Characterization of Lentiviral Vector Production Using Microwell Suspension Cultures of HEK293T-Derived Producer Cells

Abstract

ProSavin(®) is a lentiviral vector (LV)-based gene therapy for Parkinson's disease. ProSavin(®) is currently in a Phase I/II clinical trial using material that was generated by transient transfection of adherent human embryonic kidney (HEK)293T cells. For future large-scale productions of ProSavin(®), we have previously reported the development and characterization of two inducible producer cell lines, termed PS5.8 and PS46.2. PS46.2 has been successfully adapted to grow in suspension cultures. The present study describes the creation of a small-scale (<2 ml) microwell-based experimental platform for the parallel investigation of ProSavin(®) production using suspension-adapted PS46.2. This is combined with statistical design of experiments (DoE) techniques to enable rapid characterization of the process conditions that impact cell growth and LV production. The effects of postinduction period, microwell liquid fill volume, and concentration of inducer (doxycycline) on ProSavin(®) titer and the particle:infectivity (P:I) ratio was investigated using three rounds of DoE, in order to identify appropriate factor ranges and optimize production conditions. We identified an optimal "harvest window" between approximately 26-46 hr within which maximal titers of around 6×10(4) transducing units (TU)/ml were obtained (an approximately 30-fold improvement compared to starting microwell conditions), providing that the fill volume was maintained at or below 1 ml and the doxycycline concentration was at least 1.0 μg/ml. Insights from the microwell studies were subsequently used to rapidly establish operating conditions for ProSavin(®) production in a 0.5-L wave bioreactor culture. The information presented herein thus aids the design and evaluation of scalable production processes for LVs.

Bioprocess considerations for T‐cell therapy: Investigating the impact of agitation, dissolved oxygen, and pH on T‐cell expansion and differentiation

“…To analyse the data, viable cell concentration and percentage of CD8+ T CM cells from each run were entered into the Design Expert software. Data for each response factor was transformed as appropriate to approximate normal distribution (Guy, McCloskey, Lye, Mitrophanous, & Mukhopadhyay, 2013). The automatic model selection tool was used to keep only significant terms in the model.…”

Development of a high‐throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design

“…Scale‐down bioprocesses form the backbone of rapid bioprocess design, allowing for cost‐effective and rapid development of biopharmaceutical production processes. Specifically, small‐scale fermentation process design combined with multivariate data analysis techniques and subsequent process upscaling has proven to be effective for characterizing and optimizing industrial‐scale operations …”

“…Specifically, small-scale fermentation process design combined with multivariate data analysis techniques and subsequent process upscaling has proven to be effective for characterizing and optimizing industrial-scale operations. [1][2][3][4] Pichia pastoris, recently reassigned to the genus Komagataella, is a methanol assimilating yeast that has been receiving increased industrial attention over the past two decades due to its potential to produce a wide array of complex biopharmaceuticals. [5][6][7][8][9][10][11][12] P. pastoris offers the potential of heterologous protein secretion, however depending on physical and chemical properties of the protein of interest, this is not always a viable option.…”

The Committee for Advanced Therapies' of the European Medicines Agency Reflection Paper on Management of Clinical Risks Deriving from Insertional Mutagenesis

“…Such vector-based gene therapy medicinal products (GTMP) have been widely used to treat monogenic disorders, cancer, and infectious diseases (Rossi et al, 2007;Naldini, 2011). Integrating vectors are typically employed for ex vivo gene transfer in different cell types, such as hematopoietic stem cells (HSCs), lymphocytes, and epidermal cells (Mavilio et al, 2006;Ott et al, 2006;Aiuti et al, 2009;Cartier et al, 2009;Ciceri et al, 2009;Boztug et al, 2010;Cavazzana-Calvo et al, 2010;Hacein-Bey-Abina et al, 2010;Gaspar et al, 2011a;Candotti et al, 2012), although they are also used for in vivo approaches (Balaggan and Ali, 2012;Guy et al, 2013).…”