Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA
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ORIGINAL ARTICLE
Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA Xin-An Lu . Ting He . Zhihai Han . Yanping Ding . Liang Zhao . Guanghua Liu . Floris De Smet . Xiaojun Huang . Danqing Chen . Feifei Qi . Xiangyu Zhao
Received: 18 March 2020 / Accepted: 16 October 2020 Ó Springer Nature B.V. 2020
Abstract The supercoiled circular (SC) topology form of plasmid DNA has been regarded to be advantageous over open circular or linearized analogue in transfection and expression efficiency, and therefore are largely demanded in the biopharmaceutical manufacturing. However, production of highpurity SC plasmid DNA would result in high manufacturing cost. The effect of SC proportion in plasmid DNA on the quality of packaged lentiviral vectors has never been reported. In this study, we established an Xin-An Lu, Ting He and Zhihai Han are co-first authors. X.-A. Lu T. He Y. Ding L. Zhao G. Liu D. Chen F. Qi (&) Immunochina Pharmaceuticals Co., Ltd, 100089 Beijing, China e-mail: [email protected]
efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serumfree media, and the lentiviral titer was not associated with the proportion of SC plasmid DNA. Plasmids DNA with different proportion of SC, open-circular, and linearized forms were prepared using the thermal denaturation method, and were transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, but not from HEK293SF cells, was significantly impaired when the proportion of SC plasmid DNA decreased from 60–80% to South Street of Xizhimen, Xicheng District, 100044 Beijing, China e-mail: [email protected]
Z. Han Department of Pulmonary and Critical Care Medicine, The Sixth Medical Center of PLA General Hospital, 100048 Beijing, China F. De Smet Sartorius Stedim North America Inc., 565 Johnson Avenue, Bohemia, New York 11716, USA X. Huang X. Zhao (&) Peking University People’s Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, No. 11
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Cytotechnology
30–40%. Further decrease of SC plasmid proportion to 3% led to a dramatic reduction of lentiviral titer no matter the packaging cell line was. However, lentiviral vectors from HEK293SF cells still showed a high titer even when the proportion of SC plasmid DNA was 3%. This study demonstrated that extremely high proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at least under our manufacturing process. Keywords Supercoiled circular plasmid DNA Proportion Lentiviral vector Titer Suspension cell culture
Introduction Adoptive transfer of genetically engineered cells has become a promising strategy for immunotherapy and gene therapy. Vectors (including both viral and nonvira
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