Comparability and biosimilarity
Comparability has been an important aspect of biotherapeutic development since the advent of the well-characterized biotherapeutic
product guidelines established in the 1990s. These formalized FDA guidelines were a boon to the development of biotherapeutics
because they offered a pragmatic pathway to characterizing multiple elements and trying to relate them to the clinic. As time
has progressed, many default techniques, such as peptide mapping and glycan profiling, have become increasing automated. Indeed,
the automation of these techniques routinely uncovers important issues such as sequence variation in biosimilar candidates
or variations in glycosylation profiles (11, 16, 17).
With the advent of HDX–MS, the comparability toolbox can be extended further into the realm of HOS. Comparability in HOS was
aptly demonstrated by a study of a commercial growth hormone that was covalently attached to a polyethylene glycol (PEG) polymer
(i.e., PEGylated) (18, 19). In that study, the authors used HDX–MS to analyze granulocyte colony stimulating factor (G-CSF)
upon PEGylation. PEGylation is known to reduce immunogenicity and antigenicity and may prolong circulating time (20). FDA
has approved a number of biotherapeutics that are PEGylated, and many more are believed to be in the pipeline (18, 19).
PEGylation may induce conformational changes, steric interferences, and changes in electrostatic binding (21). The researchers
performed a number of experiments to obtain an overall picture. Three undeuterated experiments and two complete HDX experiments
were performed for each protein. In total, 10 separate experiments were performed for all forms of G-CSF within two consecutive
days, which helps illustrate the importance of automation and robotics.
Significant differences in deuterium incorporation were induced by PEGylation of G-CSF, but the researchers concluded that
the changes were quite small and that PEGylation did not result in gross conformational rearrangement of G-CSF (5). The raw
differences between specific peptides in the protein chain were charted automatically; if the value of differences exceeded
an empirically-determined amount (i.e., 1.5 Da), it was considered significant. The decisions about significant differences
are also captured by performing triplicate acquisitions, thus providing a proper basis for judgment. Controls were also used
with totally separate duplicate runs of G-CSF or PEGylated G-CSF to demonstrate that the differences observed in the plots
of PEGylated versus non-PEGylated were real.
With HDX–MS, this type of study can be directly matched to clinical trial data, especially in situations for which it is known
that HOS changes may occur. For this study, the authors commented that the data complexity was greatly reduced though a data
processing and presentation format that was designed to facilitate their comparison process. The study demonstrated the immediate
utility of HDX–MS for comparability studies as well as protein therapeutic characterization in the biopharmaceutical industry.
In 2012, FDA released guidelines on biosimilars that specifically called for studies of HOS, following earlier testimony to
the US Congress (22, 23). Many national guidelines have, until now, tended to use an all-encompassing description such as
"state-of-the-art" technology. The FDA guidelines of 2012 potentially reflect the rapidly expanding set of analytical tools
for biotherapeutics, as well as an understanding that techniques to study HOS are increasingly routine.
Even in studies of insulin, which is an extremely well-characterized protein, there is recent evidence for the importance
of characterizing HOS to match an innovator product's efficacy. In a case in which a biosimilar insulin was well-characterized,
the primary structure, downstream processing aspects, and manufacturing processes were well understood. Unfortunately, the
submission failed when clinical data indicated different pharmacokinetics that may well have been predicted by HOS studies
(24). The need to characterize HOS is of worldwide concern, as is clear from the wide variety of geographies where such studies
are taking place, including Japan (25).
HOS studies have benefitted greatly from advances in technology, informatics, and the robustness of the analytical LC–MS toolkit.
In addition, the wider scientific understanding of what is achievable has meant a recent quantum leap in the demand for—and
development of—HDX–MS. Like a genie released from its bottle, this powerful capability cannot be put back in the darkness.
It will only continue to grow and further inform our understanding of protein biotherapeutics.
St. John Skilton, PhD, is senior manager of late-stage development for pharmaceutical life sciences at Waters, tel: 508.377.8234.