Scalable Production of AAV Vectors in Orbitally Shaken HEK293 Cells

Abstract

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells.

Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies

“…4 As the clinical potential of rAAV is being recognized, challenges in the scale-up of its biomanufacturing process without compromising potency, purity, and safety are becoming increasingly relevant. 5 , 6 , 7 …”

“…4 As the clinical potential of rAAV is being recognized, challenges in the scale-up of its biomanufacturing process without compromising potency, purity, and safety are becoming increasingly relevant. [5][6][7] The development of manufacturing platforms for biopharmaceuticals requires identification of the critical quality attributes (CQAs) of the product and characterization of the functional relationship between CQAs and process parameters. Defined as "a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality," 8 a CQA is an attribute-broadly corresponding to the product's identity, potency, purity, or safety-with severe impacts on product quality and efficacy if outside its target range.…”

Improvement of Cerebral Ischemia-Reperfusion Injury via Regulation of Apoptosis by Exosomes Derived from BDNF-Overexpressing HEK293

“…Human embryonic kidney cells are where a line of cells known as HEK293 cells are acquired. HEK293 cells hardly exhibit endogenous receptors needed for extracellular ligands that are commonly used to express foreign genes and moderately simple to transfect [11,12]. And it is easier to cultivate than stem cells.…”

Effect of design geometry, exposure energy, cytophilic molecules, cell type and load in fabrication of single-cell arrays using micro-contact printing

“…Human umbilical vein endothelial cells (HUVECs) are used as a laboratory model system for the study of the function and pathology of endothelial cells (e.g., angiogenesis) 40 . Adeno-associated virus (AAV) AAV-293 cells are Human embryonic kidney 293 cells (HEK293) optimised for packaging of AAV virions and support high levels of viral protein expression 41 Studies performed on cancer-derived cell lines like U87 (Glioblastoma 42 ), OHS (Osteosarcoma 43 ), SW480 (Colon 44 ), PC3 (Prostate 45 ), Hep-G2 (Liver 46 ), and AY-27 (Bladder 47 ), enable researchers to test hypotheses and thus improve the efficacy of cancer treatments.…”