Developing a novel pipeline to determine monoclonal antibody solution structures and stabilities in bioprocessing conditions to optimise manufacture

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BACKGROUND: Monoclonal human IgG1 and IgG4 antibodies have become important therapeutics in the biotechnology industry. Primarily engineered for efficacy against specific targets, antibodies are highly susceptible to aggregation in downstream bioprocess environments, particularly as antibody concentrations increase up to final product concentrations of 50-200 mg/ml. This represents a major challenge for the biotechnology industry where aggregates can seriously compromise safety. It is increasingly important to understand how to manage antibody aggregation, and this is a major theme addressed in the twice-yearly BRIC meetings organised by the BBSRC, attended by the proposers. A direct elucidation of the physical parameters that initiate protein aggregation would allow the design of more robust lead candidates that aggregate less. These factors include buffer pH, temperature (T), buffer ionic strength (I), and antibody concentration (c). A systematic study of the effects of these four factors on antibody solution structures and their prospensity to form large oligomers will reveal the key stages towards their aggregation and suggest avenues for more rational protein engineering to block it. We have developed new methods using scattering and ultracentifugation to determine atomistic solution structures for IgG antibodies (Rayner et al, 2013, 2014, 2015). This year, our methods have been improved further by the incorporation of advanced structural modelling methods in the CCP-SAS project. For example, experimentally, our tests showed that an IgG4 antibody changed its conformation in two steps at pH 3, then pH 7, to form a compact structure that precipitates, and we could model all these conformational changes (Perkins, unpublished). We are thus ideally poised to undertake this timely project within the remit of the BBSRC.AIMS: (1) By studying the solution conformations of two human monoclonal IgG1 and IgG4 antibodies as a function of pH, T, I and c, we will construct phase diagrams of antibody conformation and aggregation propensity. (2) Using accurate atomistic modelling of the data, we will determine antibody structures in these different conditions to correlate major conformational changes with experimental conditions. (3) The inclusion of four other IgG monoclonals will enable us to develop a pipeline for the rapid assessment of new antibody products.EXPERIMENTAL PLAN: Fujifilm will provide gram amounts of monoclonal IgG1 and IgG4 for experiments, and on-site training on the manufacturing processes for IgG monoclonals. Using the high-throughput X-ray beamline BM29 at the ESRF synchrotron, Grenoble, France, with 96-well microplates, the effects of pH, T, I and c will be explored rapidly and systematically to yield phase diagrams. If required, high-throughput neutron data collection on instrument D22 at the ILL, Grenoble, France, will supplement the X-ray data sets. Analytical ultracentrifugation at UCL will reveal conformational or oligomerisation changes at selected pH, T, I and c values. Our new CCP-SAS modelling software (Hui et al 2015, 2016) via high performance computing will generate as many as 700,000 randomised but physically-accurate conformations. This structural library will be compared with each data set to identify the best fit in each buffer, hence identifying the conformation. Ultimately, we will design a pipeline of operations to characterise newly-developed antibody structures rapidly for their manufacturability.