Countdown to Zero: Overcoming Downstream Processing’s Top Five Challenges for Viral Vectors & Exosomes

Imagine this in your not-too-distant future: you’re one of the thousands of discovery researchers involved in lead candidate ID for gene therapy, CAR-Ts, or regenerative medicine. Your team has finally found “the chosen one” and now it’s time to tech transfer this valuable and complex biomaterial to a group with the processing skillset to convert it into doses.

As you press ahead with IND-enabling studies for your viral or exosome/extracellular vesicle product, you’re also soon to reach out for that helping hand, either in-house or at a dedicated CDMO. On the other hand, be ready to count the five ways your bioprocess pencil sketch might run into unpredictable detours. While it becomes optimized into a robust and reliable SOP, these issues are filter fouling, tangential flow filtration membrane gel polarization, uneven availability of affinity systems, scalability issues with size exclusion chromatography, and finally, uncertain effects of exposure to high salt gradients during anion exchange chromatography.  Thankfully, each of these problems is solvable with both existing and emerging process innovations. We’ll briefly discuss these here.

Downstream processing (DSP) begins when the material of interest is ready for harvest after secretion from the cell culture (i.e., retro-/lentivirus and exosomes) or lysis of the cellular material (i.e., adenovirus or AAV). The objective? To efficiently isolate this particulate material from the soupy conditioned medium or lysate via clarification, concentration, purification, polishing, and dose filling (see Figure 1). Whether the upstream process (USP) involves MSCs to obtain exosomes or HEK293s for the bioproduction of AAV, adenoviral, HSV, lentiviral, or γ-retroviral vectors, one hopes for a pothole-free ride through routine DSP. The similarities between extracellular vesicles (EVs; sometimes called exosomes) and lentivectors are almost uncanny, 1, 2, 3 and so there is considerable cross-pollination between their harvest and isolation methods. 4, 5, 6

Downstream Processing Diagram

Figure 1 (above), plug-and-play downstream processing steps can be tailored toward the unique needs of each clinical grade viral vector or extracellular vesicle product. Dashed lines represent alternate paths between process activities.

Processing of viral vectors involves additional diligence beyond extracellular vesicle preps due to the absolute need—where applicable—to remove and rigorously monitor the doses for replication-competent viruses, helper viruses, or exosomes. Compared with lentivirus particles, extracellular vesicles may be found in higher concentrations in conditioned media, some of which may contain viable virus themselves. 3, 7 In addition, viruses require complex “upstream” cell and molecular toolkits such as shuttle vectors, packaging cell lines, and recombination and/or transfection protocols. For our purposes here, we’ll overview the challenges common to both extracellular vesicles and viruses.

Although extracellular vesicle preparation and scale up borrows from decades of accumulated wisdom via the gene therapy community, ongoing efforts to standardize, scale-up, characterize, and quality control the final products 8, 9, 10 are far from uniform in common practice. For viruses and extracellular vesicles, transition from benchtop to clinical grade at industrial scale often involves replacement of low-volume ultracentrifuges with instruments that perform tasks such as tangential flow filtration (TFF), chromatography (e.g., affinity, size exclusion, ion exchange), and larger dead-end filtration systems. Scale up enables massive reductions in cost-per-dose, as well as increases in quality and fewer deviations from the target product profile. But this demands investment in development time that is precisely tailored to each product. Also, any major change in process will necessitate efforts to show that the GMP product will yield comparable or better activity.

