High added value products
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Design criteria for bulk product
Order of importance
•
•
•
•
Concentration
Productivity
Yield/ Conversion
Quality
Low added value products
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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
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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
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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
– 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
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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
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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)
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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!!)
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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
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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…)
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Chromatography
STREAMLINE™
BPG™
INdEX™
FineLINE™
CHROMAFLOW™
BioProcess™ Stainless Steel
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Filtration
Reverse Osmosis
Nanofiltration
Microfiltration
Ultrafiltration
0.001
0.01
103
10
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
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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
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Thank you for your attention
Any questions…………?
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