Rapid In-Process Monitoring of Lentiviral Vector Particles by High-Performance Liquid Chromatography

Transfiguracion, Julia; Tran, Michelle Yen; Lanthier, Stéphane; Tremblay, Sonia; Coulombe, Nathalie; Acchione, Mauro; Kamen, Amine

doi:10.1016/j.omtm.2020.08.005

Cited by 11 publications

(13 citation statements)

References 26 publications

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“…The GTA and ddPCR data in LVs also reveals a difference. Indeed, the functional viral titer is lower than the VG titer as previously documented in Transfiguracion et al [ 51 ]. This underlines the difficulty in assessing absolute quantification of EVs and LVs, but it also underlines the heterogeneous nature of EVs and LVs.…”

Section: Discussionmentioning

confidence: 65%

“…This method lacks specificity and often leads to overestimation of the total particles measured. A method for in-process LV quantification was recently published [ 51 ] involving High-Performance Liquid Chromatography (HPLC). Although the authors optimized the method for minimizing the impact of EVs, they did acknowledge the presence of EVs in the quantification of LV particles and their proportion could not be estimated since the measure of a sample with no LV particles falls outside of the claimed linear range of the method.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of Extracellular Vesicles Secreted in Lentiviral Producing HEK293SF Cell Cultures

Minh

Star

Stupak

et al. 2021

Viruses

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Lentiviral vectors (LVs) are a powerful tool for gene and cell therapy and human embryonic kidney cells (HEK293) have been extensively used as a platform for production of these vectors. Like most cells and cellular tissues, HEK293 cells release extracellular vesicles (EVs). EVs released by cells share similar size, biophysical characteristics and even a biogenesis pathway with cell-produced enveloped viruses, making it a challenge to efficiently separate EVs from LVs. Thus, EVs co-purified with LVs during downstream processing, are considered “impurities” in the context of gene and cell therapy. A greater understanding of EVs co-purifying with LVs is needed to enable improved downstream processing. To that end, EVs from an inducible lentivirus producing cell line were studied under two conditions: non-induced and induced. EVs were identified in both conditions, with their presence confirmed by transmission electron microscopy and Western blot. EV cargos from each condition were then further characterized by a multi-omic approach. Nineteen proteins were identified by mass spectrometry as potential EV markers to differentiate EVs in LV preparations. Lipid composition of EV preparations before and after LV induction showed similar enrichment in phosphatidylserine. RNA cargos in EVs showed enrichment in transcripts involved in viral processes and binding functions. These findings provide insights on the product profile of lentiviral preparations and could support the development of improved separation strategies aimed at removing co-produced EVs.

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Section: Discussionmentioning

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Characterization of Extracellular Vesicles Secreted in Lentiviral Producing HEK293SF Cell Cultures

Minh

Star

Stupak

et al. 2021

Viruses

Self Cite

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“…CAR-encoding lentiviral particles were produced by transient transfection of Lenti-X 293T cells (TaKaRa) as described previously ( 21 ). Briefly, the CAR-expressing plasmid, along with 3 packaging plasmids (pLP1, pLP2, and pLP/VSVG), was transfected into 293T cells by polyetherimide (PEI, Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

IL-7 and CCR2b Co-Expression-Mediated Enhanced CAR-T Survival and Infiltration in Solid Tumors

Zhang

Han

et al. 2021

Front. Oncol.

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Chimeric antigen receptor T (CAR-T) cells are not effective in solid tumor treatment due to reduced invasion and expansion, and short survival time. This study aimed to explore whether interleukin (IL)-7 and CCR2b expression could improve GD2-CAR-T cell survival and infiltration in neuroblastoma and melanoma treatment. IL-7 and CCR2b were inserted into the classical second-generation CAR structure to construct 7×2b CAR. The 7×2b CAR-T cell phenotypes were evaluated by flow cytometry and the chemokine levels by ELISA. The 7×2b CAR-T cell migration and anti-tumor abilities were detected by Transwell assay and animal experiments in vivo. We report that compared with that of CAR-T cells, 7×2b CAR-T cell IL-7 secretion and CCR2b expression did not affect the T cell surface expression of CAR or CAR-T specificity and efficacy against tumor cells. The 7×2b CAR-T cells could induce IFN-γ secretion in GD2-positive tumor cells, killing them as well as conventional CAR-T cells. Moreover, IL-7 and CCR2b co-expression enhanced the 7×2b CAR-T cell survival and migration. Similar to conventional CAR-T, 7×2b CAR-T cells could also inhibit tumor growth and increase IFN-γ, Gzms-B, and IL-2 expression. Finally, unlike in mice injected with CAR-T cells, CD3 expression was the most abundant in the spleen and tumor tissues in mice injected with 7×2b CAR-T cells. Our study demonstrates that IL-7 and CCR2b co-expression in GD2-CAR-T cells exhibit stronger anti-tumor activity than classical second-generation CAR-T cells, shedding light on the potential novel GD2-positive neuroblastoma and melanoma treatment approach.

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“…Both methods can be very time consuming to complete and insufficient for large numbers of samples. A recent addition, which utilises high-performance liquid chromatography (HPLC) with AEX resins, can elute bands of vector, and, based on their fluorescence, estimate the number of vector particles within

total particles per mL range [ 267 ]. This method has been used to differentiate DNA from vector and is comparable to ELISA and ddPCR and allows for the application of various samples from differing aspects of the process within 6.5 min.…”

Section: Vector Characterisation and Quality Controlmentioning

confidence: 99%

Lentiviral Vector Bioprocessing

Perry

Rayat

2021

Viruses

110

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Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.

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Rapid In-Process Monitoring of Lentiviral Vector Particles by High-Performance Liquid Chromatography

Cited by 11 publications

References 26 publications

Characterization of Extracellular Vesicles Secreted in Lentiviral Producing HEK293SF Cell Cultures

Characterization of Extracellular Vesicles Secreted in Lentiviral Producing HEK293SF Cell Cultures

IL-7 and CCR2b Co-Expression-Mediated Enhanced CAR-T Survival and Infiltration in Solid Tumors

Lentiviral Vector Bioprocessing

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