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Potential aggregation prone regions in biotherapeutics: A survey of commercial monoclonal antibodies.

ABSTRACT: Aggregation of a biotherapeutic is of significant concern and judicious process and formulation development is required to minimize aggregate levels in the final product. Aggregation of a protein in solution is driven by intrinsic and extrinsic factors. In this work we have focused on aggregation as an intrinsic property of the molecule. We have studied the sequences and Fab structures of commercial and non-commercial antibody sequences for their vulnerability towards aggregation by using sequence based computational tools to identify potential aggregation-prone motifs or regions. The mAbs in our dataset contain 2 to 8 aggregation-prone motifs per heavy and light chain pair. Some of these motifs are located in variable domains, primarily in CDRs. Most aggregation-prone motifs are rich in beta branched aliphatic and aromatic residues. Hydroxyl-containing Ser/Thr residues are also found in several aggregation-prone motifs while charged residues are rare. The motifs found in light chain CDR3 are glutamine (Q)/asparagine (N) rich. These motifs are similar to the reported aggregation promoting regions found in prion and amyloidogenic proteins that are also rich in Q/N, aliphatic and aromatic residues. The implication is that one possible mechanism for aggregation of mAbs may be through formation of cross-beta structures and fibrils. Mapping on the available Fab-receptor/antigen complex structures reveals that these motifs in CDRs might also contribute significantly towards receptor/antigen binding. Our analysis identifies the opportunity and tools for simultaneous optimization of the therapeutic protein sequence for potency and specificity while reducing vulnerability towards aggregation.

SUBMITTER: Wang X

PROVIDER: S-EPMC2726584 | biostudies-literature | 2009 May-Jun

REPOSITORIES: biostudies-literature

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Potential aggregation prone regions in biotherapeutics: A survey of commercial monoclonal antibodies.

Wang Xiaoling X Das Tapan K TK Singh Satish K SK Kumar Sandeep S

mAbs 20090529 3

Aggregation of a biotherapeutic is of significant concern and judicious process and formulation development is required to minimize aggregate levels in the final product. Aggregation of a protein in solution is driven by intrinsic and extrinsic factors. In this work we have focused on aggregation as an intrinsic property of the molecule. We have studied the sequences and Fab structures of commercial and non-commercial antibody sequences for their vulnerability towards aggregation by using sequen ...[more]

PMID: 20065649

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Project description:BackgroundPotato virus Y (PVY, genus Potyvirus) causes substantial economic losses in solanaceous plants. Routine screening for PVY is an essential part of seed potato certification, and serological assays are often used. The commercial, commonly used monoclonal antibodies, MAb1128, MAb1129, and MAb1130, recognize the viral coat protein (CP) of PVY and distinguish PVYN strains from PVYO and PVYC strains, or detect all PVY strains, respectively. However, the minimal epitopes recognized by these antibodies have not been identified.Methodology/principal findingsSPOT peptide array was used to map the epitopes in CP recognized by MAb1128, MAb1129, and MAb1130. Then alanine replacement as well as N- and C-terminal deletion analysis of the identified peptide epitopes was done to determine critical amino acids for antibody recognition and the respective minimal epitopes. The epitopes of all antibodies were located within the 30 N-terminal-most residues. The minimal epitope of MAb1128 was 25NLNKEK30. Replacement of 25N or 27N with alanine weakened the recognition by MAb1128, and replacement of 26L, 29E, or 30K nearly precluded recognition. The minimal epitope for MAb1129 was 16RPEQGSIQSNP26 and the most critical residues for recognition were 22I and 23Q. The epitope of MAb1130 was defined by residues 5IDAGGS10. Mutation of residue 6D abrogated and mutation of 9G strongly reduced recognition of the peptide by MAb1130. Amino acid sequence alignment demonstrated that these epitopes are relatively conserved among PVY strains. Finally, recombinant CPs were produced to demonstrate that mutations in the variable positions of the epitope regions can affect detection with the MAbs.Conclusions/significanceThe epitope data acquired can be compared with data on PVY CP-encoding sequences produced by laboratories worldwide and utilized to monitor how widely the new variants of PVY can be detected with current seed potato certification schemes or during the inspection of imported seed potatoes as conducted with these MAbs.

Dataset Information

Potential aggregation prone regions in biotherapeutics: A survey of commercial monoclonal antibodies.

Publications

Potential aggregation prone regions in biotherapeutics: A survey of commercial monoclonal antibodies.

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