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December 08, 2022

Lentiviral vectors (LVVs) are often used to deliver genetic material for modifying cells and conferring therapeutic properties during the production of cell and gene therapies (CGT). However, CGT manufacturers continue to use legacy methods carried over from basic research. CCRM is working on overcoming the challenges of scaling up LVV production to reduce production costs and facilitate CGT's commercial production.

Some scale-up methods for both upstream process (USP) and downstream process (DSP) are described below.


USP involves culturing cells that are hosts for virus production. In transient production, cells need to be grown in sufficient quantities and are then typically transfected with four plasmids encoding viral genes. The cells then express viral proteins and nucleic acids, which are packaged into viral particles inside cells. The viral vectors are harvested from the culture and further purified using purification methods in DSP steps. CCRM’s approach to scaling up LVV production is to culture suspension-adapted cells in a bioreactor.

The following criteria should be considered when scaling up the USP:

  1. Size of the bioreactor: If cells are adherent, CCRM will adapt these cells to grow in suspension, through well-developed protocols. When cells have been adapted to grow in suspension, scale-up can be done by increasing the volume and paying attention to the key engineering fluid dynamic parameters.
  2. Scale-up parameters: Important fluid dynamic parameters to be optimized during scale-up include mixing characteristics within the bioreactor, where power input, shear and mass transfer, among others, are typical parameters. The performance of the bioreactor is often measured by the ability to transfer oxygen, which is measured by a mass-transfer parameter called kLa, and is used to ensure that sufficient oxygen is available for cells for optimal growth during scale-up.
  3. Scale-down models: Operating conditions that are scale independent – such as media composition, pH, temperature, cell density at transfection, and transfection conditions – should be tested at small scales in a design of experiments format to optimize. The optimized conditions can then be translated to a larger scale.
  4. Optimal mixing conditions: There is a vast amount of literature available on growing most of the commonly used cells in bioreactors, which can be leveraged to determine the conditions for growing the host cells. Select the small-scale reactor to be representative of the larger-scale system in terms of mixing method and impeller designs.


The current overall recovery in DSP of purified and concentrated LVV is 10-30 per cent. DSP is a complex, multi-step process and entails losses at every step. Improving each step will improve overall recovery, which will reduce manufacturing costs and increase the amount of viral vector that reaches clinics.

Consider these process innovations for scaling up DSP:

  1. Harvest and clarification:Harvesting LVV particles involves taking media from suspension bioreactor-based cultures and clarifying it by separating out host cell debris. Historically, debris was typically removed using centrifugation, which has limited ability for scale-up and may involve open processes. CCRM uses filtration methods to remove cell debris in closed systems that provide a scalable alternative to centrifugation.
  2. Ultrafiltration and diafiltration: This step simultaneously concentrates virus and removes impurities, such as host cell DNA and protein contaminants below the molecular weight cut-off of the filter. Culture media and contaminants pass through the membrane and are collected as waste, while the membrane retains the LVVs and concentrates them over time. Tangential-flow filters allow continuous processing while minimizing filter fouling. Diafiltration simultaneously allows the exchange of the buffer during the concentration to formulation buffer to increase viral stability across a range of temperatures. CCRM's unit operation can remove 80-95 per cent of host cell DNA and protein during this step. CCRM has also developed proprietary buffers to stabilize virus.
  3. DNA digestion:To satisfy regulations, LVV products must be free of host cell DNA fragments larger than 200 base pairs. When using transient transfection to make viruses, plasmid DNA used during USP must be removed. CCRM has developed methods using benzonase (an enzyme with the ability to cleave nucleic acids) in the bioreactor, which enables it to function in a controlled environment with increased digestion efficacy.
  4. Polishing:Chromatography further separates virus from any residual host DNA and protein, but some chromatography methods can result in significant losses of functional virus. CCRM uses a gentler, alternative multi-modal chromatography resin (Capto™ Core 700), which allows for simpler unit operation and better viral recovery. Viral particles are collected in the flow-through under conditions that maintain infectivity.
  5. Sterile filtration:Sterile filtration is typically done using 0.2 µm filter pore size. As LVVs are smaller in size than most microorganisms, sterile filtration poses special challenges and results in 80-90 per cent loss of virus. CCRM has worked on methods to optimize sterile filtration and has achieved yields of up to 70 per cent.

Contact us to learn more about our custom solutions for addressing your LVV product needs.

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