Optimized conditions for the production of recombinant amphotropic retroviral vector preparations

Abstract

The production and stability of recombinant retroviral vecwas reached between retroviral vector production and tors was examined at various temperatures. The two studdecay. Maximal accumulated recombinant retroviral titers ied recombinant retroviral vectors, based on different packwere five-to ten-fold increased after a medium incubation aging cell lines, exhibited a four-fold increased half-life at at 32°C as compared to 37°C. Furthermore, multiple cycles 32°C as compared to 37°C. Surprisingly, this increased of freezing and thawing of retroviral vector supernatants stability at 32°C was only observed within a very narrow hardly affected the recombinant retroviral vector titer, indetemperature window. At 30°C and 34°C, retroviral vector pendent of the presence of serum. This knowledge on half-lives were quite similar to that at 37°C. Regardless of characteristics of recombinant retroviral vectors has practithe vector half-life, retroviral vectors accumulated in the cal implications for the manufacturing of these viruses for culture medium for a period of 48 h before an equilibrium clinical gene therapy protocols.Keywords: retroviral vector titer; retroviral vector stability; gene therapyRecombinant retroviral vectors are widely used to introretroviral vectors at different temperatures, culture volumes, and producer cell densities. duce genes into cells. At present recombinant retroviral vectors are being employed in the majority of clinicalIn the design of our experiments we included intermediate storage steps in liquid nitrogen. Therefore, we gene therapy protocols. The success of such therapies is critically dependent on the transduction efficiency.first investigated the effect of repeated freezing (in liquid nitrogen) and thawing (at 37°C) of vector supernatant Among other factors, this transduction efficiency will be influenced by the titer of vector preparations employed.batches on their retroviral vector titer. The vector supernatant batches were obtained by performing a 2 h harvest Several methods have been described to obtain high recombinant retroviral vector titers. These methods from the recombinant amphotropic neo-vector producing cell line POPA/neo-4 at 37°C. One batch contained 10% include redesign of the retroviral vector, 1-3 the generation of producer cells with increased recombinant retrovirus newborn calf serum (NBCS) and a second was kept serum-free. The vector supernatant batches were divided copy numbers, 4,5 changing the retroviral vector harvest procedure by adding sodium butyrate or dexamethasone over several ampoules that were frozen and thawed for different numbers of cycles, ranging from 0 to 16 times. to the culture medium 6,7 or by using a culture temperature of 32°C instead of 37°C, 6,8,9 and down-stream conNext, the end-point retroviral vector titer was determined on NIH/3T3 cells. As can be seen in Figure 1, the first centration of retroviral vectors in the vector supernatant batch. 9,10 In general, each of these methods results in an two to four cycles had the largest impact ...

A serum-free Vero production platform for a chimeric virus vaccine candidate

“…Although the specific reasons for this thermal sensitivity are not identified, amino acid changes in viral polymerases have produced PIV3 viruses that exhibit temperature-sensitive phenotypes (Feller et al 2000;Skiadopoulos et al 1999). Other virus systems have demonstrated similar thermal sensitivity: the cultivation of retrovirus packaging cells at 32°C instead of 37°C increased the yield of infectious virus particles by a maximum of 2-to 15-fold (Kaptein et al 1997;Kotani et al 1994;Lee et al 1996).…”

Artificial capillary culture: expansion and retroviral transduction of CD4+ T-lymphocytes for clinical application

“…Non-viable particles may come from several areas: (1) Viable retrovirus, which has a half-life of 4-8 h at 37°C 21,22 remains in the ECS, becoming thermally inactivated over time and resulting in an increase of non-viable particles in the culture environment. (2) The action of ultrafiltration through the hollow fibre pores results in shearing of the viral envelope, generating viral particles incapable of infection.…”

Suspension packaging cell lines for the simplified generation of T-cell receptor encoding retrovirus vector particles

“…Pizzato et al 12 and our earlier studies reported a half-life of the amphotropic MLV pseudotype produced in CEM and PA317 packaging cells, respectively, of approximately 4 h, whereas the same pseudotype produced in FLYA13 and CCRIP packaging cells showed a slightly higher stability. 17,15 GALV and MLV-10A1 pseudotypes produced in the NIH3T3-derived packaging cell lines PG13 and PT67 have half-lives of 13 h and 14 h, respectively. 15 TCR-retrovirus produced in Db-Jurkat/GP+GALV packaging cells efficiently transduced human T-cell lines, whereas the transduction of primary human T cells was lower.…”