Effect of medium sugar source on the production of retroviral vectors for gene therapy

Abstract

The production of gene therapy retroviral vectors presents many difficulties, mainly due to vector instability and low cell productivities hampering the attainment of high titers of infectious viral vectors. The objective of this work is to increase the production titers of retroviral vectors by manipulating the sugar carbon sources used in bioreaction. Four sugars were tested (glucose, galactose, sorbitol, and fructose) on an established Moloney murine leukemia virus (MoMLV) producer cell line. Galactose and sorbitol did not support cell growth or vector production. Glucose supplemented at 25 mM supported the highest cell growth; however, the use of glucose or fructose at 83 and 140 mM have shown to improve the infectious vector titer three to fourfold. The reasons for the titer improvements were further analyzed and, although, the cell-specific productivity in viral transgene RNA and reverse transcriptase were augmented 5- and 6-fold for glucose at 140 mM and 14- and 16-fold for fructose at 140 mM, comparing with glucose at 25 mM, these increases did not seem sufficient to account for the 14- (140 mM glucose) and 32- (140 mM fructose) fold increment obtained for the infectious particles-specific productivity. Further accounting the enhancement in the titers was the improvement in the viral stability, the half-life of the vectors was enhanced by 30-60%. This resulted in a product quality with a superior ratio of infectious to total particles, thus reducing the most problematic contaminant in the production of retroviral vectors, non-infectious retroviral particles.

Removal of envelope protein‐free retroviral vectors by anion‐exchange chromatography to improve product quality

“…In addition, RVs biological activity is intrinsically very susceptible to environmental conditions, mainly temperature [6,7]. Consequently, retroviral supernatants have a large proportion of noninfectious vectors, the ratio of total to IPs usually being above 100:1 [7,8]. The presence of noninfectious vectors at such high levels may result in transduction efficiency inhibition, due to interference with viral receptors [9], and also constitute an extra bio-burden that might produce some immunological response in the patients [7, 10 -12].…”

Effect of osmotic pressure on the production of retroviral vectors: Enhancement in vector stability

“…Figure 2 and Table I strongly confirm that, independently of the osmotic agent used, the stability of the vector increased with increasing osmotic pressures; the enhancement of stability reaches a maximum near 450 mOsm/kg. The increments verified in the vector stability were not due to chemical protective effects per se: (i) because the infectivity kinetics assays were performed in diluted medium without osmotic agents to ensure that the intrinsic vector stability was being measured and (ii) because it was previously demonstrated that, in the range of sugar concentrations used, there was no chemical stabilizing influence (Coroadinha et al, 2006). The biophysical explanation for this viral stability enhancement can be obtained from the viral membrane lipid composition.…”

“…A second strategy to increase the medium osmolality was the use of the alternative sugar source fructose (instead of glucose) at high concentrations. It originates low amounts of lactate and has been shown previously to enhance the vector production and stability at high concentrations (Coroadinha et al, 2006;Gény-Fiamma et al, 2004;Merten et al, 2001b). Figure 1 depicts 24 h production titers obtained for the different media containing different osmolytes and osmolalities, after a 4 day period of cell adaptation to the medium used.…”

“…Previous studies from our laboratory have shown an important increase in the intrinsic MoMLV retroviral vector stability when production was carried out at high concentrations of glucose or fructose (Coroadinha et al, 2006). Such high concentrations of sugars can have the drawback of leading to the production of high amounts of lactate.…”

Retroviral vectors for clinical immunogene therapy are stable for up to 9 years

“…This property is dictated by the host cell and the virus production conditions, including temperature, 6,7 osmotic pressure and producer cell line metabolism. [8][9][10] In addition, downstream processing, formulation and storage temperature further determine the half-life of retroviral vectors. As a result, the variation in reported vector half-lives, which is generally represented by infectious titers, can be significant and range from 2 to 9 h, 8 to 300 h and 19 days to 18 months when stored at 37, 4 and À80 1C, respectively.…”