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Various types of Monoclonal Antibody (mAb) products including intact mAbs, mAb fragments, engineered variants, and
Antibody-Drug Conjugates (ADCs) are being developed for the treatment of cancer and other diseases due to their excellent
biocompatibility and high selectivity. The proliferation of monoclonal antibody therapeutics and their susceptibility to various
biochemical modifications has highlighted the importance of characterizing these highly heterogeneous products for their safety
and efficacy. Hydrophobic Interaction Chromatography (HIC) is a technique for separation of proteins and has been widely used
as an orthogonal method to size exclusion chromatography and ion exchange chromatography for the characterization of mAb
variants. MAbs, mAb fragments and antibody-drug conjugate samples were diluted with mobile phase A and injected on to MAbPac
HIC columns. MAb fragment was prepared by papain digestion and oxidized mAb was prepared by adding hydrogen peroxide or
2,2��?-azobis (2-amidinopropane) dihydrochloride (AAPH). Antibody-drug conjugate mimic sample was prepared by reduction of
disulfide bonds followed by addition of thiol-reactive drug mimics. Here, we introduce a new family of HIC columns designed for
mAb analysis. Three different ligand chemistries-polyamide, amide and butyl-were developed for the analysis of a wide range of
mAb samples. MAb aggregates, mAb fragment, oxidized mAbs, and antibody-drug conjugates were investigated using these new
HIC columns. Using the polyamide column, mAb aggregates as well as hydrophilic variants of the mAb were separated. For the mAb
fragment analysis, two mAb samples were digested with papain and were separated with all three HIC columns. The amide column
gave the best separation of Fab and Fc fragments of both mAbs. For oxidized mAb variant analysis, a mAb sample was oxidized using
hydrogen peroxide and was injected without further processing. The amide column was able to detect two oxidation variants. For the
final application, a cysteine-linked antibody-drug conjugate mimic sample was analyzed. The novel butyl column baseline separated
all the drug-to-antibody ratio (DAR) species with a 20 minute gradient. Together, these results demonstrate the complementary
selectivity of these columns which is critical for the characterization of various types of mAb products including ADCs.
Biography
Robert van Ling has been working in the field of protein characterization and proteomics. Currently acting in his role as European Support Expert, he focuses on the chromatographic workflows and chemistries for mAb and protein therapeutics, such aggregation studies, charge variant and oxidation monitoring.