Accumulation of succinimide in a recombinant monoclonal antibody in mildly acidic buffers under elevated temperatures.
OBJECTIVE
The purpose of this paper was to identify the location of a succinimide and determine the rate of its formation and hydrolysis in a recombinant human monoclonal IgG2 antibody aged in mildly acidic buffers at elevated temperatures.


METHODS
Cation exchange (CEX) HPLC separated multiple Main Peaks and high levels (up to 50%) of basic variants, the identification of which was an analytical challenge and required several complementary techniques. The relative abundance of the CEX basic variants was used to quantify the percentage of succinimide and to study the rates of its formation and hydrolysis.


RESULTS
Mass decrease by approximately 18 Da for intact antibodies from the CEX basic fractions suggested succinimide formation from aspartic acid as the major modification. Reversed-phase HPLC/MS of the reduced and trypsin-digested samples detected an isoaspartate 30 (isoD30) in the light chain peptide A25-R37. Direct evidence that isoD30 was from succinimide was obtained by performing succinimide hydrolysis in H2(18)O followed by tryptic digestion in H2(16)O.


CONCLUSIONS
Succinimide formation increased as pH became more acidic, whereas its hydrolysis was faster as pH became neutral and alkaline. Succinimide hydrolysis in a denatured sample was estimated to have completed in less than 2 h, but approximately three days for a similar pH but without denaturant. These observations suggest that protein conformation affects succinimide hydrolysis.
Chu
Grace C
GC
Department of Pharmaceutics, Amgen, Inc., One Amgen Center Drive, MS 8-1-C, Thousand Oaks, California 91320, USA.
Chelius
Dirk
D
Xiao
Gang
G
Khor
Hui K
HK
Coulibaly
Sururat
S
Bondarenko
Pavel V
PV
D000143:Acids / Q000737:chemistry*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013388:Succinimides / Q000737:chemistry*
D000143:Acids; D000911:Antibodies, Monoclonal; D002021:Buffers; D011994:Recombinant Proteins; D013388:Succinimides; C032620:succinimide