C‐terminal lysine variants in fully human monoclonal antibodies: Investigation of test methods and possible causes

Abstract

The C-terminal lysine variation is commonly observed in biopharmaceutical monoclonal antibodies. This modification can be important since it is found to be sensitive to the production process. The methods commonly used to probe this charge variation, including IEF, cIEF, ion-exchange chromatography, and LC-MS, were evaluated for their ability to effectively approximate relative percentages of lysine variants. A monoclonal antibody produced in a B cell hybridoma versus a CHO cell transfectoma was examined and it was determined that the relative amount of incorporated C-terminal lysine can vary greatly between these two production schemes. Another case study is shown whereby a different monoclonal antibody is subject to some minor process changes and the extent of lysine variation also exhibits a significant difference. During these studies the different methods for determining the extent of variation were evaluated and it was determined that LC-MS after trypsin digestion provides reproducible relative percentage information and has significant advantages over other methods. The final section of this work investigates the possible origins of this modification and evidence is shown that carboxypeptidase B or another basic carboxypeptidase causes this variation.

Structural and functional characterization of the trifunctional antibody catumaxomab

“…Information about modifications like oxidation, [73][74][75] deamidation, [76][77][78] isomerization, 79,80 C-and N-terminal variants, [81][82][83] as well as some unexpected modifications [84][85][86] would not have been possible without the development of new mass spectrometers with increased sensitivity, resolution, mass accuracy, and mass range. However, "older" techniques such as SDS-PAGE or SEC still provide useful information and are important for routine analysis in a Good Manufacturing Practice (GMP) environment.…”

Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry

“…The charge related heterogeneity of the mAbs can be resolved through ion exchange chromatography (IEC) or other chargebased separation methods, e.g., isoelectric focusing (IEF). 4,5 Charge variants of a recombinant humanized IgG1 were previously isolated at Genentech using cation exchange displacement chromatography and reported to have similar in vitro potency or in vivo pharmacokinetics (PK) in rats. 3 The marginal changes in MAb1, a human IgG1 monoclonal antibody produced in a NS0 cell line, exhibits charge heterogeneity because of the presence of variants formed by processes such as N-terminal glutamate cyclization, C-terminal lysine truncation, deamidation, aspartate isomerization and sialylation in the carbohydrate moiety.…”

In vivo characterization of therapeutic monoclonal antibodies

“…Although glutaminyl cyclase, an enzyme that catalyzes this conversion, is found in human blood [15], the formation of pyroglutamate from glutamate of mAbs in vivo is a pH dependent non-enzymatic reaction [14,16], as it replicated the rate constant in PBS in vitro. The C-terminal lysine residue of a mAb heavy chain is susceptible to be removed by unidentified basic carboxypeptidase during protein expression [17]. Partial removal of the C-terminal lysine leads to charge heterogeneity.…”