Continuous integrated antibody precipitation with two‐stage tangential flow microfiltration enables constant mass flow

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.

Continuous precipitation for monoclonal antibody capture using countercurrent washing by microfiltration

“…The product purity was calculated from the SEC chromatograms as the ratio of the monomer peak area to the sum of all peak areas. Note that the ZnCl 2 appeared to have some interaction with the column, resulting in an unexpected peak tailing as reported previously by Burgstaller et al…”

“…A 17.5 wt% PEG solution was added between the two static mixers to achieve final concentrations of about 2.3 g/L for the mAb, 10 mM for the ZnCl 2 , and 7 wt% for the PEG. The total residence time in the tubular precipitation reactor was only 30 s, which is much smaller than the 2.5 to 22.5 min used by Burgstaller et al The more efficient precipitation in this work is likely because of the sequential addition of the ZnCl 2 and PEG (these were added simultaneously by Burgstaller et al); both studies were performed with approximately the same mAb concentration (between 2 and 3 g/L).…”

“…The residence times in each step were estimated assuming a linear variation of the flow rate through the hollow fiber modules (from initial feed flow rate to exit retentate flow rate). This gives a total residence time of 140 s (<2.5 min) for the entire system compared to as much as 20 min (or more) for just the precipitation step in the work by Burgstaller et al…”

“…The objective of this study was to examine some of the key design variables in a fully integrated continuous precipitation process for the purification of a mAb using the ZnCl 2 –PEG system. However, in contrast to Burgstaller et al, the ZnCl 2 and PEG were added sequentially (instead of at the same time) to better control the yield and morphology of the precipitated mAb. The precipitates were then directly and continuously dewatered using a hollow fiber membrane module without any retentate vessel (hold tank) as used by Burgstaller et al In addition, the washing step was performed using a two‐stage countercurrent configuration without any ZnCl 2 or PEG to significantly reduce the buffer requirements/costs while enhancing removal of host cell proteins (HCPs).…”

“…However, in contrast to Burgstaller et al, the ZnCl 2 and PEG were added sequentially (instead of at the same time) to better control the yield and morphology of the precipitated mAb. The precipitates were then directly and continuously dewatered using a hollow fiber membrane module without any retentate vessel (hold tank) as used by Burgstaller et al In addition, the washing step was performed using a two‐stage countercurrent configuration without any ZnCl 2 or PEG to significantly reduce the buffer requirements/costs while enhancing removal of host cell proteins (HCPs). We also employed an inline resolubilization step using a low pH glycine buffer, providing a final product stream that would be suitable for subsequent purification as part of a fully integrated continuous downstream process.…”