How to produce a drug from a bacterium Flashcards
Factors to consider when developing a large scale production process
Recombinant protein expression system (seed stock- provenance of starting material)
Production media
Downstream processing
Production facilities- dedicated production facilities, bio-security
Parameters that need to be optimised with fermentation/ large scale production
Growth conditions- temperature, pH, media components, oxygen demand, rate and nature of the stirring of growing cells
Design of bioreactor: sterility, containment of organisms, monitoring, protein/cell harvesting
Harvesting of recombinant protein
Secreted proteins- easy to purify
Soluble proteins trapped inside cell- release by cell lysis
Insoluble proteins form inclusion bodies- release by cell lysis
Refold protein
Methods of cell lysis
Enzymes
Sonication
French press
Recovery of insoluble proteins
Over-exression of recombinant proteins can lead to the formation of insoluble structures known as inclusion bodies
To recover the protein from the inclusion body you need to denature the protein
Finally the protein need to refold
Protein solubilisation and refolding
Urea unfolds protein
Makes the protein soluble
Dialysis removes urea so protein can refold itself
Purification- chromatography
Several steps are typically required, combined approaches most powerful
Ion exchange, size exclusion, hydrophobic interactions, removal of LPS
Considerations: each step increases purity or removes contaminant but ten fold loss of material
Characterisation of the recombinant protein
Physical: SDS-PAGE, reversed phase HPLC, amino acid analysis, n-terminal sequencing, sugar analysis etc.
Biological: activity assays- enzyme activity, binding properties, cellular response, immunogenicity
Formulation, filling and finishing
Formulation- the protein is transferred to a final formulation
Filing step- final format filling e.g. vial
Finishing- product must be inspected for colour, clarity etc. as issue if acceptable for release; a sample is analysed to ensure if meets physical and biological specifications- final product characterisation
Final product charaterisation
Characteristic which are examined: proper protein content, pH, excipient levels, osmolarity etc., sterility, lack of antigenicity, physical appearance
Stability- determine acceptable storage/ shelf life
Packaging- is the vial or container free of problems, is the label on right and is it correct
GLP
GLP embodies a set of principles that provides a framework within which laboratory studies are planned, performed, monitored, recorded, reported and archived.
GLP helps assure regulatory authorities that the data submitted are a true reflection of the results obtained during the study and can therefore be relied upon when making risk/ safety assessments
GCP
GCP is a set of internationally recognised ethical and scientific quality requirements which must be observed for designing, conducting, recording and reporting clinical trials that involve the participation of human subjects.
Compliance with this provides assurance that the rights, safety and well-being of trial subjects are protected, and that the results of the clinical trials are credible and accurate.
