Clarisolve 20ms

For harvest volumes less than 5 L, the harvest material was clarified of whole cells and cell debris by centrifugation at 3000 rpm for 30 min, followed by 0.8/0.2 µm sterile filtration (Sartorius Stedim, Germany). Alternatively, for larger volumes, the harvest was subjected to a depth filtration train consisting of Clarisolve 20MS followed by Millistak + F0HC filters (MilliporeSigma, Burlington, MA) with a subsequent 0.8/0.2 µm sterile filter. The depth filters were arranged in series and equilibrated with 1X PBS. The cell culture harvest was pumped through the filters at a 60 LMH feed flux based on the F0HC filter area and chased with 1X PBS. Clarified harvest was stored at 2–8 °C for further development activities.

For harvest volumes less than 5 L, the harvest material was clarified of whole cells and cell debris by centrifugation at 3000 rpm for 30 minutes, followed by 0.8/0.2 μm sterile filtration (Sartorius Stedim, Germany). Alternatively, for larger volumes, the harvest was subjected to a depth filtration train consisting of Clarisolve 20MS followed by Millistak+ F0HC filters (MilliporeSigma, Burlington, MA) with a subsequent 0.8/0.2 μm sterile filter. The depth filters were arranged in series and equilibrated with 1X PBS. The cell culture harvest was pumped through the filters at a 60 LMH feed flux based on the F0HC filter area and chased with 1X PBS. Clarified harvest was stored at 2–8°C for further development activities.

Cell culture harvest was clarified by a two-stage depth filtration train of Clarisolve 20MS and F0HC (MilliporeSigma, Burlington, MA) followed by sterile filtration using a Sartopore 0.8/0.2 μm filter (Sartorius Stedim, Goettingen, Germany), operated in tandem. All chromatography was performed on an AKTA AVANT 25 (GE Healthcare). Clarified harvest was loaded onto a Protein A column (TOYOPEARL AF-rProtein A HC-650F, Tosoh Bioscience, King of Prussia, PA) and eluted with 100 mM glycine–HCl, pH 3.5. The material was then conditioned to 185 mM sodium chloride using 5 M NaCl stock solution (Lonza, Walkersville, MD) and titrated to pH 9.0 using 2 M glycine-OH, pH 10.5. The conditioned material was then purified via TOYOPEARL NH2-750F anion-exchange chromatography (Tosoh Biosciences), operated in flow-through mode using 50 mM Tris–HCl pH 8.0, 200 mM NaCl as the column equilibration and load chase buffer.