Characterization of humanized anti-TAC, an antibody directed against the interleukin 2 receptor, using electrospray ionization mass spectrometry by direct infusion, LC/MS, and MS/MS.
Characterization of a humanized monoclonal antibody (Hu-anti-TAC) directed against a surface protein expressed on T-lymphocytes was performed with an electrospray mass spectrometer. Capillary reversed-phase liquid chromatography (LC)/mass spectrometry (MS) and direct infusion MS were utilized along with tandem MS/MS analysis to confirm the sequence and to determine the sources of heterogeneity in Hu-anti-TAC. The MS analysis was performed on disulfide-reduced and trypsin-digested samples of the antibody. Two forms of diantennary carbohydrate structures were identified and found to be consistent with those reported for the human IgG1 framework. The analysis demonstrated that the N-terminus was modified by conversion of a glutamine residue to pyroglutamic acid. Another source of heterogeneity was the partial removal of the C-terminal lysine residue and was confirmed by mass calculations of tryptic peptides followed by MS/MS sequencing. This study demonstrates that the high sensitivity of electrospray mass spectrometry when combined with capillary chromatography can allow detailed characterization of microgram samples of high molecular weight proteins such as antibodies.
Lewis
D A
DA
Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080-4990.
Guzzetta
A W
AW
Hancock
W S
WS
Costello
M
M
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D015375:Receptors, Interleukin-2 / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry
D000911:Antibodies, Monoclonal; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins