Photochemical degradation of citrate buffers leads to covalent acetonation of recombinant protein therapeutics.
Protein Sci
; 19(11): 2152-63, 2010 Nov.
Article
en En
| MEDLINE
| ID: mdl-20836085
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Acetona
/
Proteínas Recombinantes
/
Citratos
/
Anticuerpos Monoclonales
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Protein Sci
Asunto de la revista:
BIOQUIMICA
Año:
2010
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos