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How To Scale-Up Lentiviral Vector Production Part 2: Considerations for Downstream Processing  

In Part 1 of our series on lentiviral vector (LVV) manufacturing we covered scale-up of upstream processing steps. In this post we will look at the key steps in downstream processing (DSP). The industry “gold-standard” for recovery of purified and concentrated LVV is 10-20 percent. Improving on this low recovery is an opportunity to reduce the cost of manufacturing and get viral vectors into the hands of researchers who need them.  

Below we break down DSP into unit operations and understand how process innovations are contributing to scalability at each step. 

DSP Is a Multi-Step Process Resulting in Purification and Concentration of LVV  

  1. DNA Digestion: Benzonase is an enzyme with the ability to cleave nucleic acids. To meet regulatory requirements, the final LVV product must be free of DNA fragments larger than 200. If using a transient transfection method to make the virus, DNA from plasmids used to transfect cells during upstream processing (USP) are one source of contamination that needs to be removed in this step. Further, DNA is sticky and can cause problems at the later stages of DSP. To build a scalable and closed process, the team at CCRM has developed a novel method in which benzonase is added directly to the bioreactor where cells are producing virus. This also allows the benzonase to operate in the controlled environment of the bioreactor where constant mixing from the impeller increases digestion efficacy.  
  2. Harvest & Clarification: In conventional LVV production systems, adherent cells in culture generate viral vectors. Therefore, harvesting media that contains LVV particles is as simple as removing media and transferring the virus-containing media to a new vessel. In contrast, to harvest media from suspension bioreactor-based culture, you must separate the free-floating cells from the media. Legacy DSP methods for LVV purification often use centrifugation, which is an open process and limited in its ability to process large volumes of media. At CCRM, we have established a method for using depth filters to eliminate cells and debris. Our method has been designed with single-use filters, reagent bags and sterile connectors or weldable tubing to allow for a closed system process. Most importantly, depth filtration offers a scalable alternative to centrifugation, which until now has been the method of choice for removal of cells and debris.  
  3. Ultrafiltration and Diafiltration: The purpose of this step is to simultaneously concentrate the virus and remove host cell DNA and protein contaminants. Tangential-flow filters, in which liquid flows parallel to the membrane surface, allow continuous processing for long periods of time without clogging the filter’s pores, as media sweeps away particles that may cause blockage. Culture media and contaminants, which are smaller than the pores, will pass through the membrane and be collected as waste, while the LVV (that are larger than the pores) will be retained by the membrane and thus concentrate over time. Diafiltration exchanges the cell culture media with a formulation buffer that increases viral stability across a range of temperatures. We have designed a unit operation that can remove 80-95 percent of DNA and protein.  Free Download
  4. Polishing: In this unit operation, chromatography is used to further purify virus and remove residual DNA and protein. Anion exchange (AEX) chromatography is a commonly used method for purifying antibodies and proteins. However, because of the requirement for a high salt buffer – a condition that rapidly degrades virus – AEX chromatography may result in a significant loss of functional virus. Our team has tested a gentler, alternative chromatography resin: Capto™ Core 700. New to the industry and designed with viral purification in mind, this resin allows for simpler unit operation and better recovery of virus without the use of high salt concentrations.  
  5. Sterile Filtration: This unit operation poses one of the most significant challenges for LVV production because the 0.2 µm pore size of the filter required to remove microorganisms is similar in size to viral particles.  This results in significant loss of virus at this step – sometimes up to 80 or 90 percent! Significant process innovation is required to optimize this step. At CCRM, we have made important progress by optimizing filter material, layout, surface area and pre-conditioning to accomplish up to 70 percent recovery at the 1L scale.  

Innovation and Customization for Improved LVV Recovery  

Producing amounts of LVV required for industrial manufacture of cell and gene therapies is a significant technical bottleneck and cost driver. CCRM has become adept at re-purposing equipment and reagents typically used for other applications in bio-manufacturing to develop custom solutions for LVV manufacturing. Our goal is to create a reproducible, closed, scalable process, improving upon the current industry standard, to generate recovery rates of 20-30 percent. 

Our approach, and the resulting in-house expertise, has allowed us to be flexible in the processes we design. Part of that is recognizing that customers have different priorities with respect to the concentration versus total recovery of the final product. Our team is continually innovating and building the capability to manipulate different unit operations to tailor processes to different needs.  

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