Efficient concentration of lentiviral vectors using tandem tangential flow filtration

Introduction

The VSV-G-pseudo-inactivated lentiviral vector is one of the indispensable biological experimental tools. Unlike the previous γ-retroviral vector, the lentiviral vector can transduce non-dividing cells and can carry more The more transgenic cassettes, the longer the transgene expression time in the cells, the higher the titer, and the lower the genotoxicity.

In general, the carrier titer in the supernatant of the toxigenic cell is sufficient to transduce the conventional cell line, but the primary cell is more difficult to transduce, and the carrier needs to be concentrated to obtain a higher concentration. In addition, some vectors are necessary steps for the use of lentiviral vectors by integrating genetic elements that reduce titers, and most cell types are not tolerant to toxin-producing cell growth media or secreted proteins thereof.

Ultracentrifugation is one of the common methods for concentrating virus particles, but its concentration factor is low, and the amount of each treatment is also small, and the cumbersome multi-step operation reduces the recovery rate of virus particles. Centrifugal filtration is a relatively simple method of concentration, but the filter itself captures a large amount of carrier trapped, causing losses. In contrast, tangential flow filtration (TFF) is simple in operation, low in loss, and can effectively reduce metabolites and secreted proteins of toxins through the washing process, but the traditional one-step TFF concentration is only 50-100 times. In this experiment, the two-step TFF was used to successfully concentrate the 5.5L lentiviral vector stock to about 1 mL, and the recovery rate was over 97%. The quality analysis of the final product showed that the concentrated lentiviral vector can effectively transduce primary human CD34+ hematopoietic stem/progenitor cells and primary human fibroblasts without significant toxicity.

experiment

The 293T cells were transfected with a transfection mixture containing pCCL vector plasmid, gag/pol expression plasmid and envelope expression plasmid pMD.G (VSV-G), and induced by sodium butyrate after transfection. The LV medium was recovered in two portions, passed through a 0.8 μm filter, and the filtrate was stored in a sterile container.

Tangential flow filtration uses the IIi tangential flow filtration system developed by Spectrum Pure KrosFlo (product number: SYR2-U20-01N). In the first step, the system is equipped with flow path 1 (FP1, product number: EZ-M1- 500S-260-01N-1, containing 500kD hollow fiber filter (fiber number 320, fiber inner diameter 0.5mm, membrane surface area 615cm2)), in the second step operation, the system is equipped with flow path 2 (FP2, product number: EZ- CHIL07-01-1, containing a 500 kD hollow fiber filter (fiber number 12, fiber inner diameter 0.5 mm, membrane surface area 40 cm2).

Check the integrity of the flow path (FP1) before filtration, completely wet the flow path with DPBS, run the system until DPBS drains, close the port and valve, run the pump until the inlet pressure reaches about 5 psi, open the filtrate port, due to air Do not seep through the wetted filter fibers. If the assembly is complete, the pressure drop should not exceed 0.01 psi/s.

After passing the integrity test, the concentration of LV-containing medium was started. The inlet pressure was monitored throughout the process and maintained below 6 psi. In the first step, 5.5L of the feed liquid can be concentrated to 50 mL, that is, concentrated about 100 times. The concentrated carrier was then subjected to constant washing with 1000 mL of washing buffer containing fetal bovine serum. After that, the system flow path was changed to FP2. After the integrity test, the 50 mL concentrate obtained in the first step was further concentrated to 1 mL, that is, the total concentration was 5000 times. The inlet pressure at this step is maintained below 9 psi.

Subsequently, titers were confirmed by HT-29 cell vector transduction experiments and absolute quantitative analysis was performed using multiplex real-time PCR techniques. At the same time, the primary human CD34+ hematopoietic stem/progenitor cells were transduced with the final concentration vector, and the transduction efficiency of the vector after concentration was confirmed. In addition, a composite STEMCCA vector directed against induced pluripotent stem cell (iPSC) production was constructed and concentrated in the same manner to examine its ability to transduce and reprogram primary human fibroblasts.

result

The use of TFF for LV concentration yields a very high concentration factor and recovery, and the freeze-thaw experiments on LV after concentration show good stability of the final product.

In order to test the quality of concentrated LV, the transduction experiment of primary human CD34+ hematopoietic stem/progenitor cells was carried out. The results showed that the transduction was very successful, and the transduction success rate increased significantly with the increase of carrier concentration without obvious toxicity. . In the primary human fibroblast transduction experiment, the vector containing the high-efficiency STEMCCA element can effectively transduce and reprogram the primary human fibroblasts to form induced pluripotent stem cells, and the success rate increases with the increase of the carrier concentration.

discuss

Using two-step tangential flow filtration, LV can be concentrated at high magnification and high recovery rate, and the process is stable and reproducible. The transduction and expression experiments on CD34+ showed that the concentrated product had no obvious toxicity, and the high concentration STEMCCA vector preparation and induction experiments proved that the process can be used for the production and concentration of large-scale complex carriers.

The content of this article is the editor's translation. If there is any inconvenience, please forgive me. For details, please refer to the original text.

Original: Cooper, AR, Patel, S., Senadheera, S., et al., Highly efficient large-scale vector concentration by tandem tangential flow filtration. Journal of Virological Methods, 2011, 177: 1-9.

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8D NLS

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