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Rational design of recombinant therapeutic proteins can help to maximise their therapeutic potential. Here, we describe an in silico approach for the identification of engineered mutants of a human immunoglobulin G1 (IgG1) as potential therapeutics. A structure-based homology model of the variable region of the Fab was built and the accessibility of the disulphide-bonded loops to a highly reducing environment was evaluated by examining the surface area covered by accessible side-chains in the unbound form of the model. Subsequently, a structure-based sequence design algorithm was applied, starting with a pool of naturally occurring solvent-accessible wild-type sequence variants. The target was to generate mutant variants of the Fab with reduced thermostability, thus enabling the engineered Fab fragments to retain their reduced unfolding propensity in an environment less reducing than that normally present in the circulation. The mutations were identified by scanning the sequence against a database of surface-accessible side-chains, and the mutations were evaluated for their structural stability and disulphide bond propensity. The results indicate that four double mutants show a strong reduction in thermostability, as judged by changes in the melting temperature, and two of the double mutants exhibit a strong propensity for disulphide bond formation, as judged by a high ratio of solvent-accessible cysteines. Predictions of thermostability and disulphide-bond propensity for individual variants in the library were examined and compared with experimental results. One of the double mutants was shown to be a better candidate for downstream production than its corresponding single-mutant parent, as judged by quality-control tests such as N-terminal sequence analysis and the melt aggregation profile. Furthermore, a comparison of single and double mutants is made in relation to biological activity, as measured in vitro using a human IgA-Fc receptor-dependent proliferation assay.PRODUCTS & SERVICES

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