title: Discovery of a chemical modification by citric acid in a recombinant monoclonal antibody.
abstract: Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called "acidic species" that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity.
authors.affiliation: Protein Analytics, Process Sciences, AbbVie Bioresearch Center , Worcester, Massachusetts 01605, United States.
authors.forename: Chris
authors.initials: C
authors.lastname: Chumsae
authors.forename: Liqiang Lisa
authors.initials: LL
authors.lastname: Zhou
authors.forename: Yang
authors.initials: Y
authors.lastname: Shen
authors.forename: Jessica
authors.initials: J
authors.lastname: Wohlgemuth
authors.forename: Emma
authors.initials: E
authors.lastname: Fung
authors.forename: Randall
authors.initials: R
authors.lastname: Burton
authors.forename: Czeslaw
authors.initials: C
authors.lastname: Radziejewski
authors.forename: Zhaohui Sunny
authors.initials: ZS
authors.lastname: Zhou
mesh_terms: D000588:Amines / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D019343:Citric Acid / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D000588:Amines; D000911:Antibodies, Monoclonal; D010455:Peptides; D011994:Recombinant Proteins; D019343:Citric Acid