Advances in LC/MS for the Characterization of Biotherapeutics

Biotherapeutics exemplified by monoclonal antibodies (mAbs) are large complex molecules that are recombinantly expressed by cellular fermentation. While the primary sequence of the protein remains the same. Post translational modifications such as glycosylation are dependent on the cell lines chosen, the fermentation media, conditions, and length of fermentation. Clipping, deamidation, oxidation, etc. may occur during fermentation, purification, and storage. Biotransformations may take place in vivo after administration of the therapeutic agent. Traditionally these modifications are observed analytically by a bottom-up approach, whereby the protein is proteolytically digested with enzymes such as trypsin to generate short peptides that are much easier to separate chromatographically and characterize by mass spectrometry. This is approach is well established, reliable, and highly effective. However, the relationship of multiple modifications and heterogeneity are lost through this approach Intact mass measurement allows direct analysis of proteins which can provide greater insights into multiple modifications within a single protein molecule. This aspect of heterogeneity may be lost when analyzing via a bottom-up approach. The inherent difficulty of intact mass analysis of large therapeutic proteins is that they are harder to chromatograph under conditions that are compatible with mass spectrometry ion sources and the multiple charge states resulting from electrospray ionization increases the spectral complexity in addition to the underlying heterogeneity of the protein. Reverse phase chromatography provides good resolution and peak shape while the denaturing conditions afford a more efficient and therefore sensitive ionization. Size exclusion chromatography (SEC) has lower resolution and is a non-focusing separation technique. However, this can be conducted under native conditions (1) that while less sensitive/efficient than denaturing conditions results in a smaller number of charge states at higher m/z, simplifying the spectra (Figure 1). PL-26 Advances in LC/MS for the Characterization of Biotherapeutics