title: When Good Intentions Go Awry: Modification of a Recombinant Monoclonal Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbic Acid.
abstract: With the advent of new initiatives to develop chemically defined media, cell culture scientists screen many additives to improve cell growth and productivity. However, the introduction or increase of supplements, typically considered beneficial or protective on their own, to the basal media or feed stream may cause unexpected detrimental consequences to product quality. For instance, because cultured cells are constantly under oxidative stress, ascorbic acid (vitamin C, a potent natural reducing agent) is a common additive to cell culture media. However, as reported herein, a recombinant monoclonal antibody (adalimumab) in cell culture was covalently modified by xylosone (molecular weight 148), an oxidative product of ascorbate. Containing reactive carbonyl groups, xylosone modifies various amines (e.g., the N-termini of the heavy and light chains and susceptible lysines), forming either hemiaminal (+148 Da) or Schiff base (imine, +130 Da) products. Our findings show, for the first time, that ascorbate-derived xylosone can contribute to an increase in molecular heterogeneity, such as acidic species. Our work serves as a reminder that additives to cell culture and their metabolites may become reactive and negatively impact the overall product quality and should be carefully monitored with any changes in cell culture conditions.
authors.affiliation: †Protein Analytics, Process Sciences Department, AbbVie Bioresearch Center, Worcester, Massachusetts 01605, United States.|§Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States.
authors.forename: Chris
authors.initials: C
authors.lastname: Chumsae
authors.affiliation: ‡Cell Culture, Process Sciences Department, AbbVie Bioresearch Center, Worcester, Massachusetts 01605, United States.
authors.forename: Patrick
authors.initials: P
authors.lastname: Hossler
authors.affiliation: †Protein Analytics, Process Sciences Department, AbbVie Bioresearch Center, Worcester, Massachusetts 01605, United States.
authors.forename: Haly
authors.initials: H
authors.lastname: Raharimampionona
authors.affiliation: †Protein Analytics, Process Sciences Department, AbbVie Bioresearch Center, Worcester, Massachusetts 01605, United States.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: ‡Cell Culture, Process Sciences Department, AbbVie Bioresearch Center, Worcester, Massachusetts 01605, United States.
authors.forename: Sean
authors.initials: S
authors.lastname: McDermott
authors.affiliation: ‡Cell Culture, Process Sciences Department, AbbVie Bioresearch Center, Worcester, Massachusetts 01605, United States.
authors.forename: Chris
authors.initials: C
authors.lastname: Racicot
authors.affiliation: †Protein Analytics, Process Sciences Department, AbbVie Bioresearch Center, Worcester, Massachusetts 01605, United States.
authors.forename: Czeslaw
authors.initials: C
authors.lastname: Radziejewski
authors.affiliation: §Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States.
authors.forename: Zhaohui Sunny
authors.initials: ZS
authors.lastname: Zhou
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D001205:Ascorbic Acid / Q000737:chemistry* / Q000378:metabolism; D018929:Cell Culture Techniques; D007661:Ketoses / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D007661:Ketoses; D011994:Recombinant Proteins; C067906:threo-pentos-2-ulose; D001205:Ascorbic Acid