5. What the Foul!? Filter Fouling by Non-EV/Virus Impurities

A frequent initial step in extracellular vesicle or virus purification involves one or more filtration steps (≥0.45mm to 100 mm) of the conditioned media or lysate to clarify it free from debris, aggregates, and stray cells. As the waste material accumulates on the inlet side of the filter, it begins to cake and block the flow, generating an overpressure in a phenomenon known as fouling. Fouling reduces the process efficiency due to manual filter changes and increases overall hands-on time needed for the step. With aseptic filtrations, there is also loss of product yield due to disposing the hold-up volume along with the old filter. A pre-clarification, low-G centrifugation step is sometimes performed with smaller volumes of conditioned media to mitigate this issue. However, centrifugation does not always scale well for higher media volumes from multi-liter bioreactors. The typical development activity involves empirically testing which filter (retention rate, brand, material) is a best fit for the job. As always, navigate a tradeoff:  larger filters to handle more loading (i.e., lower volume per surface area) are more expensive upfront, but smaller units under higher loading are more likely to foul, necessitating the hassle of in-process filter swaps. In the final analysis, however, a correctly optimized filtration will opt for no filter changes. This is a superior use of time as well as cost, because two smaller filters are usually more expensive than a single filter with double the surface area in terms of both direct consumable price as well as the yield loss reflected in cost per dose.

It’s plausible that an enzymatic (e.g., nuclease) additive to the cell culture medium could minimize some of the filmy, mucoid residues that can clog filters. Today, benzonase is the exemplar, and it’s perhaps not even toxic to live cells in culture. 11 In adenovirus or AAV preps following cell lysis, benzonase treatment is routine as a preliminary step. With lentivirus preps, this nuclease is sometimes introduced immediately after clarification and prior to chromatography or diafiltration. 12 The drawback is that benzonase is historically among the costliest material expenses for virus prep; some accordingly opt to add it later in the process, after concentration, when flow volumes are lower. Later addition of benzonase demands its removal as a drug residual, however. Therefore, a quest for additional low-cost, high-activity, Agency-friendly enzymes to break down miscellaneous varieties of process-interrupting “crud” remains of keen interest to downstream processing experts.

4. When the Gelling Gets TFF, the TFF Gets Gelling

Tangential flow filtration (TFF; also called cross flow filtration) is a cornerstone in bioprocessing for clarifying, concentrating, and washing biomolecules all at once, and has been used for viral vector downstream processing since the 1990s 13 and extracellular vesicles since the late ’00s. 5, 14, 15 This process may increase the bioparticle of interest’s concentration by orders of magnitude. Modular TFF componentry can be flexibly brought into different stages of the downstream processing for different purposes—for example, early-on for clarification and/or later for diafiltration and buffer exchange. With operator leverage over permeate flux rates, cycle segment timing, or directionality in response to real-time pressure monitoring of feed, retentate, and permeate, this massively scalable industrial process (also used by the dairy industry) enables unique possibilities for optimization beyond less sophisticated separation methods.

Since TFF directs a parallel feed stream across the membrane surface, the continuous sweeping fluid action might intuitively seem to break up gel-forming debris. TFF is thus modeled to exhibit different kinetics vs. dead-end or normal flow filters (NFFs). 16 Despite this, membrane polarization is par for the course, especially with cells cultured in protein-rich media and/or cell types that are professional secretors of matrix proteins (e.g., MSCs). In fact, the declining “diminishing returns” slope displayed on curves of TMF (transmembrane flux) vs. product concentration is a “fingerprint” of any unique input feed material. The key is to zero in on the right balance between adequate transmembrane TMP for a good flux, but low enough to slow the fouling.

A fouled (i.e., polarized) TFF membrane appears as a slimy gel-like coating, and it involves the gradual trapping of aggregates that lower the effective molecular weight cutoff, reducing final purity. Degraded TFF membrane function could necessitate extra downstream processing steps that would also diminish yield along the quest for desirable purity or selectivity. To diminish this hazard, some have envisaged adoption of alternating tangential flow (ATF) from perfusion cell culture into TFF DSP systems. This is where the flow might be periodically reversed in pulses, effectively backflushing the membranes and dislodging aggregates back into the retentate stream, prolonging useful process time. 5, 17, 18 Stating the obvious, however, any TFF bioprocess optimization for virus or EV-sized particles must necessarily screen for the membrane with the largest pore size to allow longest unclogged filter life. Yet pore size must also be small enough to retain the particles of interest.

