DEAMIDATION AND ISOASPARTIC ACID FORMATION IN ANTIBODIES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The normal degradation pathway for recombinant glycoprotein pharmaceuticals, such as monoclonal antibodies (mAbs), can lead to a variety of undesired products that can adversely affect the ability of the protein to function efficiently. Among the degradation pathways for antibodies is the deamidation of asparagine residues to isoaspartic acid and aspartic acid (typically in a 3:1 ratio, respectively). The deamidation of asparagine in proteins and peptides occurs through a succinimide intermediate followed by partial hydrolysis of the succinimide ring to form the products. Deamidation occurs most frequently at asparagine residues that are followed in the amino acid sequence by a non-steric amino acid such as glycine or serine. A typical IgG1 mAb contains 15  20 asparagine residues on each heavy chain and 4 - 7 asparagine residues on each light chain. However, not all of the asparagine residues are expected to be deamidated since they are not all adjacent to a compact amino acid and the three-dimensional structure of the antibody can aid in protection against degradation at some locations. In addition, isomerization of aspartic acid to isoaspartic acid can also occur through a similar pathway and is both undesirable and considerably more difficult to detect. The presence of deamidation and isoaspartic acid is a concern for biopharmaceutical manufacturing, as these degradation products may lead to tertiary structural changes and loss of activity, especially if present in the CDR regions of either the light or heavy chain. The recently published results by OConnors research group showed that differentiation of aspartic and isoaspartic acid residues is possible using electron capture and electron transfer dissociation. This offers the possibility that rigorous structural characterization of these modifications in monoclonal antibodies may be routinely possible. Furthermore, recent results from the OConnor group suggest that actual site-specific quantitation of the Asn:Asp:isoAsp ratio may be possible as well.