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Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody.

ABSTRACT: The effects of secondary structure on asparagine (N) deamidation in a 22 amino acid sequence (369-GFYPSDIAVEWESNGQPENNYK-390) of the crystallizable (Fc) fragment of a human monoclonal antibody (Fc IgG1) were investigated using high-resolution ultra performance liquid chromatography with tandem mass spectrometry (UPLC/MS). Samples containing either the intact Fc IgG (approximately 50 kD) ("intact protein"), or corresponding synthetic peptides ("peptide") were stored in Tris buffer at 37 degrees C and pH 7.5 for up to forty days, then subjected to UPLC/MS analysis with high energy MS1 fragmentation. The peptide deamidated only at N(382) to form the isoaspartate (isoD(382)) and aspartate (D(382)) products in the ratio of approximately 4:1, with a half-life of approximately 3.4 days. The succinimide intermediate (Su(382)) was also detected; deamidation was not observed for the other two sites (N(387) and N(388)) in peptide samples. The intact protein showed a 30-fold slower overall deamidation half-life of approximately 108 days to produce the isoD(382) and D(387) products, together with minor amounts of D(382). Surprisingly, the D(382) and isoD(387) products were not detected in intact protein samples and, as in the peptide samples, deamidation was not detected at N(388). The results indicate that higher order structure influences both the rate of N-deamidation and the product distribution.

SUBMITTER: Sinha S

PROVIDER: S-EPMC2776945 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody.

Sinha Sandipan S Zhang Lei L Duan Shaofeng S Williams Todd D TD Vlasak Josef J Ionescu Roxana R Topp Elizabeth M EM

Protein science : a publication of the Protein Society 20090801 8

The effects of secondary structure on asparagine (N) deamidation in a 22 amino acid sequence (369-GFYPSDIAVEWESNGQPENNYK-390) of the crystallizable (Fc) fragment of a human monoclonal antibody (Fc IgG1) were investigated using high-resolution ultra performance liquid chromatography with tandem mass spectrometry (UPLC/MS). Samples containing either the intact Fc IgG (approximately 50 kD) ("intact protein"), or corresponding synthetic peptides ("peptide") were stored in Tris buffer at 37 degrees C ...[more]

PMID: 19544580

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Project description:BackgroundMany antibody crystal structures have been solved. Structural modeling programs have been developed that utilize this information to predict 3-D structures of an antibody based upon its sequence. Because of the problem of self-reference, the accuracy and utility of these predictions can only be tested when a new structure has not yet been deposited in the Protein Data Bank.MethodsWe have solved the crystal structure of the Fab fragment of RAC18, a protective anti-ricin mAb, to 1.9 Å resolution. We have also modeled the Fv structure of RAC18 using publicly available Ab modeling tools Prediction of Immunoglobulin Structures (PIGS), RosettaAntibody, and Web Antibody Modeling (WAM). The model structures underwent energy minimization. We compared results to the crystal structure on the basis of root-mean-square deviation (RMSD), template modeling score (TM-score), Z-score, and MolProbity analysis.FindingsThe crystal structure showed a pocket formed mainly by AA residues in each of the heavy chain complementarity determining regions (CDRs). There were differences between the crystal structure and structures predicted by the modeling tools, particularly in the CDRs. There were also differences among the predicted models, although the differences were small and within experimental error. No one modeling program was clearly superior to the others. In some cases, choosing structures based only on sequence homology to the crystallized Ab yielded RMSDs comparable to the models.ConclusionsMolecular modeling programs accurately predict the structure of most regions of antibody variable domains of RAC18. The hypervariable CDRs proved most difficult to model, particularly H chain CDR3. Because CDR3 is most often involved in contact with antigen, this defect must be considered when using models to identify potential contacts between antibody and antigen. Because this study represents only a single case, the results cannot be generalized. Rather they highlight the utility and limitations of modeling programs.

Dataset Information

Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody.

Publications

Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody.

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