C-terminal lysine processing of human immunoglobulin G2 heavy chain in vivo

• Published in: Biotechnology and bioengineering Vol. 108; no. 2; pp. 404 - 412
• Main Authors: Cai, Bing, Pan, Hai, Flynn, Gregory C
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.02.2011; Wiley; Wiley Subscription Services, Inc
• Subjects: Amino acids; Biological and medical sciences; Biotechnology; carboxypeptidase; Cells; clearance; critical quality attribute; Enzymes; Fundamental and applied biological sciences. Psychology; Genes; Half-Life; Health. Pharmaceutical industry; human antibody; Human Experimentation; Humans; Immunoglobulin G - administration & dosage; Immunoglobulin G - metabolism; Immunoglobulin Heavy Chains - administration & dosage; Immunoglobulin Heavy Chains - metabolism; Industrial applications and implications. Economical aspects; Lysine - metabolism; Monoclonal antibodies; Peptides; posttranslational modification; Production of active biomolecules; Protein Processing, Post-Translational; quality by design; Recombinant Proteins - administration & dosage; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacokinetics; Drug; Human; Heavy peptide chain; Immunoglobulins; Enzyme; Antibody; critical quality attribute; quality by design; Carboxypeptidase; Clearance; IgG2; Biopharmaceuticals; Posttranslational modification; In vivo; Recombinant protein; human antibody
• ISSN: 0006-3592; 1097-0290; 1097-0290
• DOI: 10.1002/bit.22933

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. Biotechnol. Bioeng. 2011;108: 404-412.