How to Scale Up Lentiviral Vector Production

Scale Up Lentiviral vector (LVV) Lentiviral vector & AAV manufacturing we have covered scaling up of processing steps in upstream. In this blog, we will examine the main steps involved in downstream processing (DSP). In the industry, the "gold standards" for the recovery of concentrated and purified LVV ranges from 10-20 percent. Making improvements to this recovery can lower the manufacturing costs and put viral vectors in researchers who require them.

Below, we break down DSP to unit operations and discover how new processes can contribute to scalability every step.

DSP is a multi-step process that results in the purification and concentration of the LVV

DNA Digestion: The Benzonase enzyme is an enzyme that can cut nucleic acids. To satisfy regulatory specifications that the finished LVV product has to have DNA-free fragments that are larger than 200 Bp. If you use a transient transfer method to generate the virus, the DNA of plasmids used for transfection in the process of the upstream process (USP) is one cause of contamination that must be eliminated during this process. Furthermore, DNA is sticky and could cause problems at the end of DSP. To create a flexible and secure method, the team of CCRM has created a unique technique in which benzonase can be directly added to the bioreactor, where cells produce viruses. It also permits the benzonase enzyme to function within the controlled environment of the bioreactor, where continuous mixing by the impeller improves the efficiency of digestion.

Harvest and Clarification: In traditional LVV production systems cells that are adherent in culture create virus vectors. So the process of harvesting media that contain LVV particles can be as easy as removing the media and then moving the virus-containing media into the new vessel. For instance, to collect media from a bioreactor that is based on suspension to harvest it, you need to remove the free-floating cells and the medium. Traditional DSP strategies for LVV purification typically utilize centrifugation, an open procedure, and is limited in its capacity to process massive amounts of media. At CCRM we have created an approach to use depth filters to remove particles and cells. The method we have developed has been created using single-use filters, reagent bags, sterile connectors, or weldable tubing that allows for an enclosed system. In addition, depth filtration provides a flexible alternative to centrifugation which has until now been the most popular method to eliminate particles and cells.

Diafiltration and Ultrafiltration This step is to simultaneously isolate the virus as well as remove the host cell's DNA and proteins that are contaminating the host cell. Tangential flow filters, where fluid flows in parallel with the membrane's surface, permit uninterrupted processing for lengthy periods without blocking the pores of the filter since the media removes particles that can cause obstruction. Media and contaminants from a culture that is smaller than the pores will flow through the membrane and get taken away as waste, whereas the LVV (that is larger than pores) are retained by the membrane, and will accumulate over time. Diafiltration replaces the cell-culture media with a formulation buffer which enhances the stability of viral cells over a wide temperature range. We've designed a process that removes 80-95 percent of protein and DNA.

Polishing: in this operation, chromatography can be used to further clean the virus and get rid of the remaining DNA and protein. The process of anion exchange (AEX) Chromatography is a method that is widely used to purify proteins and antibodies. But, due to the need for a high salt buffer - which quickly degrades viruses - AEX is a process that could result in significant loss of virus function. Our team has evaluated an alternative, gentler chromatography resin called Capto(TM) Core 700. The first of its kind in the market and developed to purify viral infections in mind This resin facilitates a more simple unit operation and more efficient recuperation of viruses, without the need for high salt concentrations.

Sterile Filtration operation presents one of the greatest issues to LVV production because the 0.2 um pores of the filter that is required to eliminate microorganisms are comparable to viruses. This leads to a significant reduction of virus in this stage - often up to 90 or 80 percent! A significant amount of process innovation is needed for optimizing this step. At CCRM we have made significant progress in improving the filter material, layout surface area, and conditioning to achieve 70 percent or more recovery on the 1L scale.

Innovation and Customization to Improve Recovery of LVV

The production of large quantities of LVV to make industrial cell and gene therapies is an important technological bottleneck and a cost driver. The CCRM team has developed a skill of using reagents and equipment that are normally employed in other areas of bio-manufacturing, to create customized solutions for the manufacture of LVV. We aim to develop an accurate, reliable closed, scalable, and scalable process that is superior to the current standard in the industry, to produce recoveries of 20-30.

Our methodology and our in-house expertise have allowed our company to become flexible with the methods we develop. One of the reasons is that we recognize that different customers have different needs in terms of concentration and the total recuperation of final products. Our team is always creating new ways to innovate and develop the ability to control different unit operations to adapt processes to various demands.

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