1. Downstream Processing
BIOREACTORS AND DOWNSTREAM PROCESSES M
DOWNSTREAM PROCESSING OF BIOLOGICAL MOLECULES
cristiana.boi@unibo.it
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2. R.G. Harrison et al. Bioseparations Science and Engineering, 2nd Ed. Oxford U. Press, NY, 2015.
3. • Solubility
– Precipitation, crystallization
• Hydrophobicity
– Hydrophobic interaction chromatography (HIC)
• Charge
– Ion exchange chromatography (IEC)
• Size
– Gel filtration chromatography (GF), membrane
filtration
• Specific binding ability
– Affinity chromatography
Cristiana Boi
Protein Purification Steps Based on
Chemical and Biological Properties
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4. • Define objectives
– Purity, activity, quantity required
• Develop analytical assays
– Concentration, activity, purity
– UV, SDS-PAGE, ELISA, HPLC
• Define target protein and impurity characteristics
– pI, pH stability, size, hydrophobicity, etc.
• Remove, inactivate proteases early
• Reduce sample volume
– Smaller equipment, faster
• Use different technique at each step
• Minimize number of steps
Cristiana Boi
Steps in Process Development
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5. Cristiana Boi
Steps in Process Development
Yield Y1Y2...YN
Where Yi is the yield of the single process step
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6. • Preparation, Extraction, Clarification
– Remove cell debris, cell lysis, filtration
– Separation of insolubles
• Capture
– Isolate product, concentrate, stabilize
• Intermediate purification
– Remove bulk impurities
• Polishing
– Final purity
• Formulation
– Dosage form
Cristiana Boi
General Purification Strategy
http://www.gelifesciences.com
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7. Cristiana Boi
Purification Stages
R.G. Harrison et al. Bioseparations Science and Engineering, 2nd Ed. Oxford U. Press, NY, 2015.
CONFIDENTIAL: NOT FOR DISTRIBUTION
8. Cristiana Boi
Generic Protein Separation Process
Bioreactor
Cell Lysis and
Separation
Product
Capture
Cell debris
HCP
Impurities
Centrifugation, Filtration, Flocculation
Precipitation,
Filtration,
Column capture
Purification
Chromatography
IEC, HIC, Affinity
HCP
Impurities
HCP
Impurities
Polishing
Chromatography
Gel, Affinity
Impurities
Pathogen
Removal
Nanofiltration
Formulation
Final
Product
Buffer exchange, diafiltration
Buffer exchange, diafiltration
No inclusion bodies
Buffer exchange
diafiltration
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