Extracellular Vesicle Isolation by a Tangential-Flow Filtration-Based Large-Scale Purification Method.

Extracellular vesicle (EV) isolation from conditioned cell culture medium has been a challenging topic. It is particularly difficult to obtain pure and intact EVs at a large scale. The commonly used methods such as differential centrifugation, ultracentrifugation, size exclusion chromatography, polyethylene glycol (PEG) precipitation, filtration, and affinity-based purification each have their advantages and limitations. Here, we present a tangential-flow filtration (TFF) based, multi-step purification protocol that combines filtration, PEG precipitation, and Capto Core 700 multimodal chromatography (MMC) to isolate EVs at high purity from large volumes of cell culture conditioned medium. Inserting the TFF step before PEG precipitation removes proteins, which may aggregate in subsequent steps and co-purify with EVs.

Large-scale heparin-based bind-and-elute chromatography identifies two biologically distinct populations of extracellular vesicles.

Purifying extracellular vesicles (EVs) has been challenging because EVs are heterogeneous in cargo yet share similar sizes and densities. Most surface marker-based affinity separation methods are limited to research or diagnostic scales. We report that heparin chromatography can separate purified EVs into two distinct subpopulations as ascertained by MS/MS: a non-heparin-binding (NHB) fraction that contains classical EV markers such as tetraspanins and a heparin-binding (HB) fraction enriched in fibronectins and histones. Both fractions were similarly fusogenic but induced different transcriptional responses in endothelial cells. While EVs that were purified by conventional, non-affinity methods alone induced ERK1/2 phosphorylation and Ki67, the NHB fraction did not. This result suggests heparin chromatography as an additional novel fractionation step that is inherently scalable, does not lead to loss of material, and separates inflammatory and pyrogenic EVs from unreactive EVs, which will improve clinical applications.

High throughput chromatography and analytics can inform viral clearance capabilities during downstream process development for biologics.

High throughput process development (HTPD) using liquid handling robotics and RoboColumns is an established methodology in downstream process development to screen chromatography resins and optimize process designs to meet target product profiles. However, HTPD is not yet widely available for use in viral clearance capability of the resin due to a variety of constraints. In the present study, a BSL-1-compatible, non-infectious MVM model, MVM-VLP, was tested for viral clearance assessment with various resin and membrane chromatography operations in a HTPD mode. To detect the MVM-VLP in the high throughput experiments, an electrochemiluminescence immunoassay (ECLIA) assay was developed with up to 5 logs of dynamic range. Storage time suitability of MVM-VLP solutions in various buffer matrices, in the presence or absence of a glycoprotein vaccine candidate, were assessed. Then, MVM-VLP and a test article monoclonal antibody (mAb) were used in a HTPD design that included commercially available ion exchange media chemistries, elucidating a wide variety of viral clearance ability at different operating conditions. The methodologies described herein have the potential to be a part of the process design stage in biologics manufacturing process development, which in turn can reduce risk associated with viral clearance validation studies.

Advances in Purification of SARS-CoV-2 Spike Ectodomain Protein Using High-Throughput Screening and Non-Affinity Methods.

The spike (S) glycoprotein of the pandemic virus, SARS-CoV-2, is a critically important target of vaccine design and therapeutic development. A high-yield, scalable, cGMP-compliant downstream process for the stabilized, soluble, native-like S protein ectodomain is necessary to meet the extensive material requirements for ongoing research and development. As of June 2021, S proteins have exclusively been purified using difficult-to-scale, low-yield methodologies such as affinity and size-exclusion chromatography. Herein we present the first known non-affinity purification method for two S constructs, S_dF_2P and HexaPro, expressed in the mammalian cell line, CHO-DG44. A high-throughput resin screen on the Tecan Freedom EVO200 automated bioprocess workstation led to identification of ion exchange resins as viable purification steps. The chromatographic unit operations along with industry-standard methodologies for viral clearances, low pH treatment and 20 nm filtration, were assessed for feasibility. The developed process was applied to purify HexaPro from a CHO-DG44 stable pool harvest and yielded the highest yet reported amount of pure S protein. Our results demonstrate that commercially available chromatography resins are suitable for cGMP manufacturing of SARS-CoV-2 Spike protein constructs. We anticipate our results will provide a blueprint for worldwide biopharmaceutical production laboratories, as well as a starting point for process intensification.