Elucidation of Two Major Aggregation Pathways in an IgG2 Antibody
Nicholas Van Buren, Douglas Rehder, Himanshu Gadgil, Masazumi Matsumura, Jaby Jacob
Journal of Pharmaceutical Sciences
wo major aggregation pathways observed in an IgG2 molecule are described. Different aggregate species generated by long-term incubation of the antibody at 37°C were collected by a semi-preparative size exclusion chromatography method. These purified species were analyzed extensively by denaturing size-exclusion chromatography methods. The major aggregation pathway at low pH (4.0) resulted in the formation of both dimers and high molecular weight (HMW) aggregates. It was found that these dimers and HMW aggregates contain antibody molecules that have a peptide bond cleavage between an aspartic acid and proline residue in the CH2 domain. Evidence that unfolding of the CH2 domain may be driving the aggregation at low pH is presented. At higher pH (pH – 6.0), formation of a dimer having approximately 75% covalent character was the major aggregation pathway while formation of higher molecular weight aggregates were largely suppressed. The covalent dimer consisted of both disulfide linked antibody molecules and another species (∼26%) that was formed due to nondisulfide covalent bonds between two heavy chains. At pH – 5.0, both dimer and higher molecular weight aggregates were formed and the aggregation pathway was a combination of the major pathways observed at pH – 4.0 and 6.0. The dimer species formed at pH – 5.0 had a larger contribution from covalent species—both disulfide and nondisulfide linked, while the HMW aggregate contained a higher percentage of molecules that had the peptide bond cleavage in the CH2 domain. The dimer formed at pH – 6.0 was found to have identical secondary and tertiary structure as the intact antibody molecule. However, the dimer and higher molecular weight aggregate formed at pH – 4.0 have altered secondary and tertiary structure.
Circular dichroism, Secondary structure, Tertiary structure, Aggregation, Antibodies, Pharmaceutical, Biochemistry