Process intensification for lentiviral vector manufacturing using tangential flow depth filtration

Tona, Robert M.; Shah, Reeti; Middaugh, Kimberly; Steve, Justin; Marques, João Carlos; Roszell, Blair R.; Jung, Cindy

doi:10.1016/j.omtm.2023.02.017

Cited by 10 publications

(25 citation statements)

References 40 publications

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“…Low TMP values (below 0.3 psi) for both TFDF runs, even after virus infection, high cell retention efficiencies (>99%), and a low turbidity in the permeate (>95% reduction) indicated minimal particle breakthrough. Previous studies already demonstrated the applicability of the TFDF system for continuous harvest of AAV and LV (Mendes et al 2022 ; Tona et al 2023 ; Tran and Kamen 2022 ). As expected, we also achieved equal concentrations of infectious virus in both the bioreactor and permeate sample, taken at the same time.…”

Section: Discussionmentioning

confidence: 99%

“…Product retention of hollow-fiber membranes is a well-known challenge of ATF-based cell culture productions, preventing continuous harvest of, e.g., viral particles (Genzel et al 2014 ; Hadpe et al 2017 ; Nikolay et al 2020 ; Nikolay et al 2018 ; Tona et al 2023 ; Vázquez-Ramírez et al 2019 ). To evaluate the applicability of hollow-fiber membranes for the production of fusogenic oncolytic viruses forming large muti-nucleated syncytia, a commercially available 0.65-μm mPES membrane with a lumen size bigger than previously observed syncytia (120–140 μm) was selected, connected to a mATF, and characterized for virus retention.…”

Section: Discussionmentioning

confidence: 99%

“…By the establishment of perfusion cultures, where depleted medium is continuously exchanged with fresh medium, while the cells are retained in the bioreactor by the use of a cell retention device, even higher cell concentrations can be achieved. Process intensification in perfusion mode to increase virus titers, cell-specific virus yields (CSVYs), space-time yield (STY), and volumetric virus productivity (VVP) have already been elaborated for different viruses and vectors such as for Zika virus (Nikolay et al 2018 ), influenza A virus (IAV) (Wu et al 2021 ), lentiviral vectors (LV) (Tran and Kamen 2022 ; Tona et al 2023 ), adeno-associated virus (AAV) (Mendes et al 2022 ), rVSV-COV-2 (Yang et al 2023 ), rVSV-NDV (Göbel et al 2023a ), and modified vaccinia virus Ankara (Gränicher et al 2021a ; Gränicher et al 2021b ). However, by targeting higher cell concentrations, the CSVY might be decreased (high-cell density effect) thus lowering the STY and VVP (Bock et al 2011 ; Nadeau and Kamen 2003 ).…”

Section: Introductionmentioning

confidence: 99%

“…For high-cell concentration cultivations, membrane-based systems, i.e., alternating tangential flow (ATF) and tangential flow filtration (TFF) modules are widely employed. One major drawback of these systems is the risk of filter fouling (despite the self-cleaning backflush of the ATF) and retention of virus particles, which leads to unwanted accumulation of virus inside the bioreactor within the cell environment until full harvest of the bioreactor broth is possible (Genzel et al 2014 ; Hadpe et al 2017 ; Nikolay et al 2020 ; Nikolay et al 2018 ; Tona et al 2023 ; Vázquez-Ramírez et al 2019 ). As virus release typically leads to cell lysis, cell debris and DNA increase over time, equally increasing viscosity of the culture broth and further increasing the risk of membrane clogging.…”

Section: Introductionmentioning

confidence: 99%

“…This has been recently demonstrated for the production of IAV defective interfering particles utilizing a tubular membrane (VHU, pore size ~10 μm) coupled to an ATF system (Hein et al 2021 ). Using tangential flow depth filtration (TFDF) perfusion modules (pore size 2–5 μm), continuous harvest of LV as well as AAV has been already shown (Mendes et al 2022 ; Tona et al 2023 ; Tran and Kamen 2022 ). This allows for shorter residence times of infectious virus particles and the possibility to immediately store harvested material in cooled tanks to increase virus stability and, thus, virus yields and productivity.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration

