![]() ![]() As first unit operation in DSP, clarification removes remaining cells, debris and other larger impurities. CM are either stored until DSP or are directly processed. During downstream processing (DSP), conditioned media (CM) are processed to obtain concentrated and purified EV products. During upstream processing (USP), cells are isolated, stored (cell banking) and expanded furthermore, EV-containing conditioned media are produced. Like for other biotherapeutic agents, the manufacturing of EV products can be subdivided in the upstream and downstream processing and the subsequent quality control, each of them containing several unit operations. For translating them effectively into the clinics, scalable production processes fulfilling good manufacturing practice (GMP) are needed. ![]() ![]() Similar results were obtained without an initial ultrafiltration step.Įxtracellular vesicles (EVs) especially of mesenchymal stem/stomal cells (MSCs) are increasingly considered as biotherapeutic agents for a variety of different diseases. Purification of the rVSV-S virus with CaptoTM Core 700 resulted in viral infectivity above 85% for this step, with the efficient removal of host cell proteins, consistent with regulatory requirements. rVSV-S cannot enter the inner pores of the resin and is collected in the flow-through eluent. Finally, a highly efficient chromatography purification process with CaptoTM Core 700 resin, which does not require binding and the elution of the virus, is described. The large virus and spike protein binds very strongly to the high surface area of the membrane adsorbents, resulting in poor virus recovery (<15%), while the use of packed-bed chromatography, where the surface area is smaller, achieves better recovery (up to 33%). The use of anion-exchange chromatography in all forms results in strong binding of the virus to the media, necessitating a high salt concentration for elution. ![]() Cell harvest is initially treated with endonuclease, clarified, and further concentrated by ultrafiltration before chromatography purification. Several purification strategies are evaluated using a variety of chromatography methods, including membrane adsorbers and packed-bed ion-exchange chromatography. This study reports a highly efficient, rapid one-step purification process for the production of the recombinant vesicular stomatitis virus-based vaccine, rVSV-∆G-spike (rVSV-S), recently developed by the Israel Institute for Biological Research (IIBR) for the prevention of COVID-19. Percent of cells positive for GFP expression are indicated. (E) Similar to (D) but all samples were diluted further by one-fifth and virus-infected cells were quantified flow cytometry. HEK293 cells were exposed to equivalent dilutions of AdGFP lysates or samples purified by methods 1 or 3 and at 24 hours post infection, visualized by fluorescence microscopy (GFP) and bright field (BF). (D) Coomassie blue staining shows relative adenovirus hexon proteins in purified samples. Densitometric analysis for quantities of hexon proteins is provided. Capto core 400 serial#Serial dilutions of 1 in 5 were provided for lysates and Ad-MCMV-IL-2. SDS-PAGE and Coomassie staining (left) or silver staining (right) shows recovery and purity of samples purified by Capto Core 700 resin compared to Ad-MCMV-IL-2 (contains an IL-2 transgene) CsCl purified adenovirus. (C) HEK293 cells infected with Addl70-3 (no transgene) or AdGFP (GFP transgene) and left unpurified (lysate) or purified by methods 1 or 3 as depicted in (A). Locations of major adenovirus proteins are indicated. (B) Recovery and purity of adenovirus was assessed by SDS-PAGE and Coomassie staining. Mock-and adenovirus infected lysates were frozen and thawed three times, then either left unpurified (lysate) or subjected to 4 alternative purification strategies as summarized in (A). (A,B) 10 cm2 equivalence of HEK293 cells were left uninfected (mock) or infected with adenovirus Addl309 (containing E1 and E3 regions of Ad5) until cytopathic effect exceeded 90%. Purification of adenovirus in high-throughput using Capto Core 700 in-slurry approach. ![]()
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