Just as with non-TFF filter systems, one cannot help but ponder what the accumulating “gel” is made of via a specific conditioned media context? Nucleic acids? Glycoprotein aggregates? Carbohydrates? A gemish of cellular wastes? Can this sludge’s pre-adsorption mass be selectively digested, monomerized, or solubilized, saving time and headaches during empirical test drives of higher-performance filters? In many bioprocess scenarios, a cocktail of enzyme additives might need to be determined by trial and error. We’ve learned that empirically working through parallel challenges could reap a powerful benefit on downstream processing efficiency and yield.

3. Are Your Particles Better Off in a Bind?

The contribution of Protein A (PrA) to purification of monoclonal antibody (mAb) DSP has been profound. Affinity chromatography for these biologics drugs, diagnostics, and MACS reagents facilitated final products of high purity with a measure of built-in selectivity for correctly folded antibodies. Although full-scale affinity chromatography (AC) resins and columns could also potentially streamline downstream processing and achieve greater efficiency for viral vectors and extracellular vesicles, the supply chains for these beyond benchtop grade are less solid. Moreover, the perennial challenges with any kind of elution conditions remain: how to effectively deplete the residual elution buffer, ligand, or enzyme from the final vialed product.

Of the relevant AC platforms, AAV is perhaps best served by the market, where AVB Sepharose can widely plug and play into industrial-scale AAV preparations. 19 However, AVB doesn’t capture all serotypes equally well, such as the versatile AAV-9 serotype. For lower affinity serotypes or heavily engineered synthetic capsids, it might be worthwhile to splice in a high-affinity epitope that is recognized by the industry standard’s AVB camelid nanobody as demonstrated by 20 Dr. Jim Wilson’s U. Penn group. Alternatively, the AVITAG technology system could site-specifically label viral capsids or extracellular vesicles with a biotin within a ~14-amino acid synthetic linker insertion, rendering biological nanoparticles accessible to the huge catalog of off-the-shelf, high-affinity streptavidin toolkits. 21, 22 For lentivectors, smaller scale affinity reagent systems that target VSV-pseudotyped particles are becoming more common. In addition, a generic AC resin based on heparin is a possible means to capture multiple classes of virus or extracellular vesicles. While heparin is widely available cheaply and at massive scale, one potential drawback is that it might not be selective enough to achieve elution fractions of desired purity; also, its largest source is from pigs, which may require extra precaution from bioburden concerns related to PERVs.

Synthetic biology principles might be well-suited to affinity purification solutions. If precise affinity interactions are suboptimal for industrial column matrices and resins, why not bio-pan for the best capsid or extracellular vesicle surface ligand to fit these? (Find the best set of “keys” to pick the most scalable and accessible affinity system “lock?”) Of course, such artificial display handles would need to be biocompatible and minimally immunogenic in humans. One possible source of such synthetic affinity epitopes might be short mimotopes with exclusive specificity for anti-cancer mAbs devised decades ago, such as rituximab or cetuximab, of which hundreds of kilograms have already been manufactured and administered. Moreover, herds of transgenic goats that secrete mAbs into their milk could theoretically produce orders of magnitude more “bioprocess grade” material than the IV injectable doses. 23 Mimotopes are currently being investigated in human trials as “safety switch” components for maximum in vivo control of CAR-T therapies. 24 It requires little imagination to see how they could be applied more broadly.

