title: Rapid identification of an antibody DNA construct rearrangement sequence variant by mass spectrometry.
abstract: During cell line development for an IgG1 antibody candidate (mAb1), a C-terminal extension was identified in 2 product candidate clones expressed in CHO-K1 cell line. The extension was initially observed as the presence of anomalous new peaks in these clones after analysis by cation exchange chromatography (CEX-HPLC) and reduced capillary electrophoresis (rCE-SDS). Reduced mass analysis of these CHO-K1 clones revealed that a larger than expected mass was present on a sub-population of the heavy chain species, which could not be explained by any known chemical or post-translational modifications. It was suspected that this additional mass on the heavy chain was due to the presence of an additional amino acid sequence. To identify the suspected additional sequence, de novo sequencing in combination with proteomic searching was performed against translated DNA vectors for the heavy chain and light chain. Peptides unique to the clones containing the extension were identified matching short sequences (corresponding to 9 and 35 amino acids, respectively) from 2 non-coding sections of the light chain vector construct. After investigation, this extension was observed to be due to the re-arrangement of the DNA construct, with the addition of amino acids derived from the light chain vector non-translated sequence to the C-terminus of the heavy chain. This observation showed the power of proteomic mass spectrometric techniques to identify an unexpected antibody sequence variant using de novo sequencing combined with database searching, and allowed for rapid identification of the root cause for new peaks in the cation exchange and rCE-SDS assays.
authors.affiliation: a Analytical Sciences; Amgen Inc. ; Seattle , WA USA.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Scott
authors.forename: Rich
authors.initials: R
authors.lastname: Rogers
authors.forename: Alain
authors.initials: A
authors.lastname: Balland
authors.forename: Lowell J
authors.initials: LJ
authors.lastname: Brady
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D016466:CHO Cells; D002412:Cations; D002852:Chromatography, Ion Exchange; D018388:Codon, Terminator / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D019075:Electrophoresis, Capillary; D007074:Immunoglobulin G / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping / Q000379:methods; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D002412:Cations; D018388:Codon, Terminator; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D004247:DNA
keywords: C-terminal extension; CAN, acetonitrile; CEX, cation exchange; CHO, Chinese hamster ovary; DNA, deoxyribonucleic acid; DTT, dithiothreitol; Da, Dalton; FDR, false discovery rate; HC, heavy chain; HPLC, high performance liquid chromatography; LC, light chain; MS, mass spectrometer; MS/MS, tandem mass spectrometry; MW, molecular weight; NCBI, National Center for Biotechnology Information; NCG, non-concensus glycosylation; PSM, peptide-spectrum matches; RP-UPLC, reversed phase ultra-high pressure liquid chromatography; SEC, size exclusion chromatography; TFA, trifluoracetic acid; TOF, time of flight mass spectrometer; UV, ultraviolet; aa, amino acids; mass spectrometry; ppm, parts per million; rCE-SDS, reduced capillary electrophoresis-sodium dodecyl sulfate; sequence variant