Recent advances in biologic drug development have led to creation of new formats of biologics to target diseases in new ways. A large and rapidly growing area of new biologic formats is bispecific antibodies, with over 80 formats currently in development.  One approach for generating bispecific antibodies is assembly of two half antibodies into a bispecific.  This approach can result in novel impurities which may be highly toxic and require multiple orthogonal assays to quantify.  New analytical tools are needed to increase process understanding, reduce analytical burden, enable real-time monitoring of process dynamics, and reduce yield loss to unwanted byproducts. Raman spectroscopy is an emerging technique in biopharmaceutical industry and has demonstrated value for fermentation process monitoring. This talk will cover application of in-line Raman spectroscopy to monitoring assembly of a knob-hole bispecific antibody. Partial Least Squares (PLS) modeling was used to predict assembly products from multiple assays (HPLC-SEC, HPLC-RP, mass spec, Ellman’s assay). Raman spectral preprocessing and region selection increased model accuracy. Model strength was confirmed via predictive validation. Analysis of prediction errors suggests that formation of bispecific antibodies, aggregates, and homodimers as measured by HPLC-SEC are well modeled across a range of temperatures, pH, and reductant concentrations. Accurate predictions were also made for system free thiols as measured by the Ellman’s assay.

In-line Raman spectroscopy for bispecific assembly process monitoring

253rd National Meeting (2017)

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