Production of Lentiviral Vectors Using Suspension Cells Grown in Serum-free Media

Bauler, Matthew; Roberts, Jessica K.; Wu, Chang-Chih; Fan, Baochang; Ferrara, Francesca; Yip, Bon Ham; Diao, Shiyong; Kim, Young–In; Moore, Jennifer C.; Zhou, Sheng; Wielgosz, Matthew M.; Ryu, Byoung Y.; Throm, Robert E.

doi:10.1016/j.omtm.2019.11.011

Cited by 73 publications

(77 citation statements)

References 65 publications

(124 reference statements)

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“…Significant work has been performed to find alternatives to FBS, including either supplements 32 or adapting cells to serum-free suspension culture, 16 but many producer cell lines require serum for production. Minimizing the FBS concentration during cell culture can reduce the amount of serum proteins that must be removed during purification.…”

Section: Resultsmentioning

confidence: 99%

“…While suspension systems for LVV production are being developed, 14 , 15 , 16 production of LVVs frequently occurs in adherent cells requiring an adherent cell system and serum for scale-up. 17 An adherent cell system facilitates media changes and the harvest of secreted products.…”

Section: Introductionmentioning

confidence: 99%

“…Vector production in a bioreactor allows for more consistent control of process parameters such as pH and DO, as compared to a flask-based process such as the one described in Figure 4 of Bauler et al. 16 The use of a bioreactor also allows for scaling up from the iCELLis Nano in process development to the iCELLis 500 in production, which reduces labor as compared to a flask-based process, which must be scaled out rather than scaled up. Immobilization in polyester macrocarriers protects cells from stresses associated with bubble sparging or an impeller while media circulates up through the fixed bed and is replenished with gases as it falls back to the reservoir as a thin film.…”

Section: Introductionmentioning

confidence: 99%

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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%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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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“…LVs were produced as previously described. 51 Briefly, 293T cells (ATCC CLR-11268), adapted to grow in suspension using serum-free media, were transfected with the transfer vector and helper plasmids, pCAG-kGP1-1R-AF, pCAG-vesicular stomatitis virus glycoprotein G (VSVG))-AF, and pCMV-Rev-AF expressing HIV-1 gagpol , the vesicular stomatitis virus glycoprotein, and HIV-1 Rev, respectively. Forty-eight hours later, the supernatant was harvested by a combination of centrifugation and 0.22 μm filtration to remove cell debris.…”

Section: Methodsmentioning

confidence: 99%

“…LV particles were purified by high-performance liquid chromatography (HPLC) and titered on HOS cells as previously described. 51 …”

Section: Methodsmentioning

confidence: 99%

Route of 41BB/41BBL Costimulation Determines Effector Function of B7-H3-CAR.CD28ζ T Cells

Nguyen

Okeke

Clay

et al. 2020

Molecular Therapy - Oncolytics

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B7-H3 is actively being explored as an immunotherapy target for pediatric patients with solid tumors using monoclonal antibodies or T cells expressing chimeric antigen receptors (CARs). B7-H3-CARs containing a 41BB costimulatory domain are currently favored by several groups based on preclinical studies. In this study, we initially performed a detailed analysis of T cells expressing B7-H3-CARs with different hinge/transmembrane (CD8α versus CD28) and CD28 or 41BB costimulatory domains (CD8α/CD28, CD8α/41BB, CD28/CD28, CD28/41BB). Only subtle differences in effector function were observed between CAR T cell populations in vitro . However, CD8α/CD28-CAR T cells consistently outperformed other CAR T cell populations in three animal models, resulting in a significant survival advantage. We next explored whether adding 41BB signaling to CD8α/CD28-CAR T cells would further enhance effector function. Surprisingly, incorporating 41BB signaling into the CAR endodomain had detrimental effects, while expressing 41BBL on the surface of CD8α/CD28-CAR T cells enhanced their ability to kill tumor cells in repeat stimulation assays. Furthermore, 41BBL expression enhanced CD8α/CD28-CAR T cell expansion in vivo and improved antitumor activity in one of four evaluated models. Thus, our study highlights the intricate interplay between CAR hinge/transmembrane and costimulatory domains. Based on our study, we selected CD8α/CD28-CAR T cells expressing 41BBL for early phase clinical testing.

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Characterization of key manufacturing uncertainties in next generation therapeutics and vaccines across scales

Sarkis

Shah

Papathanasiou

2023

J Adv Manuf & Process

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Viral vectors are advanced therapy products used as genetic information carriers in vaccine and cell therapy development and manufacturing. Despite the first product receiving market authorization in 2012, viral vector manufacturing has still not reached the level of maturity of biologics and is still highly susceptible to process uncertainties, such as viral titers and chromatography yields. This was exacerbated by the COVID-19 pandemic when viral vector manufacturers were challenged to respond to the global demand in a timely manner. A key reason for this was the lack of a systematic framework and approach to support capacity planning under uncertainty. To address this, we present a methodology for: (i) identification of process cost and volume bottlenecks, (ii) quantification of process uncertainties and their impact on target key performance indicators, and (iii) quantitative analysis of scale-dependent uncertainties. We use global sensitivity analysis as the backbone to evaluate three industrially relevant vector platforms: adeno-associated, lentiviral, and adenoviral vectors. For the first time, we quantify how operating parameters can affect process performance and, critically, the trade-offs among them.Results indicate a strong, direct proportional correlation between volumetric scales and propagation of uncertainties, while we identify viral titer as the most critical scale-up bottleneck across the three platforms. The framework can de-risk investment decisions, primarily related to scale-up and provides a basis for proactive decision-making in manufacturing and distribution of advanced therapeutics.

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Production of Lentiviral Vectors Using Suspension Cells Grown in Serum-free Media

Cited by 73 publications

References 65 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

Route of 41BB/41BBL Costimulation Determines Effector Function of B7-H3-CAR.CD28ζ T Cells

Characterization of key manufacturing uncertainties in next generation therapeutics and vaccines across scales

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