Integration of a perfusion reactor and continuous precipitation in an entirely membrane‐based process for antibody capture

Abstract

Continuous precipitation coupled with continuous tangential flow filtration is a cost‐effective alternative for the capture of recombinant antibodies from crude cell culture supernatant. The removal of surge tanks between unit operations, by the adoption of tubular reactors, maintains a continuous harvest and mass flow of product with the advantage of a narrow residence time distribution (RTD). We developed a continuous process implementing two orthogonal precipitation methods, CaCl2 precipitation for removal of host‐cell DNA and polyethylene glycol (PEG) for capturing the recombinant antibody, with no influence on the glycosylation profile. Our lab‐scale prototype consisting of two tubular reactors and two stages of tangential flow microfiltration was continuously operated for up to 8 days in a truly continuous fashion and without any product flow interruption, both as a stand‐alone capture and as an integrated perfusion‐capture. Furthermore, we explored the use of a negatively charged membrane adsorber for flow‐through anion exchange as first polishing step. We obtained a product recovery of approximately 80% and constant product quality, with more than two logarithmic reduction values (LRVs) for both host‐cell proteins and host‐cell DNA by the combination of the precipitation‐based capture and the first polishing step.

Residence time distribution of continuous capture of recombinant antibody by precipitation and two stage tangential flow filtration

“…For the experiment in which precipitate was directly fed to the system (steady-state emulation), the precipitated monoclonal antibody used was generated during an integrated perfusioncapture process. 33 Briefly, the CCCS was subjected to calcium chloride (CaCl 2 ) precipitation for the removal of impurities at a final concentration of 100 mmol L −1 CaCl 2 in 10 mmol L −1 MOPS, pH 7.0 by dilution of CCCS with a 1 M CaCl 2 stock solution in 0.1 mol L −1 MOPS, pH 7.0. The stream was clarified continuously by depth filtration and the monoclonal antibody was captured via PEG 6000 precipitation in a tubular reactor at a final concentration of 18% PEG 6000 in 30 mmol L −1 MOPS, pH 7.0 by dilution with a stock solution of 50% PEG 6000 in 100 mmol L −1 MOPS, pH 7.0.…”

“…The TFF stages remained identical, as the hollow fiber modules used still operate far below the critical flux in the range of 17-34 LMH. 33 For the laboratory-scale capture unit, we could describe the convoluted RTD generated via step input experiment with NaNO 3 with a TIS model, where N = 5, with a correlation coefficient of 0.96 (Fig. 4).…”

“…A pool-less integrated perfusion-capture process over several days of process campaign could be operated. 33 For this, we constructed a capture unit consisting of a skid housing a tubular reactor for continuous precipitation and two TFF stages interconnected in a feedand-bleed mode. The advantage of our system is that surge tanks are no longer required for this process, although the two-stage filtration can have a significant impact on the RTD.…”

Addition of sodium malonate alters the morphology and increases the critical flux during tangential flow filtration of precipitated immunoglobulins

“…Precipitation has been the dominant platform for plasma protein separations since the development of the Cohn fractionation process in the 1940s (Cohn et al, 1946; Foster et al, 1986). A number of recent investigations have demonstrated that precipitation can also be used for the purification of monoclonal antibodies (mAbs) (Dutra et al, 2020; Ferreira‐Faria et al, 2023; Li et al, 2019; Pons Royo et al, 2023; Recanati et al, 2023; Recanati et al, 2024), exploiting the relatively high titers of current Chinese hamster ovary (CHO) cell cultures (Liang et al, 2023; Shukla et al, 2017). The precipitated protein can be effectively dewatered and washed using tangential flow filtration (TFF), potentially providing a low‐cost platform for the continuous downstream processing of mAbs (Li et al, 2019; Minervini et al, 2023).…”