Downstream Bioprocessing of Viral Vectors

Gene therapies represent a new medical paradigm for treating patients and addressing unmet or underserved medical needs. However, with this opportunity comes clinical and manufacturing challenges that the industry must address. Our recent Ebook, “Insights on Successful Gene Therapy Manufacturing and Commercialization,” identifies challenges and opportunities to improve current operations. By working with a talented group of authors, all experts in the field of gene therapy, we developed a guide to gene therapy manufacturing. As expected, downstream bioprocessing was a large focus of the Ebook and provided an opportunity to take an in-depth look at each process step.

Viral Vectors
When looking at downstream gene therapy manufacturing, it is important to evaluate the role that viral vector selection plays in the downstream bioprocess. Viral vectors vary greatly and manufacturing is largely dependent on the type of vector being used. There are important similarities and differences between the commonly used gene therapy viral vectors, which include adenovirus (AdV), adeno-associated virus (AAV) and retroviruses (which are primarily gammaretrovirus) (RV), and lentivirus (a type of retrovirus) (LV). The differences extend beyond physical characteristics to differences in vector serotypes, which can significantly influence process conditions. The choice of upstream process and platform also impacts the downstream process, particularly purification. As a result each downstream process should be optimized with the vector type in mind as each vector type presents unique challenges and opportunities.
Despite these differences in the vectors, current downstream protocols generally have a similar workflow: a clarification step (which may require additional depth filtration due to cell lysis at time of harvest), purification (typically ion exchange or affinity chromatography), and a polishing step (additional chromatography step/size exclusion). In addition to the above, filtration, dialysis, or TFF steps are typically utilized in order to concentrate, exchange buffer, and prepare final formulation.
In the Ebook, each step in this process was carefully examined with challenges and areas for improvement described. In addition, the process as a whole was evaluated and key takeaways included:

Downstream process challenges
• Yield – Yield is an important concern in downstream vector manufacturing as loss of viral yield puts increased pressure on the upstream process and is costly and time consuming. Viral instability and loss of function is one area where yield loss can occur, particularly with LV and other enveloped viruses. Thus reducing the number of steps, and time in process, and reducing shear forces can improve overall yield for unstable vectors.
• Purity – Obtaining sufficient purity without loss of function is also a key challenge in downstream bioprocessing. Overall process design and efficiency is key to controlling impurities and effort must be taken to avoid introduction of contaminants in the downstream process, unless necessary as is the case with enzymes or processing agents. Any introduction of contaminants increases process burden, as they require additional analytical and processing steps for their removal.

Areas for advancement
• Affinity chromatography – Affinity chromatography shows great promise as it is effective, and its use could potentially enable a platform-like approach in portions of downstream. The advancement of affinity chromatography media across multiple viruses and serotypes would also be an important advancement for the industry.
• Automation and closed systems – Automation is needed in several areas to reduce both labor and costs. Closed systems serve to protect both the product and the operators. Incorporation of technologies used in recombinant antibody manufacturing, such as single-use technologies and continuous processing would also enable employment of automation and closed systems.
• Move toward more efficient methods and the development of virus-specific tools – Overall the industry needs to continue its movement away from older, tools and technologies that were developed for use with small molecules and antibodies toward more fit-for-purpose methods. Virus-specific tools are needed to advance both small scale and large scale processing.
• Analytics – Development of better or faster at-line or inline analytics for intermediate crude with lower viral concentration will benefit the industry by enabling process efficiency and lower costs. High throughput analytics could reduce process development timelines by providing a more comprehensive look at the process and in identifying areas for improvement.

Moving a downstream process to clinical and commercial manufacturing
In addition to looking at the individual process steps, several key insights were uncovered with respect to moving a downstream process to clinical and commercial manufacturing.
Transferring a vector from a developmental setting in academia, to manufacturing for clinical use, presents several considerations for downstream production development. Often the objectives and consequently choice of tools and technologies are different between academia and clinical production. Areas including consistency of operation and bioburden control are not typically focused on in most research production but are critical in clinical manufacturing. Thus, there are several areas that need further examination and possible alternative solutions including:

• Scalability – One of the major considerations when moving from research production is how to achieve the increased production scale. One approach toward implementation of greater scale is to move away from poorly scalable techniques to systems with greater throughput. For instance, it has been demonstrated that VSG-g LV vectors can be purified using Mustang Q membrane (Pall Corporation) up to a volume of 1,500 L/day and lead to vector preparations of high purity.
• GMP Compliance – Research material is not required to be GMP compliant, thus this is a major change when moving a product to clinical manufacturing. Rational design of the downstream process to simplify and increase efficiency can reduce the number of required GMP reagents and steps, which increases quality, safety and significantly lowers costs.
• Process development timelines – The timeline to develop a downstream process is dependent on many factors such as the type of vector, intended use, scale, cost considerations, and technology available. Independent of the factors involved, a reduced developmental timeline is a desirable goal as it quickens time to market and lowers cost.
• Costs considerations – Costs are a combination of many factors such as, time, labor, operating expenses, and capital expenses. Replacing traditional, academic methods that are time consuming, complex, and inefficient with approaches that are more cost effective, can lower costs with the tradeoff of required redevelopment time.
• Risk Assessment – Another important consideration is an overall evaluation of vector risks (risk assessment) and subsequent design of the upstream and downstream process to minimize that risk with available technology. Thus, an overall risk assessment followed by a mitigation design should be considered in light of the regulatory standards and patient safety that is required for clinical manufacturing.

Summary
While we identified several manufacturing challenges facing gene therapy commercialization, there is also an increasing range of options to process and obtain high titers and high-quality batches of viral vectors. Continued development of new tools and technologies that are fit-for-purpose will continue to advance manufacturing efficiency, product safety and lower costs. As we move forward, we expect to see the same kind of extensive manufacturing advancements that the monoclonal antibody industry experienced and continue to refine today.

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