Preclinical Proof-of-Concept, Analytical Development, and Commercial Scale Production of Lentiviral Vector in Adherent Cells

Abstract

The therapeutic efficacy of a lentiviral vector (LV) expressing the herpes simplex virus thymidine kinase (HSV-TK) was studied in an immunocompetent rat glioblastoma model. Intraperitoneal ganciclovir injections (50 mg/kg/day) were administered for 14 consecutive days, resulting in reduced tumor volumes as monitored by MRI. Survival analyses revealed a significant improvement among the LV-expressing HSV-TK (LV-TK)/ganciclovir-treated animals when compared to non-treated control rats. However, a limiting factor in the use of LV has been the suboptimal small-scale production in flasks. Our aim during the translation phase, prior to entering the final pre-clinical and early clinical phases, was to develop a scalable, robust, and disposable manufacturing process for LV-TKs. We also aimed to minimize future process changes and enable production upscaling to make the process suitable for larger patient populations. The upstream process relies on fixed-bed iCELLis technology and transient plasmid transfection. This is the first time iCELLis 500 commercial-scale bioreactor was used for LV production. A testing strategy to determine the pharmacological activity of LV-TK drug product by measuring cell viability was developed, and the specificity of the potency assay was also proven. In this paper we focus on upstream process development while showing analytical development and the proof-of-concept of LV-TK functionality.

Keywords: bioreactor; glioma; lentivirus; production; scale up; transfection.

Figure 1

Cell Viability of BT4C Rat Glioma Cells after GCV Treatment Measured at Days 3 and 6 after LV-TK Transduction, and the Efficacy of LV-TK/GCV in a Rat Malignant Glioma Model (A and B) BT4C cells were either non-transduced or transduced with treatment vector. Cell viability was analyzed with MTS assay 3 days (A) and 6 days (B) post-transduction. Experiments were carried out with three or more replicates. (C) BT4C tumor growth was monitored by MRI. Images represent the average tumor size of the specific time point. (D) Group average tumor volumes in pixels as measured from the MRI images. (E) Survival of the LV-TK treated rats bearing malignant gliomas. NT, non-transduced; MOI, multiplicity of infection; LV-TK, lentivirus vector expressing herpes simplex virus thymidine kinase; GCV, ganciclovir. Results are expressed as mean ± SEM, *p > 0.05, **p > 0.01, ***p > 0.001.

Figure 2

LV Productivity and Residual DNA in iCELLis Nano Runs and Large Scale Runs (A and B) vp/mL (A) and TU/mL (B) in the first iCELLis large scale run (run 1). (C) Residual DNA concentration in iCELLis Nano and large-scale runs (runs 1 and 2) before and after benzonase treatment. (D–G) vp/mL (D) and TU/mL (E) in iCELLis Nano runs 1–5 before and after the benzonase treatment, and (F) vp/mL and (G) TU/mL in the second large-scale run (run 2). AB, after benzonase; TU, transductive units; vp, viral particle.

Figure 3

Comparison between Flow Cytometry and qPCR-Based Titers Shows Correlation between the Results (Correlation Coefficient 0.62), but the GFP Titers Are Generally Increased Compared to the qPCR Values

Figure 4

Potency Assay Testing (A) Effect of LV-GFP with or without GCV. Data are presented as mean ± SEM, n = 64/128. Statistical analysis is one-way ANOVA, ***p > 0.001. (B) Effect of LV-TK with GCV. Bar plot presents mean values, line plot S-curve fitting presented as mean ± SD, n = 59–60.