pH-dependent crystal structures of a humanised therapeutic antibody Fab fragment: impact on molecular dynamics simulations and comparison to solution structures

Abstract Antibody-fragment (Fab) therapy development has the potential to be accelerated by computational modelling and simulations that predict their target binding, stability, formulation, manufacturability, and the impact of further protein engineering. Such approaches are currently predicated on starting with good crystal structures that closely represent those found under the solution conditions to be simulated. A33 Fab, is an undeveloped immunotherapeutic antibody candidate that was targeted to the human A33 antigen homogeneously expressed in 95% cases of primary and metastatic colorectal cancers. It is now used as a very well characterised testing ground for developing analytics, formulation and protein engineering strategies, and to gain a deeper understanding of mechanisms of destabilisation, representative of the wider therapeutic Fab platform. In this article, we report the structure of A33 Fab in two different crystal forms obtained at widely differing pH. These reveal important insights into the structural impact of pH on the ‘grafted’ complementarity-determining regions (CDRs) and the switch linker between the variable and the constant regions. It also enabled a direct evaluation of the impact of initial crystal structure selection on the outcomes of molecular dynamics simulations under different conditions, and their subsequent use for determining best fit solution structures using previously obtained small-angle x-ray scattering (SAXS) data. The differences in the two structures did not have a major impact on MD simulations regardless of the pH, other than a slight persistence of structure affecting the solvent accessibility of one of the predicted APR regions of A33 Fab. Interestingly, despite being obtained at pH 4 and pH 9, the two crystal structures were more similar to the SAXS solution structures obtained at pH 7, than to those at pH 4 or pH 9. Furthermore, the P 6 5 crystal structure from pH 9 was also a better representation of the solution structures at any other pH, than was the P 1 structure obtained at pH 4. Thus, while obtained at different pH, the two crystal structures may represent highly ( P 6 5 ) and lesser ( P 1) populated species that both exist at pH 7 in solution. These results now lay the foundation for confident MD simulations of A33 Fab that rationalise or predict behaviours in a range of conditions.