Liquid chromatographic RP-HPLC patterns are used to provide a fingerprint unique to an individual protein. This fingerprint comprises UV signals originating from the absorbance of peptides eluting from a reverse-phase liquid chromatography column, measured at a particular wavelength. Chromatograms produced are highly reproducible and can be used to demonstrate comparability between proteins e.g. batches of a sample and a reference, or for characterisation of different reference batches.
In order to produce RP-HPLC fingerprints (chromatograms), proteins must first be digested with a suitable enzyme or reagent and the peptides produced bound to and eluted from a stationary phase, using an organic solvent gradient. Because all proteinaceous components in the sample are digested at the same time the fingerprint can be used to assess sample purity, heterogeneity and sequence abnormality, simply by comparing the presence, absence or elution position of eluting peaks.
Liquid chromatographic patterns are a requirement of the ICH Q6B guidelines for characterisation and confirmation of biopharmaceuticals in support of new marketing applications. Reverse-phase liquid chromatography is listed as a suitable technique for providing chromatographic patterns and data on identity, homogeneity and purity.
M-Scan Brochure: Biotechnology and Pharmaceutical Analysis (8pp.)