Method Validation: Accuracy

[Blazer]

Hello all,

I've recently started a new job at a peptide manufacturer and the majority of the validations I am overseeing are for customer's peptides where there is no reference material (i.e. these are new chemical entities). I'm looking for some opinions/anecdotes/etc regarding the use of ICH regulations for accuracy, specifically 4.1.1b (comparison of results versus a 2nd well-characterized procedure) and 4.1.1c (infer once linearity, precision, specificity have been shown). I'm not sure how acceptable these methods of determining accuracy are in the eyes of an auditor. Any input or opinions you could provide would be most helpful.

Thank you.

[danko]

If your substance is unique and you can’t get a well defined standard, then you’ll need to characterize it yourself. In peptide context it would be AAS, salts, water content and of course chromatographic purity.

Best Regards

[Blazer]

Thanks Danko, I understand this and this is how we would calculate the purity of the peptide. However, validating the accuracy of the HPLC method to determine this chromatographic purity is where I'm hung up and having a hard time convincing QC/QA management about designing the experiment.

Put simply, how does one prove the accuracy of an HPLC method for a drug substance? The ICH provides three paths for drug substance accuracy validation: quantitation against a reference standard (not an option for us, obviously), infer the accuracy from the specificity, linearity and precision or use an orthogonal method that is well characterized (an example I've seen is DSC testing for purity).

[danko]

Hi again,

Yes, I was referring to the second option. It goes without saying that you can’t use the chromatographic assay result as a reference value, because that’s the aim of your experiment – to validate it against some more or less absolute number. And everybody knows that HPLC produces relative values.
So, if you calculate the mass of you peptide using AAS (amino acid sequencing) because you know the mass of every amino acid you’ll find, that would be the peptide part. Then you’ll have to determine water content either by Karl Fischer or loss on drying. If you know that there could be some salts in your substance they can be determined by Atom Absorption and finally the specificity could be deduced using chromatographic purity determination as a convenient estimate, especially if you show some separation of forced degradants etc.

Best Regards