Photochemical degradation of citrate buffers leads to covalent acetonation of recombinant protein therapeutics.
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.
Valliere-Douglass
John F
JF
Department of Analytical and Formulation Sciences, Amgen, Seattle, Washington 98119, USA.
Connell-Crowley
Lisa
L
Jensen
Randy
R
Schnier
Paul D
PD
Trilisky
Egor
E
Leith
Matt
M
Follstad
Brian D
BD
Kerr
Jennifer
J
Lewis
Nathan
N
Vunnum
Suresh
S
Treuheit
Michael J
MJ
Balland
Alain
A
Wallace
Alison
A
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
D000911:Antibodies, Monoclonal; D002247:Carbon Isotopes; D002951:Citrates; D007097:Imines; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D000096:Acetone; D007501:Iron; D005998:Glycine