C-terminal lysine processing of human immunoglobulin G2 heavy chain in vivo.
Although human IgG heavy chain genes encode a C-terminal lysine, this residue is mostly absent from the endogenous antibodies isolated from serum. Some low but variable level of C-terminal lysine is present on therapeutic antibodies expressed in mammalian cell culture systems. Here, we monitored the C-terminal lysine processing of a recombinant human IgG2 antibody after intravenous injection into human subjects. Peptide mapping of the therapeutic antibody isolated from serum samples by affinity purification was used to quantify the C-terminal lysine levels over time in vivo. The C-terminal lysine residue was found to be rapidly lost in vivo with a half life of about an hour (62 min). In vivo C-terminal lysine processing could be reproduced in vitro, but at a faster rate, by incubating in human serum. Pretreated serum, under conditions used to inactivate carboxypeptidase U, generated in vitro C-terminal lysine processing rates that more closely matched those in vivo. Endogenous IgG, isolated from human blood, contained very low levels of C-terminal lysine (∼0.02%), consistent with the expected circulating half life of antibodies and the calculated C-terminal lysine processing rate. Thus, the low residual IgG2 C-terminal lysine is rapidly processed in vivo and such processing likely occurs on endogenous antibodies in circulation.
Cai
Bing
B
Department of Process and Product Development, Amgen Inc., Thousand Oaks, California 91320, USA.
Pan
Hai
H
Flynn
Gregory C
GC
D006207:Half-Life; D006805:Human Experimentation; D006801:Humans; D007074:Immunoglobulin G / Q000008:administration & dosage / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000008:administration & dosage / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000378:metabolism / Q000493:pharmacokinetics
D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins; D008239:Lysine