But, of course, proteins contain a range of reactive side chains such as acidic, amide and reactive sulfur-containing and aromatic amino acids. The environment in which these can be reactive is not just limited to within the cell but can also take place during product purification, processing and storage. The end result of all of this is that the product, which could seem to be so structurally simple based on the expected amino acid sequence can actually be seen as a compound open to a wide range of chemical and biochemical modifications.
If you start to consider the possible reactions that your product could be involved in then you quickly realize that despite any pre-conceived ideas about the nature of your product, the data produced will not necessarily be one single signal for the overall product mass. In fact, it is far more likely that the intact molecular weight analysis of your product will be split between several signals which may vary considerably in relative intensity.
So how do we handle intact mass analysis of products as complex as this? Matrix Assisted Laser Desorption Ionisation mass spectrometry (MALDI-MS) is a good tool to obtain an overall mass of a PEGylated product without any issues with resolving the multitude of signals produced by LC/MS over many charged species. In a similar vein, the very high product heterogeneity associated with extreme levels of glycosylation, such as for Enbrel, can be most easily analyzed in terms of generating an average product mass for the intact species by MALDI-MS. This also enables ready identification of any dimeric species, if present.
For a more in-depth investigation of the PEG polymer profile in particular, resolution of the individual polymer species (which differ from one another by increments of a (CH2)2O unit) is necessary and is possible by LC/MS. This type of analysis of PEGylated products is frequently performed at BioPharmaSpec.
Protein molecular weight analysis can also very quickly highlight a problem with the sample such as an issue at the translation stage that may have resulted from a point mutation or frameshift within the DNA itself. Amino acid misincorporations are also possible and can contribute to sample heterogeneity. If present, these are often at very low abundance and best analyzed through specific searches as part of a peptide mapping strategy. This was discussed in an article authored by my colleague, Dr. Richard L. Easton, “Dealing with the Challenges of Sequence Variants”.
When used for intact mass analysis, mass spectrometry is a powerful tool to begin the journey of structural characterization, especially as a means of investigating the extent to which processing (both intracellular and process related) has taken place.
From this “simple” analysis we can start to ascertain the intactness of the product and potentially assign differences in major signals with possible PTMs. This gives us valuable preliminary information for the product and serves as a point from which more detailed structural investigations can take place and to which they must ultimately relate back. The next stages would be to involve more in-depth techniques such as peptide mapping to further investigate the product and to interrogate, in detail, specific structural characteristics.