PF2016 9

• objectives of downstream processing: high level purity and high yield
• impurities: cell culture medium, medium supplements, cells and endotoxins
• objective of harvesting is to separate the protein of interest from process Impurities
• harvesting mechaisnsm; centrifugation, membrane microfiltration, tangential-flow filtration, depth filtration
• centrifuge can separate cells from cell debris, shearing can damage proteins, can increase number os sub micron particles, can precede filtration
• filtration steps throughout the process can remove particulate matter, reduce bio burden and ensure sterility
• microfiltratin removes particles, sediment, algae, protozoa, bacteria
• ultrafiltration removes small colloids, viruses
• in normal filtration, liquid flows perpendicular to the filter media, material that does not pass through the filter remains on the filter surface
• in cross flow filtration, the feed solution flow parallel to the surface of the membrane. it is driven by pressure and the remainder is circulated back to the feed tank
• affinity chromotography

  separates proteins on the bases of a reversible interaction between the target protein and a specific ligand attached to a chromatography matrix. it I highly selective, high resolution and concentrates the sample
• affinity chromatography - protein a

  protein a is the gold standard. is very expensive. IgG binds to protein A at its Fc region. this is a very specific interaction and hydrophobic in nature. binds at neutral pH and elutes at acidic pH
• gel chromotography

  sepearets proteins based on molecular size. low selectivity, high resolution, does not concentrate sample, does not require specific buffer system
• ion exchange chromatography

  separates proteins based on surface charge, charged protein and oppositely charged chromatography medium, medium selectively, high resolution, concentrates sample
• > isoelectric point

  protein will bind to a positively charged anion exchanger
• <isoelectric point

  protein will bind to a negatively charged cation exchanger
• hydrophobic interaction chromotography

  separates proteins based on hydrophobicity. interaction between a protein and the hydrophobic surface of a chromatography medium
• CIPP is capture(isolate, concentrate, stabilise), intermediate purification stage (bulk impurities )and polishing stage (trace amounts of impurities)
• diafiltration is when a buffer of different composition is added to the feed reservoir at the same rate as permeate is removed. the new buffer progressively replaces the old retentive with no change in product concentration. is used to change buffers between chromatography steps
• diafiltration factor represents the extent to which the original buffer is replaced by new buffer
• sources of viral contamination include raw materials, bioreactor contamination, downstream processing(affinity column, excipient) and personnel/ environemt
• risk of viral contamination increases with changes in critical process parameters, failures of virus detection systems, limitation with detection methods and data errors
• viral inactivation methods include solvent/detergent inactivation