Rapid method for monitoring galactosylation levels during recombinant antibody production by electrospray mass spectrometry with selective-ion monitoring.
This report describes a simple and rapid method to determine the relative amounts of glycoforms differing in terminal galactose on a recombinant antibody produced in Chinese hamster ovary (CHO) cells. The method uses a single quadrupole mass spectrometer coupled to an HPLC system to quantify the glycoform amounts found on a recombinant antibody that binds to the human CD20 antigen. Samples from the recombinant antibody process are reduced and injected directly into the HPLC system where the heavy and light chain antibody fragments, as well as host-cell protein contaminants, are separated chromatographically. Mass-selective detection is performed in the selected-ion monitoring (SIM) mode to monitor the most abundant (38+) ions corresponding to the glycoforms found on the heavy chain of the recombinant antibody. Results obtained using the assay demonstrate good sensitivity, linearity and reproducibility. Comparison to a method using capillary electrophoresis (CE) of the labeled free oligosaccharides demonstrates similar quantitation of the glycoforms in the recombinant antibody. The LC-MS method provides a simple and rapid means for accurately quantifying antibody glycoforms directly from cell culture and other process samples.
Wan
H Z
HZ
Department of Manufacturing Sciences, Genentech, Inc., South San Francisco, CA 94080, USA.
Kaneshiro
S
S
Frenz
J
J
Cacia
J
J
D000818:Animals; D000917:Antibody Formation; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006224:Cricetinae; D005690:Galactose / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D011994:Recombinant Proteins; D005690:Galactose