Brian Hubbard

Use and Optimization of a Dual-Flowrate Loading Strategy To Maximize Throughput in Protein-A Affinity Chromatography

The effect of an alternate strategy employing two different flowrates during loading was explored as a means of increasing system productivity in Protein‐A chromatography. The effect of such a loading strategy was evaluated using a chromatographic model that was able to accurately predict experimental breakthrough curves for this Protein‐A system. A gradient‐based optimization routine is carried out to establish the optimal loading conditions (initial and final flowrates and switching time). The two‐step loading strategy (using a higher flowrate during the initial stages followed by a lower flowrate) was evaluated for an Fc‐fusion protein and was found to result in significant improvements in process throughput. In an extension of this optimization routine, dynamic loading capacity and productivity were simultaneously optimized using a weighted objective function, and this result was compared to that obtained with the single flowrate. Again, the dual‐flowrate strategy was found to be superior.

pH Transitions in Ion-Exchange Systems: Role in the Development of a Cation-Exchange Process for a Recombinant Protein

Unexpected transient changes in effluent pH can occur during ion‐exchange chromatography. Such changes can occur even if a column that is equilibrated with a buffer receives another solution in the same buffer and of the same pH but of a different salt concentration. An attempt is made to understand the basis for this phenomenon and apply it to the process purification of a recombinant protein on a strong cation‐exchange resin. Incomplete column equilibration was eliminated as a possible cause of these effects. Various buffering species and various salt ions were studied at different solution concentrations to investigate pH transitions on strong cation‐exchange resins. A further comparison was made between cation‐exchange resins with different backbone chemistries. On the basis of these studies, a mechanism is proposed for these phenomena based on competitive equilibria between ions from the buffer salts and H+/OH‐ ions. In addition to the equilibria between these ions and the functional groups on the resins, charged groups on the resin backbone were also found to contribute to transient pH changes. The results from this study were applied to the cation‐exchange step for a recombinant protein that was sensitive to pH excursions to help maintain activity of the protein during the purification process.

Viral Clearance by Traditional Operations with Significant Knowledge Gaps (Session II): Protein A Chromatography.

Protein A chromatography is commonly used for the affinity capture of antibodies directly from harvested cell culture fluid (HCCF). The highly specific interaction between the protein A ligand and the Fc portion of antibodies and Fc fusion molecules enables significant purification factors to be

Meeting report-workshop on spike characterizations and virus removal by filtration: trends and new developments.

This workshop was held on June 3, 2013, in Berlin, Germany, in conjunction with the PDA Virus & TSE Safety Forum 2013. A total of nine speakers presented on key considerations of virus filtration, including a historical overview and emerging trends in evaluating parvovirus filters. Several talks addressed understanding the mechanism of virus capture and breakthrough by filters, as well as addressing this risk by carefully controlling transmembrane pressure. Improvements to validation studies were proposed via the use of highly purified virus preparations, more relevant models such as Chinese Hamster Ovary retrovirus-like particles, as well as new assays for virus quantification. The workshop ended with a panel discussion covering a range of topics including virus breakthrough, up-stream media treatment, virus spike preparation quality control, and regulatory expectations.