title: Photochemical degradation of citrate buffers leads to covalent acetonation of recombinant protein therapeutics.
abstract: Novel acetone and aldimine covalent adducts were identified on the N-termini and lysine side chains of recombinant monoclonal antibodies. Photochemical degradation of citrate buffers, in the presence of trace levels of iron, is demonstrated as the source of these modifications. The link between degradation of citrate and the observed protein modifications was conclusively established by tracking the citrate decomposition products and protein adducts resulting from photochemical degradation of isotope labeled (13)C citrate by mass spectrometry. The structure of the acetone modification was determined by nuclear magnetic resonance (NMR) spectroscopy on modified-free glycine and found to correspond to acetone linked to the N-terminus of the amino acid through a methyl carbon. Results from mass spectrometric fragmentation of glycine modified with an acetone adduct derived from (13)C labeled citrate indicated that the three central carbons of citrate are incorporated onto protein amines in the presence of iron and light. While citrate is known to stoichiometrically decompose to acetone and CO(2) through various intermediates in photochemical systems, it has never been shown to be a causative agent in protein carbonylation. Our results point to a previously unknown source for the generation of reactive carbonyl species. This work also highlights the potential deleterious impact of trace metals on recombinant protein therapeutics formulated in citrate buffers.
authors.affiliation: Department of Analytical and Formulation Sciences, Amgen, Seattle, Washington 98119, USA.
authors.forename: John F
authors.initials: JF
authors.lastname: Valliere-Douglass
authors.forename: Lisa
authors.initials: L
authors.lastname: Connell-Crowley
authors.forename: Randy
authors.initials: R
authors.lastname: Jensen
authors.forename: Paul D
authors.initials: PD
authors.lastname: Schnier
authors.forename: Egor
authors.initials: E
authors.lastname: Trilisky
authors.forename: Matt
authors.initials: M
authors.lastname: Leith
authors.forename: Brian D
authors.initials: BD
authors.lastname: Follstad
authors.forename: Jennifer
authors.initials: J
authors.lastname: Kerr
authors.forename: Nathan
authors.initials: N
authors.lastname: Lewis
authors.forename: Suresh
authors.initials: S
authors.lastname: Vunnum
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Treuheit
authors.forename: Alain
authors.initials: A
authors.lastname: Balland
authors.forename: Alison
authors.initials: A
authors.lastname: Wallace
mesh_terms: D000096:Acetone / Q000737:chemistry*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D016466:CHO Cells; D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002951:Citrates / Q000737:chemistry*; D006224:Cricetinae; D003412:Cricetulus; D005998:Glycine / Q000737:chemistry; D006801:Humans; D007097:Imines / Q000737:chemistry; D007074:Immunoglobulin G; D007501:Iron / Q000737:chemistry; D007553:Isotope Labeling; D013058:Mass Spectrometry; D019906:Nuclear Magnetic Resonance, Biomolecular; D055668:Photochemical Processes; D050050:Protein Carbonylation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D002247:Carbon Isotopes; D002951:Citrates; D007097:Imines; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D000096:Acetone; D007501:Iron; D005998:Glycine