This assay principle is demonstrated using M13 bacteriophage virus and human chorionic gonadotropin as model analytes. In the presence of large or artificially-expanded analytes, reporter reagents bind to analyte species to form complexes large enough to be excluded from the adsorbent core, allowing their signal to be observed. In the absence of analyte, antibody-enzyme reporter conjugates can enter the adsorbent and be captured, and their signal is lost. Capto™ Core 700 and related resins possess a noninteracting size-selective outer layer surrounding a high-capacity nonspecific mixed-mode capture adsorbent core. We introduce analyte-dependent exclusion of reporter reagents from restricted-access adsorbents as the basis of an isocratic reporter-exclusion immunoassay for viruses, proteins, and other analytes. Here, we introduce and discuss volume reducing and purification methods frequently used for viruses and analyze their value for the manufacturing of EV-based therapeutics. As EVs share several features with enveloped viruses, that are used for more than two decades in the clinics now, several principles can be adopted to EV manufacturing. Consequently, it is a major challenge in the therapeutic EV field to identify appropriate EV concentration and purification methods allowing scale up. Most of the EV preparation methods used in conventional research labs including differential centrifugation procedures are limited in their scalability. The key operations of each EV DSP is volume-reduction combined with purification of the concentrated EVs. 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.

Capto core 400