Key considerations for scaling up lentiviral production

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The use of lentiviruses as a tool for cell and gene therapies (C&GT), including CAR-T therapies, is on the rise. Lentiviral vectors (LVs) offer the ability to carry large transgenes that can be stably integrated into the host genome of the target cell. With increasing popularity, lentiviral production projects are growing in scale and often require support from specialist contract development and manufacturing organizations (CDMOs).

Moving from small-scale to large-scale production comes with an array of challenges. As larger volumes may impact downstream methods, problems can arise if the optimum lentivirus production protocol for cell proliferation is not determined. Fill-finish methods also need to be thought about carefully, as vector loss can occur due to container adsorption when using glass vials. To ensure the successful scale-up of lentiviral production projects, drug developers should therefore seek the support of CDMOs with experience and expertise in solving the challenges surrounding their development and manufacturing.

In this article, Katherine Moynihan, PhD, Vice President of Business Development and Marketing at Genezen, discusses the many challenges that can arise during lentiviral production scale-up. She advises that by using the right equipment and working with a knowledgeable team, scaling these projects can be achieved at an accelerated pace while reducing risk.

Recognizing the direction of the project and understanding the potential challenges

Before beginning the immense task of scaling up lentiviral production, the most important decision is project direction. A strong awareness of its scale and magnitude is essential. The volumes needed to be achieved by the end of the project will determine the methods and equipment used throughout. A solid understanding of the technologies available, the dosage requirements, and the potential magnitude of scale up, coupled with a detailed plan of execution are required to avoid backtracking and associated delays.

Once the scale is determined, preparations can be made for the challenges that could arise, such as vector loss or contamination. Many of these can be solved by identifying and using the most suitable equipment for the scale of the lentivirus project.

Choosing the right equipment for scaling cell proliferation

Amplification of lentiviruses requires transient transfection of the plasmids encoding the genes for viral vector production into cells, followed by cell proliferation. These producer cell lines utilized in lentiviral production are often adherent, necessitating a large surface to volume area for growth. Traditionally, 2D systems such as cell stacks have been used to provide an adhesive surface. Although cell stacks are a familiar and conventional approach to grow adherent cell lines, they require extensive manual procedures, leading to contamination risk. They also rely on facilities with a large capacity for their high surface to volume ratio. Consequently, 2D systems are not generally suitable for large scale operations.

An attractive alternative to 2D systems for adherent cell line growth at large-scale are automated, single-use, fixed-bed bioreactors. Compact fixed-beds provide a much larger surface area for cells to adhere to in lower volumes as compared with cell stack systems. Their automation and single-use properties alleviates the need for time-consuming and delicate manual operations and reduces the likelihood of contamination. The net effect is that fixed-bed bioreactors decrease both operational costs and capital investments as compared with traditional 2D methods.

When considering suitable equipment for scaling up lentivirus manufacturing, fixed-bed bioreactors should be prioritized. However, it is important to remember that many of these instruments have limited volume capacities. It is also the case that many bioreactors are built for either small or large volumes, but there are few options available for volumes in between.

Choosing the right equipment for scaling lentiviral fill-finish processes

Another important process that must be scaled for lentivirus projects is fill-finish. Appropriate fill-finish processes are essential as lentiviral vectors are inherently unstable from a pharmaceutical perspective and exposure to an adsorptive container like glass can cause vector loss1. Consequently, glass vials should not be used in their fill-finish processes and necessitate the need to screen and identify suitable containers made of plastics. Alternatively, excipients such as non-ionic surfactants could be added to minimize vector loss due to the interactions with glass.

By using fully automated, single-use bag filling and draining systems, as opposed to glass vials, fill-finish processes can be more easily scaled for lentivirus projects. Additional bags can be added to these systems to enlarge batch volume size and provide flexibility. Systems that offer continuous filling can also achieve filling of high volumes at high speed while minimizing the need for human interference and consequent contamination risk.

Being supported by those with experience and expertise

Scaling lentivirus production projects successfully relies on having the support of experts who understand what equipment is needed and why it is most suited to the project and processes.

Experts in lentivirus manufacturing will also ensure that they remain at the forefront of current research in the field and often have close partnerships with academic groups to achieve this. As regulations surrounding virus manufacturing are commonly driven by scientific findings, biologics CDMOs specializing in lentiviruses that have close ties with academic groups are more likely to predict possible changes that may impact lentivirus project scale-up.

A look to the future of lentiviral production

With the rise in popularity of lentiviruses as tools in cell and gene therapies, advancements in these technologies can be expected to increase. Adherent cell lines are likely to become less popular with further development of suspension-based production processes. Stable producer cell lines that do not rely on transient transfection could also revolutionize lentivirus scale-up by reducing costs and increasing reliability and consistency between batches.

For this reason, the support of viral vector CDMOs with expertise and experience is key to the success of scaling up lentiviral production. These companies will be able to both predict and respond quickly to scientific advances, using their expert knowledge to select the most suitable equipment and provide advice. Identifying a suitable CDMO partner with these traits can ultimately mean avoiding challenges that could delay bringing essential therapies to market.

1. Physical Characterization and Stabilization of a Lentiviral Vector Against Adsorption and Freeze-Thaw by Ozan S. Kumru

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