Post translational modifications (PTMs) are changes that occur in a protein following its production. The production process of a protein from the coding mRNA chain is known as “translation”. PTMs are a diverse group of biochemical and chemical modifications to the protein and many of these are naturally derived, such as glycosylation, disulfide bridge formation and proteolytic processing. These natural modifications take place as part of the natural biosynthetic pathways that are associated with translation.
Other PTMs are associated with chemical modifications that can take place as a result of amino acid side chain interactions with their chemical environments. These modifications are not required for protein activity and may be deleterious to the efficacy of the protein, affecting its shelf life, immunogenicity or aggregation potential. Examples of these sorts of PTM are oxidation, deamidation and glycation. They can occur during production and purification as a result of the chemical processes used during manufacture or through components present in the sample or formulation buffer.
Monitoring PTMs is therefore both necessary for protein structural characterization as required by the international regulatory guideline ICH Q6B, but also from the point of view of assessing the quality of the protein product and the manufacturing/ purification processes. Unwanted PTMs can give rise to product related impurities, which also require investigation as described in ICH Q6B. The most effective means of PTM analysis by far is mass spectrometry. A combination of intact mass analysis and, critically, peptide mapping will give significant information on the nature and location of PTMs within any given product.
From an intact mass perspective, the high accuracy of Q-TOF type instruments in conjunction with electrospray sample ionization gives accurate mass information in the intact sample. This type of analysis will also provide the first evidence for the presence of PTMs as a result of the mass or mass distribution of the sample. Mass(es) detected are a result of the sum total of all PTMs that are present on the protein chains and therefore intact mass assessment on its own is unlikely to allow a full determination of the PTMs present in a protein sample.
The technique of peptide mapping provides significant and key information on PTMs. For peptide mapping studies, including investigations into the nature and location of PTMs, mass spectrometers such as the Waters Q-TOF type instruments are invaluable as they are able to generate fragment ion data from peptides as they enter the source in real time. These fragment ions give peptide sequence information and thus confirmation of the detected peptide, providing identification for peptides that are mass shifted as the result of PTMs. Furthermore, if the PTM is present at a sufficient level, the fragment ion information produced may be able to give the precise location of the PTM within the peptide sequence.
Whilst mass spectrometry can provide significant information on the nature and location of PTMs, there are techniques that can provide orthogonal data to support these assignments. The use of orthogonal procedures for determination of structural characteristics is highly regarded by regulatory authorities in any structural investigation. Examples are the use of imaged capillary isoelectric focusing (icIEF), which provides charge-based information and can help with, for example, investigations into the presence of C-terminal Lysine on monoclonal antibody heavy chains and, more generally, deamidation of proteins. Capillary gel electrophoresis (CE-SDS) can also serve to support mass spectrometry-based investigations into larger mass PTMs such as PEGylation, glycosylation and proteolytic processing.
