My questions are about validating sampling methods, as well as analytical methods, for biopharmaceutical APIs.
How do you produce a denatured protein sample for these studies? Do you heat, add alkali, add SDS, etc?
How many “soils” are sufficient? Must they be from each processing step? Or is it sufficient to use cell culture liquid (to represent worst-case upstream processes) and purified protein (for worst-case downstream equipment)
If I have two non-specific methods (e.g. TOC and BCA), but the TOC is more sensitive, is one method sufficient?
How do you produce a denatured protein sample for these studies? Do you heat, add alkali, add SDS, etc?
Treatment method for the denaturation of protein depends on the characteristics of the protein itself. The best method would be to treat the protein sample with the cleaning agent you use for protein removal during manufacturing stage (as the cleaning agents in most of the cases remove protein contaminants from the manufacturing surfaces by degrading/denaturing bigger protein molecules to smaller water soluble molecules). Generally, denatured samples are obtained by treating the API (original protein sample) with alkali (such as NaOH) or the cleaning agent with heat (increases the protein degradation rate) for a certain period of time, following which the pH is neutralized back to stop the reaction.
How many “soils” are sufficient? Must they be from each processing step? Or is it sufficient to use cell culture liquid (to represent worst-case upstream processes) and purified protein (for worst-case downstream equipment)
Selection of worst-case impurity depends on “which of the impurities” you don’t want to appear in the subsequent product? Is it the API itself or the API degradants? In my opinion you should conduct recovery studies using both purified protein (API) and degraded/denatured protein samples. Using API for recovery studies is very straight-forward & justifiable. However, in normal manufacturing conditions it is not necessary that only API residue would be present on the surface. In order to study the effect of possible interefeneces (degradants etc.) on the recovery of API, it will be a good idea to perform recovery studies using degraded (not pure) protein sample also.
If I have two non-specific methods (e.g. TOC and BCA), but the TOC is more sensitive, is one method sufficient?
Again, as I said earlier, the choice of analytical method depends on what you intend to look for in your samples? However, TOC can still be considered a better choice for the analysis of both API and its degradants. If the impurity you are looking for is “specific” i.e. it is a known impurity then a more specific method (such as ELISA etc.) would be recommended.
Choose % recovery for the denatured API (y%). Here, I am assuming that the recovery for the denatured API would be lower than the API (logically speaking). Using denatured API for recovery studies represents the actual sampling condition (i.e. the actual status of residue on the equipment surface).