Evaluation of a Serum-free Medium for the Production of rAAV-2 using HeLa Derived Producer Cells

Abstract

During the last decade, recombinant AAVs have become of increasing interest for gene therapy. Clinical trials have been conducted following promising in vivo evaluations, thus leading laboratories to adapt their production systems for larger and higher quality demands. Classical transfection protocols seem difficult and cumbersome to adapt to a bioreactor scale. The use of stable producer cells appears as an attractive alternative, as this system requires only a single infection step to induce rAAV production. Furthermore, the switch to a serum-free medium is an interesting strategy to increase the biosafety level to satisfy clinical grade requirements for gene therapy products. Here, we have combined both approaches and evaluated different rAAV producer clones in a serum-free medium. We first evaluated the cell growth in a serum-free medium and then did a partial optimisation of the medium composition to obtain vector yields as close as possible to the yields obtained in a classical serum containing medium. Different helper viruses, multiplicity of infection, times of infection and harvest have been compared in small scale cultures in order to determine the optimal settings which were then transferred and evaluated in suspension cultures in spinner flasks. The yields obtained in this system were similar to or at most 2 times lower than those obtained in a serum-containing medium. The scale-up of such a production system as well as the use of high cell density perfusion culture systems will probably lead to considerably higher yields than those obtained in a classical process.

Engineering of baculovirus vectors for the manufacture of virion‐free biopharmaceuticals

“…This protease gene is an essential late viral gene, indispensable for the cleavage of several viral proteins, and thus necessary for the maturation and assembly of viral proteins and for release of virions from infected cells (Chen et al, 1993). In addition, the viral genome of the ΔPS‐adenovirus is replicated in infected non‐complementing target cells (Oualikene et al, 2000) and also AAV vector genome replication is enabled (Jenny et al, 2005).…”

Rational plasmid design and bioprocess optimization to enhance recombinant adeno‐associated virus (AAV) productivity in mammalian cells

“…In a further experiment, performance of anchorage‐ and serum‐dependent HEK293T cells was tested against well‐established HEK293SF‐3F6 suspension cells cultivated in a chemically defined medium . With regard to regulatory concerns when using animal‐derived components, serum‐free cultivation is a highly desirable goal for vector production . For this purpose, the new plasmid system (see above) was transfected into suspension cells.…”

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

“…Reactor scale possible Blouin et al (2004), Jenny et al (2005) A549 based producer cell, rAAV production induced by infection with a Ad ts sequently, a two stage process was developed where cells were cultured and infected at 37°C, before lowering the temperature to 33°C, 24 h post-infection. Extracellular virus production under these conditions increased 3-fold in early passage cells, and up to 10-fold in late passage cells .…”