Details
| Original language | English |
|---|---|
| Pages (from-to) | 5947-5961 |
| Number of pages | 15 |
| Journal | Applied Microbiology and Biotechnology |
| Volume | 107 |
| Issue number | 19 |
| Early online date | 5 Aug 2023 |
| Publication status | Published - Oct 2023 |
Abstract
Abstract: Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting.
Keywords
- Continuous viral vector harvesting, Gene therapy, High cell density cultivation, Murine leukemia viral vectors, Perfusion cultivation, Process intensification
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
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In: Applied Microbiology and Biotechnology, Vol. 107, No. 19, 10.2023, p. 5947-5961.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Production of retroviral vectors in continuous high cell density culture
AU - Hein, Marc D.
AU - Kazenmaier, Daniel
AU - van Heuvel, Yasemin
AU - Dogra, Tanya
AU - Cattaneo, Maurizio
AU - Kupke, Sascha Y.
AU - Stitz, Jörn
AU - Genzel, Yvonne
AU - Reichl, Udo
N1 - Funding Information: The authors would like to thank Nancy Wynserski and Claudia Best for excellent technical assistance. For discussions and additional testing of the VHU membranes the authors would like to thank Ilona Behrendt and Sven Göbel. Funding Information: Open Access funding enabled and organized by Projekt DEAL. This work was supported by a grant of the German Federal Ministry of Education and Research, funding program “Forschung an Fachhochschulen”, contract number 13FH242PX6 to JS.
PY - 2023/10
Y1 - 2023/10
N2 - Abstract: Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting.
AB - Abstract: Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting.
KW - Continuous viral vector harvesting
KW - Gene therapy
KW - High cell density cultivation
KW - Murine leukemia viral vectors
KW - Perfusion cultivation
KW - Process intensification
UR - http://www.scopus.com/inward/record.url?scp=85166911235&partnerID=8YFLogxK
U2 - 10.1007/s00253-023-12689-9
DO - 10.1007/s00253-023-12689-9
M3 - Article
C2 - 37542575
AN - SCOPUS:85166911235
VL - 107
SP - 5947
EP - 5961
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
SN - 0175-7598
IS - 19
ER -