Catalent Indiana LLC

As the global market for biosimilars expands, scientists are required to rapidly develop and optimize robust manufacturing processes quickly under constrained timelines.A typical three-column mAb purification platform features Protein A capture followed by any two of the following polishing chromatog. steps: Cation Exchange (CEX), Anion Exchange (AEX), or Hydrophobic Interaction Chromatog. (HIC).With several next generation purification media and ion exchange membranes, there are several opportunities to reduce the number of chromatog. steps and create process intensification through high-throughput platform purification schemes.In this work, multiple two-column platforms were developed that combined the use of a newer high capacity Protein A resin, CEX chromatog. or a depth filtration step, and either AEX traditional chromatog. or membrane chromatog. as a polishing step.Each platform described in this work is effective at generating the model mol. trastuzumab at < 0.5 % aggregate and < 20 ng/mg Host Cell Protein (HCP) while maintaining an overall process yield of ≥ 75%.The advancements described in each platform decrease the time required for development and tech. transfer to manufacturing scale.

The 3M Emphaze AEX Hybrid Purifier is a novel purification tool that utilizes a Q-functional hydrogel and 0.2 μm membrane to reduce impurities in a biopharmaceutical downstream process.This technol. has been reported to provide a 4-log reduction in DNA at the clarification stage, which can greatly improve the efficiency of the Protein A capture step.While the advantages provided by the Emphaze AEX Hybrid Purifier are far-reaching, its Q-functionality presents a challenge for target proteins with a low isoelec. point (pI).Proteins under these conditions will usually bind to anion exchange ligands which can neg. impact the functionality of a clarification stage filter.This study involved a Design of Experiment (DoE) based approach to investigate the effects of pH and conductivity on the turbidity, yield, and impurity clearance of the Emphaze AEX Hybrid Purifier and the efficiency of the subsequent Protein A capture step.The results were then used to determine optimum filtration conditions of one such low pI fusion protein, and whether elements of this technique can be extended to purification of a monoclonal antibody (mAb) with a higher pI.Filtration conditions for the low pI target protein were identified with a 4-fold greater yield over normal harvest filtration conditions and more than 99.6% removal of HCP post Protein A chromatog.Application of these strategic elements to mAb filtration demonstrated an extended pH range at which post Protein A HCP reduction exceeds 99.9%.For each mol. studied, filtration using the Emphaze AEX Hybrid Purifier resulted in greater HCP reduction, higher product purity, and lower turbidity than unfiltered pools.This exptl. plan provides a baseline to tailor specific application of the Emphaze AEX Hybrid Purifier to each individual protein or antibody.

Protein A affinity capture of IgG from CHO culture is an effective purification step with relatively high purity and yield.However, substantial amounts of host cell proteins still bind non-specifically to the resin and co-elute with the antibody.This presentation describes conditions designed to further reduce host cell proteins and streamline monoclonal antibody purification processes.Of the parameters tested, a secondary wash was found to reduce host cell proteins greater than 20% in the Protein A low pH eluate.A membrane filtration step was demonstrated to reduce host cell proteins by 10-fold.A Design of Experiments approach was used to screen a mixed mode resin as a 2^nd chromatog. step and optimal conditions were developed.As a result of decreased host cell proteins, a typical three-column purification process for monoclonal antibody products can now be reduced to a two-column process, thus significantly simplify manufacturing processes with potentially lower cost.