Participants at a recent downstream processing meeting sponsored by Sartorius Stedim Biotech considered a host of new technologies to speed and streamline protein purification, including membrane chromatography, nanofiltration, ultraviolet irradiation for viral inactivation, and ultracentrifugation concentration technology.
“Downstream has an ongoing processing bottleneck, which continues to affect biomanufacturers, especially those retrofitting existing facilities to accommodate modern fermentation processes,” explained Uwe Gottschalk, Ph.D., vp for purification technologies at Sartorius Stedim Biotech.
There is a major push, at present, to deal with increasing titers during purification. As the antibody titer increases over 1 gram per liter the cost benefit levels out, so going over 5 grams per liter yields little economic benefit in per gram outlay. As the yield in grams per liter increases, the same equipment is employed, whereas buffers and other material requirements expand along with antibody yield. This means that processing equipment must be able to handle the larger amounts, otherwise a significant bottleneck will develop.
Sartorius-Stedim Biotech offers a range of modular purification tools that can be used together so that all the process steps, including clarification, crossflow, membrane chromatography, and viral clearance, can proceed efficiently.
Column chromatography has become the central enabling technology of bioprocessing, according to Dr. Gottschalk, who cited the many improvements that have been made in chromatography media over the years, ranging from diffusive to perfusive particles to monoliths and stacked membranes. He specifically discussed the use of UV inactivation, depth filtration, nanofiltration, and membrane chromatography for the final steps of antibody purification.
In looking toward the future, Dr. Gottschalk believes that crystallization and precipitation will eventually supersede chromatography for the penultimate stages of purification. The crystallization-precipitation approach would represent a major financial advantage, since it would allow the elimination of protein A or other expensive affinity reagents. “By optimizing the various steps in the purification process we should realize an overall 10-fold productivity increase and a significant reduction in costs,” he stated.
Alahari Arunakumari, Ph.D., senior director for process development at Medarex, also spoke on the need to move beyond affinity ligands in protein (especially antibody) purification. Her team is working on the optimization of fully human monoclonal antibody purification processes. She cited protein production levels as high as 13.8 g/L in CHO cell culture, establishing the need for maximum efficiency at the downstream processing end.
Improvements in the company’s processing protocols include precipitation of contaminants or impurities followed by ion-exchange chromatography and a change from resin to Q membrane chromatography, resulting in significant increases in loading capacity—up to a 20 g/mL increase in load protein concentration for viral clearance.
“Further improvements in our system are being put in place,” Dr. Arunakumari continued. “These include increasing the binding capacity on the ion-exchange column so as to minimize the required number of cycles and increasing the load conductivity on Q membrane chromatography so that no dilution is needed. Moreover we have reduced the dilution factor to accommodate the tank sizes and raised the formulation concentrations.”