Lentiviral vector | Filtration | Gene therapy

Enhancing the Purification of Lentiviral Vectors for Clinical Applications

Advances in filtration enhance the purification of lentiviral vectors for clinical applications

November 11, 2021

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Regenerative medicine and gene therapy has been one of the biggest success stories in medicine over the last twenty years. Following a series of early setbacks, the technology has matured to the point where new therapeutics are now entering the market at an astonishing pace.  Globally there are over 1300 industry-sponsored clinical trials currently ongoing, with approval decisions expected on a record 18 products this year alone, 10 of which are for brand new products for which there is currently no similar approved product in the market. 2021 has already seen four product approvals, of which three are gene therapy or gene-modified cell therapies (Breyanzi, Abecma, and Skysona) (1). Inherited genetic diseases that were until only recently considered to be incurable, forever locked in the unlucky patient’s genetic code, are now being unlocked as the technological promise of gene therapy is transitioned to clinical practice.


This increase in the number of new products passing through development and into the clinic has driven an equal explosion in the tools and techniques required. Amongst these tools, lentiviral vectors have become increasingly popular as they are able to stably integrate their genomic payload into the genome of both dividing and non-dividing cells, they are able to deliver a large payload of up to 9 kb, and have low rates of insertional mutagenesis compared to other vectors (2). This has made lentivirus the vector of choice for stable and sustained long-term gene expression. However, the development of rapid, efficient, and robust downstream processes to cope with the low stability of this virus has slowed the clinical to market transition for therapeutics leveraging lentiviral vectors.


In a recent (2021) publication (2), Moreira et al., describe the development of a novel purification method with high recovery rates that enhances lentivirus vector purification with the ability to accelerate the development of therapeutic products based on these vectors.


The authors used a design of experiments (DOE) approach and multivariate statistical methodology to identify and optimize the critical process parameters within a three-step viral purification protocol. Some of the key findings of the study are summarized below for each of the three stages in the purification protocol.



Six different filter types from different manufacturers were tested, utilizing different membranes, all with a 0.45 mm pore size. (Polyethersulfone (PES), polyvinylidene fluoride (PVDF), nylon, glass fiber (GF), and a polypropylene (PP) depth filter). The authors reported the highest levels of turbidity reduction with Pall Supor® PES filters (50-62% turbidity reduction) and Pall PVDF Fluorodyne® II filters (55% turbidity reduction).


One advantage of the described process is that this clarification step remains compatible with new and emerging upstream strategies such as cell culture in serum-free media, ensuring future compatibility for the purification process so that it does not need to be revised to accommodate the inevitable upstream process changes and improvements.


Anion Exchange Chromatography


The study reports initial evaluation of the chromatographic step with four different media including Pall Mustang® Q. The two critical performance parameters of transducing particles, recovery yield (TPRY) and volumetric concentration factor (VCF), were measured for all four media. While reported results for TPRY remained similar across all the media, the authors indicate VCF was significantly different, with the highest results reported for Mustang Q (VCF 100).


Based on the initial results above, AcroPrepTM Advance 96-well filter plates with Mustang Q membrane, together with an alternative 96-well filter plate, were then progressed into a full factorial DOE for three critical operating parameters: amount of viral load, elution pH, and elution NaCl concentration. The study reports results of the experiment showed that the AcroPrep filter plates with Mustang Q membrane gave higher overall recovery rates, with a higher virus load at higher NaCl concentrations, while the recovery levels with alternative plates were not only lower, but the effect of NaCl concentration was much smaller.




Sterilizing filtration of concentrated and diafiltered lentiviral particles is a critical step in the purification process. The authors noted that this typically results in the loss of up to half of the purified lentiviral particles with a substantial negative impact on the overall yield and cost of the manufacturing process. However, in this study sterilizing filtration was performed with Pall Supor EKV Mini KleenpakTM syringe filters which the authors reported as leading to a ‘very satisfactory result’ and an almost complete recovery of the lentiviral particles.




When used in conjunction with the optimized clarification, and sterilization processes described, the authors concluded that the AcroPrep Advance 96-well filter plates with Mustang Q membranes achieved the highest level of performance and were able to achieve recovery yields of close to 90%, higher than those previously described in the literature.


This work represents an important step forward in the development of rapid, efficient, and robust downstream processes that reduce the technical challenges for cell and gene therapies utilizing lentiviral vectors.


Learn more about the AcroPrep Advance 96-well filter plates with Mustang Q, as well as the other Pall filters used in the study on our website.



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