biopharmaceuticals 20jan09 - 2 - ian marison dcu
TRANSCRIPT
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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?