Aspartate Isomerization in the Complementarity-Determining Regions of Two Closely Related Monoclonal Antibodies
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- 作者：Aditya A. Wakankar ; Ronald T. Borchardt ; Charles Eigenbrot ; Steven Shia ; Y. John Wang ; Steve J. Shire ; Jun L. Liu
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：February 13, 2007
- 年：2007
- 卷：46
- 期：6
- 页码：1534 - 1544
- 全文大小：277K
- 年卷期：v.46,no.6(February 13, 2007)
- ISSN：1520-4995
文摘
The aspartic acid residues (Asp) present in the complementarity-determining regions (CDRs)of the light chains of two recombinant monoclonal antibodies (MAbs), MAb I and MAb II, are highlysusceptible to isomerization due to the presence of glycine residues (Gly) on their C-terminal ends. Aspisomerization in these MAbs leads to formation of the isoaspartate (IsoAsp) and the cyclic imide (Asu)variants of these MAbs. Both MAb I and MAb II, employed in this study, elicit their pharmacologicalresponses through binding human IgE. The formation of the MAb variants as a result of Asp isomerizationsignificantly reduces the binding affinities of these antibodies to IgE, thereby reducing their potencies.Here we report on significant differences in the susceptibility of the MAb I and the MAb II to Aspisomerization. The molecular basis for these differences in rates of Asp isomerization was elucidated.The effect of primary sequence on Asp isomerization was evaluated using pentapeptide models of theMAbs, which included the labile Asp residues and their neighboring amino acid residues. The separationof the parent MAbs and pentapeptides from their isomerization products was achieved using hydrophobicinteraction chromatography (HIC) and rp-HPLC, respectively. Structural characterization of the MAbswas performed using differential scanning calorimetry (DSC), circular dichroism (CD), and X-raycrystallography. Our investigations demonstrate that the differences in the Asp isomerization rates betweenMAb I and MAb II can be attributed to structural factors including the conformational flexibility and theextent of solvent exposure of the labile Asp residue.