Increased Fab thermoresistance via VH-targeted directed evolution

Protein Eng Des Sel. 2015 Oct;28(10):365-77. doi: 10.1093/protein/gzv037. Epub 2015 Aug 16.


Antibody aggregation is frequently mediated by the complementarity determining regions within the variable domains and can significantly decrease purification yields, shorten shelf-life and increase the risk of anti-drug immune responses. Aggregation-resistant antibodies could offset these risks; accordingly, we have developed a directed evolution strategy to improve Fab stability. A Fab-phage display vector was constructed and the VH domain targeted for mutagenesis by error-prone PCR. To enrich for thermoresistant clones, the resulting phage library was transiently heated, followed by selection for binding to an anti-light chain constant domain antibody. Five unique variants were identified, each possessing one to three amino acid substitutions. Each engineered Fab possessed higher, Escherichia coli expression yield, a 2-3°C increase in apparent melting temperature and improved aggregation resistance upon heating at high concentration. Select mutations were combined and shown to confer additive improvements to these biophysical characteristics. Finally, the wild-type and most stable triple variant Fab variant were converted into a human IgG1 and expressed in mammalian cells. Both expression level and aggregation resistance were similarly improved in the engineered IgG1. Analysis of the wild-type Fab crystal structure provided a structural rationale for the selected residues changes. This approach can help guide future Fab stabilization efforts.

Keywords: antibody engineering; directed evolution; phage display; protein expression; protein stability.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Antigens / immunology
  • Base Sequence
  • Directed Molecular Evolution / methods*
  • Humans
  • Immunoglobulin Heavy Chains / chemistry*
  • Immunoglobulin Heavy Chains / genetics*
  • Immunoglobulin Heavy Chains / immunology
  • Immunoglobulin Variable Region / chemistry*
  • Immunoglobulin Variable Region / genetics*
  • Immunoglobulin Variable Region / immunology
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis
  • Mutation
  • Peptide Library
  • Protein Conformation
  • Protein Stability
  • Temperature*


  • Antigens
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Variable Region
  • Peptide Library