Scalable Depth Filtration to Overcome Obstacles in AAV Manufacturing for Gene Therapy

As advanced medicines like gene therapy and gene modified cell therapy continue to gain interest, practical and scalable manufacturing solutions for AAV viral vectors are needed.

We began our three-part blog mini-series reviewing alternative adeno-associated virus (AAV) depth filtration solutions for gene therapies. Our first blog, which can be found here, provided an overview of the expanding gene therapy market, explained why clarification is a pain point in AAV manufacturing and laid out the benefits of using a combination of depth and membrane filters. In our second blog, which you can read here, we outlined an approach to AAV clarification process development using depth and membrane filters with two case studies involving the clarification of crude harvests from suspension and adherent AAV processes. In this third blog, we close our series with a look at how scalable AAV clarification solutions can facilitate large-scale AAV manufacturing for gene therapy and gene-modified cell therapies.

Every AAV Process is Different

AAV viral vector manufacturing involves complex upstream and downstream processes. AAV has multiple serotypes and each behaves differently during cell culture, clarification, and chromatographic separation. Most processes use transient transfection, but some rely on infection-replicative or producer cell processes. Both suspension and adherent cell culture are employed. Cell lysis is generally required, but not always. All these factors result in unique crude harvest compositions for each individual viral vector process.

Improving Clarification Processes with Scalable Transfection Processes

Transfection at large scale presents numerous challenges that can be managed with appropriate, robust scalable technologies. First, selection of an optimal transfection reagent that is suitable for large-scale AAV production is essential. For instance, new transfection reagents designed specifically for AAV vector production in suspension-based systems may provide two to three times higher yields than commonly used generic polyethylene imine (PEI) reagents.

Second, plasmid DNA (pDNA)/PEI complexes must be generated under controlled conditions to ensure they form at the size required for optimal transfection efficiency. Low-shear pumps must then be used to carefully transfer the large volumes of solutions containing these shear-sensitive complexes into the bioreactor without damaging them.

Equally important is the use of a scalable bioreactor platform that facilitates transfer of upstream AAV manufacturing processes from the lab to the manufacturing plant. The iCELLis^® fixed-bed technology is a prime example. The bioreactor platform allows easy transfer of adherent processes from flatware to the bioreactor environment in the iCELLis Nano bioreactor and then rapid scale up to the iCELLis 500+ bioreactor for large-scale manufacturing. This system has found wide acceptance in the industry and is used to produce approved products such as Zolgensma^◆, an AAV-based viral vector gene therapy used in the treatment of spinal muscular atrophy.

The ability to implement effective, robust transfection processes at large-scale means that crude harvests from these processes will be more consistent and contain higher yields of the desired viral vectors and fewer unwanted impurities, particularly partial and empty capsids.

Achieving High Throughput Clarification for Streamlined Processes

As discussed in the first part of this mini-blog series (which can be found here), effective clarification is essential for achieving efficient downstream AAV processes due to the high level of impurities present in the crude harvest following cell culture, transfection (or coinfection), and cell lysis.

In addition to providing sufficient cleanup of crude AAV harvests, clarification must also proceed with high throughput, high product yield, and ease of scale-up.

Pall’s well established and scalable Stax™ depth filter technologies can support an affordable and robust process resulting in high throughput and yields for new process development or scale-up of existing processes.

Several technical studies with AAV (and lentiviral) vectors have been completed with lessons learned applied to accelerating development of viral vector clarification processes. (Sidenote: for more detailed information, see “Optimizing the clarification of industrial scale viral vector culture for our gene therapy ,” a peer-reviewed technical paper published by Cell and Gene Therapy Insights.)

The key to success is developing and optimizing clarification processes early on, starting with an understanding of the crude harvest characteristics and process constraints and goals. Performing bench-top filterability studies using combinations of depth and membrane filters selected based on the harvest properties and process requirements help to identify the best option, which can then be further optimized to provide a robust, scalable, and affordable process with high throughput and yields. Verification of bench scale results at pilot scale is then achieved before moving to manufacturing scale.

A Note About Membrane Chromatography for Full/Empty Capsid Separation

Membrane technology may not only be beneficial for viral vector clarification, it could also have applications in downstream purification. One of the biggest challenges to large-scale viral vector manufacturing is to ensure that the percentage of empty capsids is minimized, and the empty/full ratio is always controlled. Doing so can be difficult because the empty/full ratio resulting from AAV upstream processes often varies from batch to batch using current production technologies.

While ultra-centrifugation is a great tool at the laboratory-scale for separating empty from full capsids, it is impractical on the manufacturing scale. Most large-scale downstream AAV purification processes rely on an anion exchange (AEX) chromatography polishing step (performed after affinity chromatography) to remove the undesirable empty capsids. However, AEX has limited capability in this regard. Further complicating the issue is the fact that different AAV serotypes (used because they target different areas of the body) behave differ­ently during chromatographic separation, so it is difficult to develop platform purification processes.

Membranes offer a promising alternative to the use of resins or monoliths for empty/full capsid separation. Using small conductivity step changes (rather than linear gradients), it is possible with the Mustang^® Q membrane to achieve distinct elution peaks for DNA-free and DNA-containing capsids.

Overcome Obstacles in AAV Manufacturing with End-to-End Solutions from Pall Corporation

As the gene therapy market grows and larger numbers of candidate products advance to late-stage clinical trials and commercialization, many new AAV and other viral-vector processes must be scaled to larger volumes. And while some of these challenges will be addressed as greater understanding of biology is gained and advances are achieved in upstream manufacturing technologies, there is still a ways to go. For instance, the development of stable, high-producing cell lines will eliminate the need for transfection and potentially provide consistent empty/full capsid ratios.

In the meantime, manufacturers must leverage existing downstream purification technologies to achieve optimized processes. And since overall yields across downstream purification steps from clarification to final filtration average approximately 30% today there is considerable room for improvement.

You can watch our AAV clarification process development videos to find out how to accelerate development for viral vectors. Please click here to watch the video on clarification of AAV suspension cell culture and click here to watch the video on clarification of AAV adherent cell culture.

^◆Zolgensma is a registered trademark of Novartis AG