title: Recombinant antibody color resulting from advanced glycation end product modifications.
abstract: Recombinant antibodies produced in Chinese hamster ovary (CHO) cells often exhibit a slight yellow-brown color, but the molecular basis for this color has remained elusive. We predicted that the color results from post-translational modifications on the antibody, because colored species were found to coelute with antibody products during size exclusion chromatography. Previously, modification by oxidation and advanced glycation end products (AGEs) had been shown to give rise to colored and fluorescent species whose spectral properties are in agreement with the spectral properties of CHO-derived recombinant antibodies. To test whether these modifications give rise to the color exhibited in our CHO-derived antibodies, we searched for 8 different oxidation and 28 different glycation and AGE modifications by mass spectrometry in a variety of samples exhibiting varying color intensities. Oxidation and glycation modifications correlated weakly with color in a subset of samples, but several AGEs exhibited a strong correlation with product color in all samples tested. This strong correlation with sample color was verified for a specific AGE, carboxymethyllysine, by ELISA, thus validating the mass spectrometry data. These data indicate that AGEs are at least partially responsible for the color seen in CHO-derived recombinant antibodies.
authors.affiliation: Protein Analytical Chemistry, Genentech , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Margaret
authors.initials: M
authors.lastname: Butko
authors.forename: Hilary
authors.initials: H
authors.lastname: Pallat
authors.forename: Armando
authors.initials: A
authors.lastname: Cordoba
authors.forename: X Christopher
authors.initials: XC
authors.lastname: Yu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D016466:CHO Cells; D002850:Chromatography, Gel; D003116:Color; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D017127:Glycation End Products, Advanced / Q000737:chemistry* / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry; D013058:Mass Spectrometry; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D017127:Glycation End Products, Advanced; D007074:Immunoglobulin G; D011994:Recombinant Proteins; C048496:N(6)-carboxymethyllysine; D008239:Lysine