C-terminal lysine variants in fully human monoclonal antibodies: investigation of test methods and possible causes.
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.
Department of Analytical Development, Medarex, Inc., 519 Route 173 West, Bloomsbury, New Jersey 08804, USA.
Lawrence W
LW
Dick
Difei
D
Qiu
David
D
Mahon
Michael
M
Adamo
Kuang-Chuan
KC
Cheng
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000648:ultrastructure*; D002021:Buffers; D016466:CHO Cells; D043424:Carboxypeptidase B / Q000037:antagonists & inhibitors / Q000378:metabolism; D018929:Cell Culture Techniques; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D019075:Electrophoresis, Capillary; D005260:Female; D006801:Humans; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000032:analysis / Q000648:ultrastructure; D007525:Isoelectric Focusing; D008239:Lysine / Q000032:analysis*; D010449:Peptide Mapping / Q000379:methods*; D010940:Plant Proteins / Q000494:pharmacology; D011480:Protease Inhibitors; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D000911:Antibodies, Monoclonal; D002021:Buffers; D007143:Immunoglobulin Heavy Chains; D010940:Plant Proteins; D011480:Protease Inhibitors; D043424:Carboxypeptidase B; D008239:Lysine