Better Downstream Processing
Improved yields of proteins at the upstream processing end have driven a demand for better performance at the downstream component. Uwe Gottschalk, Ph.D., vp of purification technologies at Sartorius (www.sartorius.com), has investigated membrane chromatography as an alternative to conventional packed column chromatography for protein purification.
Most antibody purification protocols involve the use of protein A columns as the primary capture mechanism, which typically can generate a 98% pure product. The subsequent treatment of the antibody calls for polishing and removal of DNA, endotoxins, proteases, and viral contaminants. But, conventional packed-bed chromatography requires columns with large diameters to permit high-volumetric flow rates and avoid a process bottleneck.
Because packed columns are configured for speed and not binding capacity, they will not be utilized to their maximum capability. In recent years, Q membrane chromatography has been re-evaluated for process scale due to the increased demands of the industry. Stacked 15-layer Q membranes have been successfully used for large-scale antibody production and viral removal.
For characterization studies, Dr. Gottschalk and his group evaluated different scale-down models, which were redesigned and carefully examined using different conditions. Scale-down modeling is essential in the biotech industry for establishing the performance of a process, but often yields a poor representation of the real life situation.
Since the original Sartobind Q 75 membrane generated higher backpressure, two new scale down devices, Sartobind Q 125 (3.5 mL) and eventually Sartobind Q 40 (1 mL) were designed in collaboration by Sartorius and Joe Zhou, Ph.D., of Amgen (www.amgen.com). The device mimics the liquid flow path found in the larger-scale module and represents an ideal scale-down tool for validation studies.
Using this approach, a Mab-processing capacity of 3 kg/M2 (11 kg/L) was achieved, which represents one membrane absorber for the polishing of each 10,000 L of bioreactor capacity. Because of their high cost and disposable nature, the Sartobind Q membranes do not economically outperform the Q column until all the categories, including hardware investment, labor, and utility costs, are factored into the picture.
According to Dr. Gottschalk, the disposable Q membrane chromatography devices are robust and simple to use, as they require no column packing or cleaning validation. Moreover, he says they appear to be an optimal technology for contaminant removal, and the amount of buffer required is less than 5% of that required for packed-bed chromatography. These studies validate Q membrane chromatography as a viable alternative to Q column chromatography for the polishing involved in late-stage antibody production.
“We like to think of ourselves as artists,” says Dr. Gottschalk. “Michelangelo claimed that he liberated a beautiful statue from its surrounding marble, and we think of bioprocessing in the same way, so we use purification technology to free an antibody from its contaminating viruses and detritus.”