Abstract
Continuous precipitation is a new unit operation for the continuous capture of antibodies. The capture step is based on continuous precipitation with PEG6000 and Zn++ in a tubular reactor integrated with a two‐stage continuous tangential flow filtration unit. The precipitate cannot be separated with centrifugation, because a highly compressed sediment results in poor resolubilization. We developed a new two‐stage tangential flow microfiltration method, where part of the concentrated retentate of the first stage was directly fed to the second stage, together with the wash buffer. Thus, the precipitate was concentrated and washed in a continuous process. We obtained 97% antibody purity, a 95% process yield during continuous operation, and a fivefold reduction in pre‐existing high‐molecular‐weight impurities. For other unit operations, surge tanks are often required, due to interruptions in the product mass flow out of the unit operation (e.g., the bind/elute mode in periodic counter‐current chromatography). Our setup required no surge tanks; thus, it provided a truly continuous antibody capture operation with uninterrupted product mass flow. Continuous virus inactivation and other flow‐through unit operations can be readily integrated downstream of the capture step to create truly continuous, integrated, downstream antibody processing without the need for hold tanks.
Highlights
After 30 min of hold time, the precipitate was concentrated on a 50‐ cm2 hollow fiber module fit with a filter membrane (either 0.1 or 0.45 μm pore size; GE Healthcare), until a feed pressure of around 100,000 Pa was achieved
The capture step is based on continuous precipitation with PEG6000 and Zn++ in a tubular reactor integrated with a two‐stage continuous tangential flow filtration unit
We found that the optimal polyethylene glycol (PEG) size for antibody precipitation was PEG6000 (Sommer et al, 2014)
Summary
After 30 min of hold time, the precipitate was concentrated on a 50‐ cm2 hollow fiber module fit with a filter membrane (either 0.1 or 0.45 μm pore size; GE Healthcare), until a feed pressure of around 100,000 Pa was achieved. The cell culture broth was continuously pumped at a flow rate of 6.6 ml min−1 (transfer pump stage one) and combined with a feed stream of 1.4 ml min−1 (permeate pump stage one) of 40% PEG6000 plus 11.43 mM ZnCl2 in 50 mM Tris buffer, pH 7.0.
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