Continuous in-line diafiltration using a novel multi-channel prototype cassette for viral vector buffer exchange

In-line diafiltration (IL-DF) using 30 kDa molecular weight cutoff (MWCO) single pass tangential flow filtration (SP-TFF) cassettes has been evaluated for continuous biologics manufacturing, but there has been limited application of IL-DF in viral vector purification processes. A novel multi-channel, self-contained cassette prototype, where the DF buffer is exchanged continuously across an elongated retentate channel, was developed by Sartorius. Initial prototypes were constructed with 30 kDa polyethersulfone (PES) membranes, but a higher MWCO membrane may enable higher fluxes and improved buffer exchange (BX) while retaining the large viral vector product. This work details the evaluation of single-stage 30 kDa and 300 kDa PES membrane cassette prototypes using an oncolytic strain of Coxsackievirus A21 (CVA21), a non-enveloped, 30 nm picornavirus. IL-DF was implemented as an intermediate BX step using a clarified cell culture feed in a challenging serum-containing matrix, and targeted >90% BX into a low salt chromatography buffer. Flux excursion experiments were performed at a range of feed fluxes with a DF ratio of 1-5 and a volume concentration of 1-3-fold. Loading studies were performed at selected conditions, and mass yields in retentate flowthrough fractions and a post-use flush were analyzed by quantitative capillary western. The 300 kDa IL-DF exhibited higher membrane permeability, allowing for the operation of higher fluxes at lower TMP and achieved >90% BX at a DF ratio of just 2, reducing buffer volume requirements. Compared to the 30 kDa cassette, the 300 kDa cleared 3-5-fold more impurity protein in the retentate. High retentate mass yield was observed for the 30 kDa IL-DF, but unexpectedly, the yield of the 300 kDa IL-DF was <50% likely due to the accumulation of virus at the membrane surface that was recovered in the high flux post-run flush. Using the same CVA21 feed, conventional SP-TFF was evaluated with commercial 30 kDa and 300 kDa PES Sartocon cassettes, and similar lower retentate recovery was observed with the 300 kDa membrane. Scanning electron microscopy analysis of the retentate film layer indicated increased surface roughness of the 300 kDa PES filter that may trap large virus particles with the low crossflow of SP-TFF. These results demonstrated a high-yield BX of a viral vector with the 30 kDa IL-DF prototype, but further development with larger MWCO cassettes is necessary to minimize potential losses to the membrane surface.