Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions

Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions. Refolding was, however, efficiently prevented by combining denaturation with reduction of the intra-domain disulfide bridges, which created favourable conditions for selection of clones with improved stability. Using this strategy, scFv mutants with 8-9°C improved thermal stability and 0.8-0.9 M improved stability for guanidinium chloride were found after 4-5 enrichment cycles. The most stable mutants selected contained either Lys^H66Arg or Asn^H52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.