Identification of isomerization and racemization of aspartate in the Asp-Asp motifs of a therapeutic protein.
A thermally stressed Fab molecule showed a significant increase of basic variants in imaged capillary isoelectric focusing (iCIEF) analysis. Mass analyses of the reduced protein found an increase in -18Da species from both light chain and heavy chain. A tryptic peptide map identified two isoAsp-containing peptides, both containing Asp-Asp motifs and located in complementarity-determining regions (CDRs) of light chains and heavy chains, respectively. The approaches of hydrolyzing succinimide in H(2)(18)O followed by tryptic digestion were used to label and identify the sites of isomerization. This method enabled identification of the isomerization site by comparing the MS/MS spectra of isomerized peptides with and without (18)O incorporation. The light chain peptide L2 VTITCITSTDID(12)DDMNWYQQKPGK underwent simultaneous isomerization and recemization at residue Asp-12 after thermal stress as evidenced by the coinjection of synthetic peptide L2 with l-Asp-12, l-isoAsp-12, d-Asp-12, and d-isoAsp-12, respectively. A thermal stress study of the synthetic peptide (l-)L2 showed that the isomerization and racemization did not occur, indicating that the Asp degradation in this Asp-Asp motif is more related to the protein conformation than the primary sequence. Another isomerization site was identified as Asp-24 in the heavy chain peptide H5 QAPGQGLEWMGWINTYTGETTYAD(24)DFK. No other isomerizations were detected in CDR peptides containing either Asp-Ser or Asp-Thr motifs.
Protein Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA. zhang.jennifer@gene.com
Jennifer
J
Zhang
Holly
H
Yip
Viswanatham
V
Katta
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D022801:Complementarity Determining Regions / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007525:Isoelectric Focusing / Q000379:methods; D007536:Isomerism; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000627:therapeutic use; D013388:Succinimides / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013696:Temperature
D022801:Complementarity Determining Regions; D004151:Dipeptides; D007140:Immunoglobulin Fab Fragments; D010455:Peptides; D011506:Proteins; D013388:Succinimides; C108952:aspartyl-aspartic acid; C032620:succinimide