Biologics - Stability assessments Flashcards
Lab work
Why are stability assessments for biologics important?
Biologics are sensitive products that may undergo chemical or physical degradation
Degradation can result in a loss potency/efficacy, increased likelihood of severe side effects, or immunogenic reactions
Describe physical degradation
Aggregation – clumping or association of protein molecules or monomers together to form larger structures.
Leads to loss of potency and increased immunogenicity.
Assessed by a viscous/cloudy solution observation, reduced protein concentration after centrifugation, or size exclusion chromatography.
Unfolding/denaturing - significant structural/conformational change, may be reversible or irreversible.
Often caused by exposure to high heat or acidic/alkaline conditions, or equipment such as pumps with filtration/shear effects.
Assessed by intrinsic fluorescence spectroscopy, circular dichroism spectroscopy (measure melting point), trypsin digestion with SDS PAGE analysis, analytical ultracentrifugation or NMR.
Describe chemical degradation
Fragmentation – cleavage of proteins into smaller units/peptides, most commonly effecting therapeutic antibodies
Enzymatic fragmentation is facilitated by the action of protease which may remain in the purified protein harvest after production
Oxidation – caused by exposure of amino acids to oxygen or hypoxic conditions
Deamidation/isomerisation - amide side chains transform into carboxylic acid groups
Results in loss of ammonia and formation of succinimide intermediate, which then undergoes hydrolysis
How do you increase the stability of biologics?
Preventing aggregation – addition of sugars or salts to a protein solution, or carefully considered polymers, amino acids or non-ionic surfactants. Do not have repeated cycles of freezing-thawing as this stimulates aggregation
Increasing kinetic stability – introducing hydrophobic mutations, disulphide bonds, salt bridges or metal ions at the protein surface. Stabilises and rigidifies regions involved in unfolding
Can also use freeze-drying or lyophilisation to restrict protein mobility. This can however lead to pH changes (which will inactive proteins) if there are differential precipitation of buffers/solvent
Increase shelf-life – storage at low temperatures
What are general storage requirements for biologic vials?
Refrigerating, not freezing
Protection from light, proper ventilation
Regular cleaning/maintenance of storage and manufacturing areas
Do not shake
Do not use if discoloured
Ensure proper labelling, staff must write “date first opened” if multi-dose
What are the biophysical methods to test biologics?
Optical testing - provides info on size, shape, protein-protein interactions, and particulates. e.g. TEM, AFM
Separation testing - provides info on molecular weight, number of species and concentrations. e.g. AUC, HPLC, electrophoresis
Spectroscopic testing - provides info on molecular interactions and structure. e.g. UV-Vis, fluorescence, light scattering, MS, NMR, FTIR
Calorimetric testing - provides info on melting temperature, molecular interaction and structure. e.g. DSC
