Scalable lentiviral vector production using stable producer cell lines in perfusion mode

Lentiviral vectors (LVs) are becoming an important tool in gene and cell therapy and are being utilized in several clinical studies for rare and more frequent genetic and acquired diseases, as well as in cancer therapies. However, two major challenges need to be overcome in order to generate enough material to treat patients: First, current production platforms result in low titers (stable producer cell lines from adherent cell lines) or are not amenable to large scale production (LV produced by transfection). Next, LVs are known to have a low temperature stability. To address these two challenges, the National Research Council Canada has developed packaging cell lines and stable producer cell lines for the production of LVs which can grow in suspension in serum-free media and produce LV in the 106 TU/ml range without optimization. Furthermore, productions are performed in perfusion mode in order to operate at high cell densities and address the low LV stability. Please click Additional Files below to see the full abstrac

Scalable lentiviral vector production using stable producer cell lines in perfusion mode

Lentiviral vectors (LVs) are becoming an important tool in gene and cell therapy and are being utilized in several clinical studies against genetic and acquired diseases, as well as in cancer therapies. To address the challenges linked to the generation of preclinical and clinical supply, the National Research Council Canada has developed packaging cell lines and stable producer cell lines for the production of LVs which can grow in suspension in serum-free media and produce LV in the 106 TU/ml range without optimization. We focus on the development of perfusion processes to both intensify the process and harvest produced LV rapidly

High-performance liquid chromatographic total particles quantification of retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein

NRC publication: Ye

High yield purification of functional baculovirus vectors by size exclusion chromatography

NRC publication: Ye

Monitoring Lentiviral Vector Production Kinetics Using Online Permittivity Measurements

Lentiviral vectors (LVs) are effective delivery vehicles that have been successfully used in gene and cell therapy. LVs are most commonly produced via the transient transfection of several plasmid constructs in adherent cell cultures. Recently, we described an efficient and scalable LV production in serum-free suspension cultures. To further facilitate the translation of LV-based interventions to the clinic, the robustness of the production process needs to be ensured to ultimately achieve a specified quality and quantity of LV production lots. However, routine processes are largely empirical, and strategies to monitor LV production kinetics in real-time have not yet been described. In this work, in situ real-time permittivity measurements were assessed to document the production of LVs. Characteristic process phases that were closely associated with LV production kinetics were identified. The permittivity signal evolution was interpreted by exploiting various independent online and offline monitoring measurements. Cellular membrane properties and, to a lesser extent, cell size were the main factors contributing to the permittivity variations. It is concluded that the permittivity-related parameters can be used for the detection of viral release, allowing real-time assessment of process performance. The technology should thus greatly facilitate process development and optimization.Peer reviewed: YesNRC publication: Ye

Size-exclusion chromatography purification of high-titer vesicular stomatitis virus G glycoprotein-pseudotyped retrovectors for cell and gene therapy applications

DA - 20030811IS - 1043-0342LA - engPT - Journal ArticleRN - 0 (DNA, Viral)RN - 0 (G protein, vesicular stomatitis virus)RN - 0 (Genetic Vectors)RN - 0 (Membrane Glycoproteins)RN - 0 (Viral Envelope Proteins)RN - EC 3.1.- (Deoxyribonucleases)SB - IMNRC publication: Ye

Particle quantification of influenza viruses by high performance liquid chromatography using CIM\uae anion exchange monolithic column

A high performance liquid chromatography (HPLC) method using a strong anion exchange monolithic column based on convective interaction media) CIM\uae technology was developed for the particle quantification of influenza viruses. The virus which was specifically detected by native fluorescence eluted in 5.55 min at 1.5 M NaCl gradient in 20 mM Tris-HCl + o.01% Zwittergent, pH 8.0 in a total analysis time of 13.5 min. The linearity of a curve was demonstrated for all inluenza virus investigated with a good correlation coefficient (R2) greater than 0.995. Among all the virus investigated, the detection limit of the method ranged between 2.07x108 and 4.35x109 while the quantification limit ranged between 6.90x108 and 1.45x1010 virus particle per ml (VP/ml), respectively. The intra- and inter-assay precision of the method were less than 5% and 10% respectively. The method which was developed using sucrose cushion purified influenza viruses was shown to be suitable in the analysis of cell culture supernatants making it an ideal in-process monitoring tool to facilitate the development of influenza vaccine manufacturing processes.posterNRC publication: Ye

Retroviral vector production using suspension-adapted 293GPG cells in a 3L acoustic filter-based perfusion bioreactor

DA - 20061003IS - 0006-3592 (Print)LA - engPT - Journal ArticlePT - Research Support, Non-U.S. Gov'tRN - 0 (Viral Envelope Proteins)SB - IMNRC publication: Ye

Rapid and reliable quantification of reovirus type 3 by high performance liquid chromatography during manufacturing of Reolysin

Reolysin, a human reovirus type 3, is being evaluated in the clinic as an oncolytic therapy for various types of cancer. To facilitate the optimization and scale-up of the current process, a high performance liquid chromatography (HPLC) method has been developed that is rapid, specific and reliable for the quantification of reovirus type 3 particles. Using an anion-exchange column, the intact virus eluted from the contaminants in 9.78 min at 350 mM NaCl in 50mM HEPES, pH 7.10 in a total analysis time of 25 min. The virus demonstrated a homogenous peak with no co-elution of other compounds as analyzed by photodiode array analysis. The HPLC method facilitated the optimization of the purification process which resulted in the improvement of both total and infectious particle recovery and contributed to the successful scale-up of the process at the 20 L, 40 L and 100 L production scale. The method is suitable for the analysis of crude virus supernatants, crude lysates, semi-purified and purified preparations and therefore is an ideal monitoring tool during process development and scale-up.Peer reviewed: YesNRC publication: Ye