2. AEX Tools — More than a Small Pinch of Salt to Get By

Whereas SEC separates soluble bioparticles from impurities by size/volume, anion exchange (AEX) separates by surface charge. A positively charged resin and/or matrix selectively immobilizes negatively charged extracellular vesicles 25 or an assortment of VLPs 26 and viral particles, 27 and these are traditionally eluted along a gradient from low-salt to high salt, or alternatively, high-pH to low-pH (or both). One key advantage of AEX is its ability to very rapidly process small scale (e.g., research) to massive volume inputs (e.g., for clinical doses) and its compatibility with GMP-friendly instruments and processes. 28 Ion exchange is standard procedure, for example, as a capture step in Oxford Biomedica/Henogen’s downstream processing of a lentivirus trial product. 29, 30

The major disadvantage of AEX is that viral particles are particularly labile to high salt conditions for extended durations. 31, 32 Process parameters like high pressure or high salt may similarly reap unpredictable effects on the integrity and/or composition of extracellular vesicle subpopulations. 5, 33 Furthermore, negatively charged contaminants such as nucleic acids or proteins can flow along with extracellular vesicle or virus preparations, obstructing the binding capacity of the AEX matrix, necessitating their removal by an enzyme such as benzonase. Any elution buffer containing high salt or enzyme residuals would necessarily need to be quickly buffer exchanged through diafiltration.

1. Wait a SEC… Can Your Process Reach Scale?

Size exclusion chromatography (SEC) has been in practical use since the ‘50s for separation of complex, soluble polymers (such as proteins or vaccines) from other smaller molecular radius impurities in downstream processing. Today, it’s a common step in virus dose preparation, with increasing adoption by extracellular vesicle investigators. 34, 35 Regarding extracellular vesicles, SEC is shown to favorably enrich for EV-sized particles with putative EV-associated protein ID annotations. 35, 36 The method is broadly used for separation after volume reduction measures such as TFF. Importantly, SEC would be an effective unit operation after AC as well as AEX to remove elution residuals and re-equilibrate product into a favorable final buffer.

One key tradeoff to manage is an increase in post-eluate volume and its requirement for large volume of buffer. Furthermore, current inventories of off-the-shelf, commercially available columns are generally used only for very small-scale analytical purposes; development and commercial scale requires custom-packed columns. Scaling up a preparative SEC process is cumbersome in terms of chromatography hardware, equipment footprint, and elution volumes, all of which can result in a more costly process. By some conservative estimates, compared to AC or AEX, 50-fold more SEC resin volume is required for a given input column load. Thus, SEC requires hardware and equipment with much greater volumetric capacities. For example, a 50-liter bioreactor run that leverages initial TFF to concentrate the sample 10-fold could necessitate ~250L (50 x 5L) of buffer and >12 20L SEC columns. While the industry does support this scale at present, a future tidal wave 37 of newly marketed extracellular vesicle drugs may find the entry point for SEC to be challenging in their initial scale-up efforts.


In this blog, we’ve reviewed some of the temporary pitfalls that most downstream processing optimization may encounter in various virus or extracellular vesicle preparations. Although a process with “zero” obstacles is sheer fantasy, the ultimate result of a competent development team’s efforts will yield a process that’s reproducible and reliable, and a product whose quality attributes are within the specs of the target product profile. Through savvy process development optimization, the tricky dilemma known to any Chemistry-101 lab student (purity vs. yield) can be adequately resolved. As a gardener would tell you, the quality of a fruit harvest is improved by careful and attentive pruning.

We focused herein on “generic” issues that are common to both extracellular vesicles and viral vectors and didn’t dwell on additional items of interest to virus purification, such as the extensive prerequisite knowledge needed to contend with their biology, the separation of empty from full genome capsids, and the removal of adventitious viruses that could sneak into final doses as bioburdens. Exosomes/extracellular vesicles may not share these same issues but are faced with questions related to their relative novelty and recent arrival to the armamentarium of experimental advanced therapies.

It’s evident that process innovations need to continue to drive efficiency and cost reduction in the DSP space, for they have scarcely begun to realize their full potential. For our part, as we develop useful and novel additives and techniques for introduction into workable bioprocess flow schemes, we’ll be eagerly ready to share with you all we’ve learned.


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