RESUMEN
PURPOSE: To identify and understand isomerization products and degradation profile of different aspartate residues in an IgG1 monoclonal antibody. METHODS: Recombinant IgG1 was incubated for extended periods of time in a formulation buffer at recommended and accelerated storage temperatures. Isomerization reaction products were analyzed using ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), peptide mapping, and LC-MS. Model peptides with sequences containing specific aspartate residues in IgG1 were synthesized and incubated under accelerated conditions. Products of isomerization reactions of peptides were analyzed by reverse phase chromatography (RP-HPLC) and LC-MS. X-ray crystallography data from Fab of IgG1 were used to understand mechanism of isomerization reactions. RESULTS: A MAb containing labile Asp32-Gly sequence in CDR I region undergoes rapid isomerization reaction and leads to formation of isoaspartate (IsoAsp) and cyclic imide (Asu) forms. Isomerization of aspartate residues was observed in a non-CDR region containing Asp74-Ser sequence. Isomerization reaction at Asp74-Ser led to formation of Asu74 and trace isoAsp74. While isoAsp32 increased linearly with time, isoAsp74 did not increase during storage. Asu32 and Asu74 followed non-linear degradation kinetics and reached steady state over time. Isomerization reaction of two different model peptides containing Asp32-Gly or Asp74-Ser with neighboring amino acid sequences as those found in the MAb result in formation of IsoAsp. CONCLUSIONS: Observed levels of Asu and trace IsoAsp at the Asp74 site are unusual for typical isomerization reactions. In addition to primary sequences, pKa, solvent exposure and high order structure around aspartate residues may have influenced isomerization reaction at Asp74 in MAbI. Different degradation profiles from the two Asp residues can influence shelf life and should be carefully evaluated during product development.
