ABSTRACT
Lentiviral vectors (LVs) are a powerful tool for gene and cell therapy and human embryonic kidney cells (HEK293) have been extensively used as a platform for production of these vectors. Like most cells and cellular tissues, HEK293 cells release extracellular vesicles (EVs). EVs released by cells share similar size, biophysical characteristics and even a biogenesis pathway with cell-produced enveloped viruses, making it a challenge to efficiently separate EVs from LVs. Thus, EVs co-purified with LVs during downstream processing, are considered "impurities" in the context of gene and cell therapy. A greater understanding of EVs co-purifying with LVs is needed to enable improved downstream processing. To that end, EVs from an inducible lentivirus producing cell line were studied under two conditions: non-induced and induced. EVs were identified in both conditions, with their presence confirmed by transmission electron microscopy and Western blot. EV cargos from each condition were then further characterized by a multi-omic approach. Nineteen proteins were identified by mass spectrometry as potential EV markers to differentiate EVs in LV preparations. Lipid composition of EV preparations before and after LV induction showed similar enrichment in phosphatidylserine. RNA cargos in EVs showed enrichment in transcripts involved in viral processes and binding functions. These findings provide insights on the product profile of lentiviral preparations and could support the development of improved separation strategies aimed at removing co-produced EVs.
Subject(s)
Extracellular Vesicles/metabolism , Genetic Vectors/biosynthesis , Genetic Vectors/genetics , HEK293 Cells/metabolism , Lentivirus/genetics , Biological Transport , Cell Culture Techniques , Chromatography, Liquid , Computational Biology/methods , Culture Media, Conditioned , Exosomes , Extracellular Vesicles/ultrastructure , Humans , Lipidomics , Mass Spectrometry , Proteomics/methodsABSTRACT
The production of lentiviral vectors (LVs) in human embryonic kidney 293 (HEK293) cells using serum-free medium in a suspension culture for the transduction of chimeric antigen receptor T-cells (CAR-T) can be achieved by different methods. This chapter describes LV production by transient transfection, induction of stable packaging cell lines, and induction of stable producer cell lines.
Subject(s)
Cell Culture Techniques , Culture Media, Serum-Free , Genetic Vectors/biosynthesis , Genetic Vectors/genetics , Lentivirus/genetics , T-Lymphocytes/metabolism , Transduction, Genetic , Humans , Immunotherapy, Adoptive , Receptors, Chimeric Antigen , T-Lymphocytes/immunology , TransfectionABSTRACT
During the scale-up of several Chinese Hamster Ovary (CHO) cell monoclonal antibody production processes, significant reduction of the antibody interchain disulfide bonds was observed. The reduction was correlated with excessive mechanical cell shear during the harvest operations. These antibody reduction events resulted in failed product specifications and the subsequent loss of the drug substance batches. Several methods were recently developed to prevent antibody reduction, including modifying the cell culture media, using pre- and post-harvest chemical additions to the cell culture fluid (CCF), lowering the pH, and air sparging of the harvested CCF (HCCF). The work described in this paper further explores the option of HCCF air sparging for preventing antibody reduction. Here, a small-scale model was developed using a 3-L bioreactor to mimic the conditions of a manufacturing-scale harvest vessel and was subsequently employed to evaluate several air sparging strategies. In addition, these studies enabled further understanding of the relationships between cell lysis levels, oxygen consumption, and antibody reduction. Finally, the effectiveness of air sparging for several CHO cell lines and the potential impact on product quality were assessed to demonstrate that air sparging is an effective method in preventing antibody reduction.
