title: Characterization of antibody variants during process development: the tale of incomplete processing of N-terminal secretion peptide.
abstract: Monoclonal antibodies (mAbs) have emerged as one of the most important classes of biotherapeutics, although development of these molecules is long and arduous. A production cell line must be established, and growth conditions for the cells and purification processes for the product must be optimized. Integration of the appropriate analytical strategies in these activities is the cornerstone of Quality by Design and in-process control approaches are encouraged by the Food and Drug Administration. We report here the development of a reversed phase-high performance liquid chromatography (RP-HPLC) method to follow the presence of a mAb product-related variant observed during the purification process development. The variant eluted as a later peak on RP-HPLC, compared with the mAb control (3.25 min and 2.85 min, respectively). We isolated this hydrophobic variant and further analyzed it by mass spectrometry. We identified the variant as a mAb with an incompletely processed leader sequence attached to the N-terminus of one of the two heavy chains.
authors.affiliation: BioProcess Development, Merck Research Laboratories, Union, NJ, USA. alexandre.ambrogelly@merck.com
authors.forename: Alexandre
authors.initials: A
authors.lastname: Ambrogelly
authors.forename: Yan-Hui
authors.initials: YH
authors.lastname: Liu
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.forename: Selina
authors.initials: S
authors.lastname: Mengisen
authors.forename: Bingyi
authors.initials: B
authors.lastname: Yao
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
authors.forename: Susan
authors.initials: S
authors.lastname: Cannon-Carlson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D020033:Protein Isoforms; D021382:Protein Sorting Signals