Göbel,

Pelz,

Silva

et al. 2024

Appl Microbiol Biotechnol

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Cell culture-based production of vector-based vaccines and virotherapeutics is of increasing interest. The vectors used not only retain their ability to infect cells but also induce robust immune responses. Using two recombinant vesicular stomatitis virus (rVSV)-based constructs, we performed a proof-of-concept study regarding an integrated closed single-use perfusion system that allows continuous virus harvesting and clarification. Using suspension BHK-21 cells and a fusogenic oncolytic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV), a modified alternating tangential flow device (mATF) or tangential flow depth filtration (TFDF) systems were used for cell retention. As the hollow fibers of the former are characterized by a large internal lumen (0.75 mm; pore size 0.65 μm), membrane blocking by the multi-nucleated syncytia formed during infection could be prevented. However, virus particles were completely retained. In contrast, the TFDF filter unit (lumen 3.15 mm, pore size 2–5 μm) allowed not only to achieve high viable cell concentrations (VCC, 16.4–20.6×106 cells/mL) but also continuous vector harvesting and clarification. Compared to an optimized batch process, 11-fold higher infectious virus titers were obtained in the clarified permeate (maximum 7.5×109 TCID50/mL). Using HEK293-SF cells and a rVSV vector expressing a green fluorescent protein, perfusion cultivations resulted in a maximum VCC of 11.3×106 cells/mL and infectious virus titers up to 7.1×1010 TCID50/mL in the permeate. Not only continuous harvesting but also clarification was possible. Although the cell-specific virus yield decreased relative to a batch process established as a control, an increased space-time yield was obtained. Key points • Viral vector production using a TFDF perfusion system resulted in a 460% increase in space-time yield • Use of a TFDF system allowed continuous virus harvesting and clarification • TFDF perfusion system has great potential towards the establishment of an intensified vector production

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration

Göbel,

Pelz,

Silva

et al. 2024

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

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Generation of stable suspension producer cell lines for serum‐free lentivirus production

Klimpel,

Terrao,

Bräuer

et al. 2024

Biotechnology Journal

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The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV‐G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum‐free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self‐inactivating (SIN) LVs carrying a WAS‐T2A‐GFP construct at an average infectious titer of up to 4.64 × 107 TU mL−1 in a semi‐perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL−1 in a semi‐perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL−1 day−1 for up to 29 consecutive days in a non‐optimized 5 L stirred‐tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet‐off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

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Perfusion Process Intensification for Lentivirus Production Using a Novel Scale‐Down Model

Klimpel,

Pflüger‐Müller,

Cascallana

et al. 2024

Biotech & Bioengineering

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Process intensification has become an important strategy to lower production costs and increase manufacturing capacities for biopharmaceutical products. In particular for the production of viral vectors like lentiviruses (LVs), the transition from (fed‐)batch to perfusion processes is a key strategy to meet the increasing demands for cell and gene therapy applications. However, perfusion processes are associated with higher medium consumption. Therefore, it is necessary to develop appropriate small‐scale models to reduce development costs. In this work, we present the use of the acoustic wave separation technology in combination with the Ambr 250 high throughput bioreactor system for intensified perfusion process development using stable LV producer cells. The intensified perfusion process developed in the Ambr 250 model, performed at a harvest rate of 3 vessel volumes per day (VVD) and high cell densities, resulted in a 1.4‐fold higher cell‐specific functional virus yield and 2.8‐fold higher volumetric virus yield compared to the control process at a harvest rate of 1 VVD. The findings were verified at bench scale after optimizing the bioreactor set‐up, resulting in a 1.4‐fold higher cell‐specific functional virus yield and 3.1‐fold higher volumetric virus yield.

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Process intensification for lentiviral vector manufacturing using tangential flow depth filtration

Cited by 10 publications

References 40 publications

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration

Generation of stable suspension producer cell lines for serum‐free lentivirus production

Perfusion Process Intensification for Lentivirus Production Using a Novel Scale‐Down Model

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