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Viral Aggregation in Downstream Processing of Lentiviral Vectors
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May 10, 2022

Our previous post on Considerations for Downstream Processing provides an overview of each unit operation. A critical aspect of scaling-up production of LVV is the optimization of each unit operation in downstream processing (DSP) to improve the recovery of a purified and concentrated virus.

While some degree of purification is achieved in every unit operation, polishing involves the removal of major impurities such as residual host cell DNA and protein. This post examines the utility of multi-modal chromatography as a novel strategy during polishing to improve LVV purity.

Removal of Impurities Is Accomplished Using Chromatography

The goal of polishing is to maintain infectivity while recovering as much virus as possible. Optimizing this step means achieving a delicate balance between these two requirements. Chromatography is the primary method used because of its ability to separate, identify and purify various constituents of a heterogeneous mixture. Anion exchange (AEX) chromatography is a commonly-used method for the purification of proteins. However, this method is not easily adaptable to LVV manufacture because of the requirement for a high salt buffer – a condition that greatly reduces the infectivity of fragile LVV particles. While AEX is not ideal for LVV purification it has some advantages, including “harsher” buffer conditions that can overcome the tendency of viral particles to stick to the chromatography column; excellent removal of host cell protein and DNA impurities; and, the ability to achieve a concentrated solution at the end of the unit operation. But immediate dilution of the product is required to prevent loss of infectivity due to high salt concentration, often greatly reducing the concentration of LVV.

LVV-Friendly Modifications to Chromatography

To optimize polishing for improved recovery of LVV, our team has tested a gentler, alternative chromatography resin: Capto™ Core 700. This work is an excellent example of how process innovation in cell and gene therapy (CGT) is improving upon technology originally developed in the biopharmaceutical industry for the production of monoclonal antibodies and vaccines. Capto™ Core 700 is a multi-modal chromatography resin composed of beads with ligand-activated cores and inactive outer shells that work via size exclusion, ion exchange, and hydrophobic interactions. This resin binds impurities (e.g., protein and DNA) within the positively charged and hydrophobic cores while excluding viruses due to the 700 kDa pores of the outer shell. Ultimately, LVV can be collected in the flow-through under gentle conditions that help maintain infectivity and higher recovery.

One potential limitation of using Capto™ Core 700 is that depending on the process parameters, it may yield a diluted final product and, therefore, a subsequent concentration step might be required to overcome this. However, every additional step in DSP results in the loss of the infective virus, which is a common problem in viral DSP.

The unit operation for polishing ideally yields a highly concentrated solution with low salt, high infectivity, high contaminant clearance, and low levels of viral particle aggregation. At CCRM, we have developed a polishing process that yields LVV solutions with these characteristics and can be customized towards LVV recovery or purity depending on the client’s needs. Ensuring this, however, requires continued innovation and process improvements that ultimately contribute to more affordable and accessible LVV for CGT developers.

Contact us to learn how our process development team can help.

 

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