Formation of pyroglutamic acid from N-terminal glutamic acid in immunoglobulin gamma antibodies.
The status of the N-terminus of proteins is important for amino acid sequencing by Edman degradation, protein identification by shotgun and top-down techniques, and to uncover biological functions, which may be associated with modifications. In this study, we investigated the pyroglutamic acid formation from N-terminal glutamic acid residues in recombinant monoclonal antibodies. Almost half the antibodies reported in the literature contain a glutamic acid residue at the N-terminus of the light or the heavy chain. Our reversed-phase high-performance liquid chromatography-mass spectrometry method could separate the pyroglutamic acid-containing light chains from the native light chains of reduced and alkylated recombinant monoclonal antibodies. Tryptic peptide mapping and tandem mass spectrometry of the reduced and alkylated proteins was used for the identification of the pyroglutamic acid. We identified the formation of pyroglutamic acid from N-terminal glutamic acid in the heavy chains and light chains of several antibodies, indicating that this nonenzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37 and 45 degrees C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyroglutamic acid at pH 6.2 and increased formation of pyroglutamic acid at pH 4 and pH 8. The half-life of the N-terminal glutamic acid was approximately 9 months in a pH 4.1 buffer at 45 degrees C. To our knowledge, we showed for the first time that glutamic acid residues located at the N-terminus of proteins undergo pyroglutamic acid formation in vitro.
Department of Pharmaceutics, Amgen, Inc., Thousand Oaks, California 91320, USA. dchelius@amgen.com
Dirk
D
Chelius
Kay
K
Jing
Alexis
A
Lueras
Douglas S
DS
Rehder
Thomas M
TM
Dillon
Alona
A
Vizel
Rahul S
RS
Rajan
Tiansheng
T
Li
Michael J
MJ
Treuheit
Pavel V
PV
Bondarenko
D000478:Alkylation; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D018698:Glutamic Acid / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007142:Immunoglobulin gamma-Chains / Q000378:metabolism*; D008969:Molecular Sequence Data; D011761:Pyrrolidonecarboxylic Acid / Q000032:analysis* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D013696:Temperature; D013997:Time Factors
D000911:Antibodies, Monoclonal; D007142:Immunoglobulin gamma-Chains; D018698:Glutamic Acid; D011761:Pyrrolidonecarboxylic Acid