One of the simplest and quickest analytical tests that can be performed is intact mass analysis, or intact molecular weight analysis. This is a great way to get meaningful information on your product to check that the molecule’s structure is as expected.
In our labs, we provide our clients with the most meaningful data for their product by using on-line liquid chromatography coupled to a high sensitivity, high resolution, mass spectrometer such as a Q-TOF (quadrupole time of flight) type instrument. However, even a relatively simple test like this needs careful consideration and an awareness of what can happen in biological systems so that nasty shocks are avoided!
Intact mass analysis using MS can be a very simple screening technique, but often there is greater complexity than appreciated when the data are analyzed.
The major factor that needs to be considered in terms of what can lead to such an increase of sample complexity is that of post-translational modifications (PTMs). As the name suggests, these modifications take place following translation of the DNA, via mRNA, to the protein product. For the most part the modifications are not templated in the protein thus the specifics of the PTMs cannot be predicted. But these post translational modifications of one form or another are highly likely to take place and can add significantly to product heterogeneity.
Common PTMs include glycosylation, not just N-linked glycosylation, but also the less often encountered O-linked glycosylation. Furthermore, N- and/or C-terminal modifications (pyroglutamic acid, acetylation, clipping/truncations) can be encountered and are very frequent on IgGs in particular.
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.
Intact Molecular Weight data obtained from analysis of a monoclonal antibody, Herceptin (Trastuzumab)
It is not just post translational modifications that give product heterogeneity of course and this can be taken to extremes in the case of PEGylated proteins. These products, which have an intrinsically heterogeneous polyethylene glycol moiety chemically added to them to enhance (amongst other things) product stability and half-life, can have hundreds of signals for an intact sample as a result of heterogeneity in PEG chain length.
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.
MALDI-MS molecular weight data obtained from analysis of Enbrel showing the monomeric and dimeric forms of the product.
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.
Learn more about appropriate intact molecular weight analyses for your product. Talk to a scientist today.
