Liquid Chromatographic Patterns
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 (RP-HPLC) is listed as a suitable technique for providing chromatographic patterns and data on identity, homogeneity and purity. 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.
Size exclusion chromatography (SEC) is a liquid chromatographic technique in which particles are separated based on their size, or hydrodynamic volume. Particles of different size elute at different rates through the column, the larger eluting first. SEC is a particularly useful technique for large molecules, such as proteins and polymers. Since SEC measures hydrodynamic volume, rather than molecular weight, it may be used to assess the tertiary structure of a protein. SEC will differentiate between proteins with the same sequence/molecular weight, but with different molecular size due to variations in folding. This may be used as a comparative technique in batch release. It is a recommended technique in the ICH Q6B guidelines for assessment of molecular weight or size, and also for liquid chromatographic patterns.
Ion Exchange Chromatography (IEX / IEC) - in this technique proteins including antibodies, small nucleotides and amino acids may be separated depending on their net charge. Either cation or anion exchange chromatography may be employed, retaining positively-charged or negatively charged analyte ions, respectively. Since proteins have numerous functional groups containing positive and/or negative charges, ion exchange chromatography is a useful method for purification and separation of proteins. It is one of the recommended techniques for liquid chromatographic patterns in the ICH Q6B guidelines.