Leveraging light chain binding avidity for control of mispaired byproducts during production of asymmetric bispecific...

Many biotherapeutic formats leverage antibody light chain affinity chromatography to enable robust manufacturing processes and to streamline process development. These include multi-specific antibody and antibody fragment platforms which are often designed for specific capture purification methods that can provide efficient removal of commonly expressed product-related impurities. Recently, several accounts of product-related impurity separation by leveraging binding avidity during affinity chromatography have been described in the literature. However, a more comprehensive evaluation of avidity-based separations, particularly for light chain affinity media with specificity for constant regions of antibody light chains, is valuable for development of emerging multi-specific and fragment antibody formats. Results in this work demonstrate the capability of camelid antibody-based light chain affinity media to separate asymmetric bispecific antibody heterodimers from impurities possessing more than one light chain of the same class that the media binds to, including mispaired variants, aggregates, and fragment impurities. Largest resolution for respective mispaired species were provided by CaptureSelect KappaXP and LambdaXP chromatography media. The addition of elution modifiers provided increased impurity separation, with CaptureSelect KappaXP requiring up to 500 mM concentrations of elution modifiers to produce substantial improvements to resolution, and LambdaXP showing much higher sensitivity. Isocratic elution methods developed for lambda light chain affinity chromatography media provided near complete removal of mispaired variants, and substantial removal of aggregates and fragment impurities. Addition of just 20 mM of elution modifiers such as NaCl are shown to drive increased binding strength and separation of heterodimer species from impurities on CaptureSelect LambdaXP. These results provide scalable and transferable methods for product-related impurity control for various biotherapeutic modalities by lambda light chain affinity chromatography.