Triple light chain antibodies: factors that influence its formation in cell culture.
THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species--Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression--evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC)--correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production--evaluated as the ratio of the cell-specific production rate of GSH (q(GSH)) to the cell-specific production rate of THIOMAB (q(p))--corresponded to decreased 3LC levels. In time-lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and q(GSH)/q(p) ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high q(GSH)/q(p) ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity.
Early Stage Cell Culture, Genentech, Inc., 1 DNA Way, MS 32, South San Francisco, California 94080-4990, USA. gomez.natalia@gene.com
Natalia
N
Gomez
Abigail R
AR
Vinson
Jun
J
Ouyang
Mary D H
MD
Nguyen
Xiaoying-Nancy
XN
Chen
Vikas K
VK
Sharma
Inn H
IH
Yuk
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D016466:CHO Cells; D018929:Cell Culture Techniques; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D007147:Immunoglobulin Light Chains; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005978:Glutathione; D003545:Cysteine