Comparison of Different Bioreactor Systems for the Production of High Titer Retroviral Vectors

Abstract

Improved, human-based packaging cell lines allow the production of high-titer, RCR-free retroviral vectors. The utility of these cell lines for the production of clinical grade vectors critically depends on the definition of optimal conditions for scaled-up cultures. In this work, a clone derived from the TE Fly GALV packaging cell (Duisit et al. Hum. Gene Ther. 1999, 10, 189) that produces high titers of a lacZ containing retroviral vector with a Gibbon Ape Leukemia Virus envelope glycoprotein was used. This clone can produce (2-5) x 10(6) PFU cm(-3) in small scale cultures and has been evaluated for growth and vector production in different reactor systems. The performances of fixed bed reactors [CellCube (Costar) and Celligen (New Brunswick)] and stirred tank reactors [microcarriers and clump cultures] were compared. The cells showed a higher apparent growth rate in the fixed bed reactor systems than in the suspension systems, probably as a result of the fact that aggregation and/or formation of clumps led to a reduced viability and reduced growth of cells in the interior of the clumps. As a consequence, the final cell density and number were in average 3- to 7-fold higher in the fixed bed systems in comparison to the suspension culture systems. The average titers obtained ranged from 0.5 to 2.1 x 10(7) PFU cm(-3) for the fixed bed and microcarrier systems, while the clump cultures produced only (2-5) x 10(5) PFU cm(-3). The differences in titers reflect cell densities as well as specific viral vector production rates, with the immobilization and microcarrier systems exhibiting an at least 10-fold higher production rate in comparison to the clump cultures. A partial optimization of the culture conditions in the Celligen fixed bed reactor, consisting of a 9-fold reduction of the seeding cell density, led to a 5-fold increased vector production rate accompanied by an average titer of 3 x 10(7) PFU cm(-3) (maximum titer (4-5) x 10(7) PFU cm(-3)) in the fixed bed reactor. The performance evaluation results using mathematical models indicated that the fixed bed bioreactor has a higher potential for retroviral vector production because of both the higher reactor productivity and the lower sensitivity of productivity in relation to the changes in final retrovirus titer in the range of 3 x 10(6) to 15 x 10(6) PFU cm(-3).

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

“…Kang et al (2000) showed that daily vector production was approximately 30 times greater in a packed bed bioreactor (packed with FibraCel discs) than a microcarrier system utilizing 3 g l À1 Cytodex. Similarly, Merten et al (2001a) showed a 116% increase in the maximum virus titer and a 400% increase in daily vector production when comparing a microcarrier system (5 g l À1 cytodex 1) and a CelliGen packed bed bioreactor containing 70 g of FibraCel discs. Evaluation of a fluidized bed bioreactor (Cytopilot) was recently carried out by .…”

“…Production can be further enhanced through optimization of the culture conditions. Secondly, viruses are particles not small molecules, and have a very short half-life, typically 2-8 h at 37°C (Le Doux et al 1999;Merten et al 2001a). Maintaining their capability to infect target cells is essential.…”

“…Both types have been used to culture retrovirus producing cells. The solid carriers Cytodex 1 and 3 have been used to culture PA317 cells (Wu et al 2002) and the former has been used to culture TE Fly GA18 cells (Merten et al 2001a). The PA317 cultures were reported as achieving a maximum virus titer of 3.1 and 3.4 · 10 6 IP ml À1 on Cytodex 1 and 3, respectively, and TE Fly GA18 cells produced 2.0 · 10 7 IP ml À1 .…”

“…Packed bed bioreactors have demonstrated superior performance over microcarrier cultures for both wCRIP MFG-lacZ cells and TE Fly GA18 cells (Merten et al 1996(Merten et al , 2001a. Kang et al (2000) showed that daily vector production was approximately 30 times greater in a packed bed bioreactor (packed with FibraCel discs) than a microcarrier system utilizing 3 g l À1 Cytodex.…”

“…In addition to packed bed and fluidized bed bioreactors, fixed bed culture systems, such as the CellCube, have been used for retrovirus production (Kotani et al 1994;Merten et al 2001a;Wikstro¨m et al 2004). The performance of the CellCube is reported to be comparable with the Celligen packed bed reactor, with each systems producing an average of 2.1 · 10 7 and 0.55 · 10 7 IP ml À1 , respectively (Merten 2004).…”