Influence of Mildly Acidic pH Conditions on the Production of Lentiviral and Retroviral Vectors

Holic, Nathalie; Seye, Ababacar; Majdoul, Saliha; Martin, Samia; Merten, O.‐W.; Galy, Anne; Fenard, David

doi:10.1089/humc.2014.027

Cited by 16 publications

(10 citation statements)

References 15 publications

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“…Based on the drop in pH post-induction for the GPRTG-EF1α-hγ c -OPT stable cell line in cell factories and studies that have shown transient LVV production to increase at lower pH values, 29 , 31 the effect of maintaining a range of pH set points throughout the vector production phase was investigated. Reactors were inoculated at 15,000 cells/cm 2 (8 × 10 7 cells total) and maintained at pH 7.2 for 5 days.…”

Section: Resultsmentioning

confidence: 99%

Lentiviral Vector Production from a Stable Packaging Cell Line Using a Packed Bed Bioreactor

Powers

Drury

Hoehamer³

et al. 2020

Molecular Therapy - Methods & Clinical Development

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Self-inactivating lentiviral vectors (LVVs) are used regularly for genetic modification of cells, including T cells and hematopoietic stem cells for cellular gene therapy. As vector demand grows, scalable and controllable methods are needed for production. LVVs are typically produced in HEK293T cells in suspension bioreactors using serum-free media or adherent cultures with serum. The iCELLis® is a packed-bed bioreactor for adherent or entrained cells with surface areas from 0.53 to 500 m 2 . Media are pumped through the fixed bed and overflows, creating a thin film that is replenished with oxygen and depleted of CO 2 as media return to the reservoir. We describe the optimization and scale-up of the production of GPRTG-EF1α-hγ c -OPT LVV using a stable packaging cell line in the iCELLis Nano 2-cm to the 10-cm bed height low compaction bioreactors (0.53 and 2.6 m 2 surface area) and compare to the productivity and efficacy of GPRTG-EF1α-hγ c -OPT LVV manufactured under current Good Manufacturing Practice (cGMP) using 10-layer cell factories for the treatment of X-linked severe combined immunodeficiency. By optimizing fetal bovine serum (FBS) concentration, pH post-induction, and day of induction, we attain viral yields of more than 2 × 10 7 transducing units/mL. We compared transduction efficiency between LVVs produced from the iCELLis Nano and cell factories on healthy, purified CD34 + cells and found similar results.

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Section: Resultsmentioning

confidence: 99%

Lentiviral Vector Production from a Stable Packaging Cell Line Using a Packed Bed Bioreactor

Powers

Drury

Hoehamer³

et al. 2020

Molecular Therapy - Methods & Clinical Development

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“…Lower pH has been shown to increase LV production. 26 Therefore, in runs 6–10 pH was decreased to 7.0 after PEI-mediated transfection. Lower pH had a positive impact on functional titer, with the total TU exceeding 1E+10 for the first time ( Table 2 ).…”

Section: Resultsmentioning

confidence: 99%

“…Lower pH levels close to pH 6 have been shown to increase vesicular stomatitis virus envelope glycoprotein G pseudotyped LV productivity. 26 As both cells and LV particles are sensitive to pH changes, the correct pH for LV production is determined by finding the optimal pH value for efficient LV production in the cells in question. In control flasks, pH decreased below 7.0 by the end of culture because of lack of pH control, which indicates that the pH could be decreased also in the bioreactor.…”

Section: Discussionmentioning

confidence: 99%

Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor

et al. 2017

Gene Ther

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Lentiviral vectors (LVs) are promising tools for gene therapy. However, scaling up the production methods of LVs in order to produce high-quality vectors for clinical purposes has proven to be difficult. In this article, we present a scalable and efficient method to produce LVs with transient transfection of adherent 293T cells in a fixed-bed bioreactor. The disposable iCELLis bioreactors are scalable with a large three-dimensional (3D) growth area range between 0.53 and 500 m2, an integrated perfusion system, and a controllable environment for production. In this study, iCELLis Nano (2.67–4 m2) was used for optimizing production parameters for scale-up. Transfections were first done using traditional calcium phosphate method, but in later runs polyethylenimine was found to be more reliable and easier to use. For scalable LV production, perfusion rate control by measuring cell metabolite concentrations in the bioreactor leads to higher productivity and reduced costs. Optimization of cell seeding density for targeted cell concentration during transfection, use of low compaction fixed-bed and lowering the culture pH have a positive effect on LV productivity. These results show for the first time that iCELLis bioreactor is scalable from bench level to clinical scale LV production.

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“…In their study, they use PALL iCELLis, a compact fixed-bed bioreactor with an integrated perfusion system, comparing yields achieved with either CaPho precipitation or PEI transfection methods, within a range of pH and under glucose and lactate concentration monitoring. In both transfection methods, they confirmed the critical role of cell density and pH in the efficiency of productivity,57 showing higher production at high cell-density and mildly acidic pH, with vp cm–2 (vp= viral particle) between 1.31E+08 and 4.85E+08 using iCELLis Nano fixed-bed bioreactors.…”

Section: Lentiviral Vectors Manufacturingmentioning

confidence: 55%

Curing Hemoglobinopathies: Challenges and Advances of Conventional and New Gene Therapy Approaches.

Motta

Ghiaccio

Cosentino

et al. 2019

Mediterr J Hematol Infect Dis

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Inherited hemoglobin disorders, including beta-thalassemia (BT) and sickle-cell disease (SCD), are the most common monogenic diseases worldwide, with a global carrier frequency of over 5%.1 With migration, they are becoming more common worldwide, making their management and care an increasing concern for health care systems.BT is characterized by an imbalance in the α/β-globin chain ratio, ineffective erythropoiesis, chronic hemolytic anemia, and compensatory hemopoietic expansion.1 Globally, there are over 25,000 births each year with transfusion-dependent thalassemia (TDT). The currently available treatment for TDT is lifelong transfusions and iron chelation therapy or allogenic bone marrow transplantation as a curative option. SCD affects 300 million people worldwide2 and severely impacts the quality of life of patients who experience unpredictable, recurrent acute and chronic severe pain, stroke, infections, pulmonary disease, kidney disease, retinopathy, and other complications. While survival has been dramatically extended, quality of life is markedly reduced by disease- and treatment-associated morbidity.The development of safe, tissue-specific and efficient vectors, and efficient gene-editing technologies have led to the development of several gene therapy trials for BT and SCD. However, the complexity of the approach presents its hurdles. Fundamental factors at play include the requirement for myeloablation on a patient with benign disease, the age of the patient, and the consequent bone marrow microenvironment. A successful path from proof-ofconcept studies to commercialization must render gene therapy a sustainable and accessible approach for a large number of patients. Furthermore, the cost of these therapies is a considerable challenge for the health care system. While new promising therapeutic options are emerging,3,4 and many others are on the pipeline,5 gene therapy can potentially cure patients. We herein provide an overview of the most recent, likely potentially curative therapies for hemoglobinopathies and a summary of the challenges that these approaches entail.

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Influence of Mildly Acidic pH Conditions on the Production of Lentiviral and Retroviral Vectors

Cited by 16 publications

References 15 publications

Lentiviral Vector Production from a Stable Packaging Cell Line Using a Packed Bed Bioreactor

Lentiviral Vector Production from a Stable Packaging Cell Line Using a Packed Bed Bioreactor

Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor

Curing Hemoglobinopathies: Challenges and Advances of Conventional and New Gene Therapy Approaches.

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