Liquid chromatographic profiling is the use of chromatographic techniques to separate molecules by different physicochemical properties. It forms a key part of any protein, glycoprotein or peptide structural characterization investigation and it is a requirement of ICH Topic Q6B.
ICH Q6B is a regulatory document that details the requirements for protein characterization and is the approach accepted by the regulatory agencies. Regarding liquid chromatography analyses, ICH Q6B states:
Chromatographic patterns and data on the identity, homogeneity, and purity can be obtained by size exclusion chromatography, reverse-phase liquid chromatography, ion exchange liquid chromatography, affinity chromatography or other suitable procedures.
Therefore, as the guidelines state, liquid chromatographic profiles provide important information on the purity of the molecule and are used as a mechanism for separation of different species within a sample. These isolated species can then be collected for further structural and physicochemical characterization if necessary.
Chromatographic Techniques at BioPharmaSpec
BioPharmaSpec has extensive experience in employing a comprehensive range of chromatographic techniques to separate molecules by different physicochemical properties. We do this for our clients so that they can fulfil the ICH Q6B requirement for structural characterization, but also as a purity and impurity assessment of their product. For a full assessment of the purity of a sample, different chromatographic separation procedures are used so that any components not resolved by one procedure are likely to be resolved based on another chromatographic technique.
Reverse phase chromatography
This technique separates molecules using the interaction of the protein, glycoprotein or peptide with a hydrocarbon coated stationary phase. A liquid gradient of increasing organic composition is used to promote elution of each component in the sample. The hydrocarbon chain lengths on the stationary phase can vary and are chosen depending on the nature of the components under investigation.
Ion exchange chromatography
This technique separates molecules based on their overall charge. Proteins, glycoproteins and peptides can carry either a nett negative or nett positive charge, depending on the amino acid sequence, post-translational modifications and glycosylation. Therefore, we can use different stationary phases, cationic or anionic, for separation of positively charged or negatively charged species respectively. Separation of charged species is brought about through the use of either salt or pH gradients in the liquid phase.
Ion exchange chromatography is complementary to imaging capillary isoelectric focusing (icIEF) which is used for charge isoform analysis. In this chromatographic form of charge based separation, fractions can be collected for further analysis, something which is not possible with icIEF.
Size exclusion chromatography (SEC)
During SEC, molecules are separated based on size as they pass through a gel column composed of beads with a defined pore size. In basic terms, larger molecules will take a shorter path through the matrix due to exclusion or limited access of pore space compared to smaller molecules. Smaller molecules can move through the pores and around the beads and thus have a longer pathlength through the column.
Size exclusion chromatography can also be linked with a multi angle laserlight scattering detector (SEC-MALS). This allows an assessment of aggregation within the sample.
Hydrophobic interaction chromatography (HIC)
During HIC, molecules are separated based on hydrophobicity/ hydrophilicity. This is achieved through the association of exposed hydrophobic domains within the protein with the stationary phase as a high salt buffer. A decreasing salt gradient is used to elute components, with more hydrophobic species eluting with lower salt concentrations.
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