Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector
| Autor: | Richard Jude Samulski, Stephen M Soltys, Joshua C Grieger |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
viruses Genetic Vectors Biology medicine.disease_cause Culture Media Serum-Free law.invention 03 medical and health sciences Bioreactors law Batch Cell Culture Techniques Drug Discovery Cell Adhesion Genetics medicine Humans Vector (molecular biology) Molecular Biology Adeno-associated virus Pharmacology Vascular Endothelial Growth Factor Receptor-1 HEK 293 cells Transfection Dependovirus Virology HEK293 Cells 030104 developmental biology Cell culture Recombinant DNA Molecular Medicine Original Article Cell bank |
| Zdroj: | Molecular Therapy. 24:287-297 |
| ISSN: | 1525-0016 |
| Popis: | Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 10(5) vector genome containing particles (vg)/cell or greater than 1 × 10(14) vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1-6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (90% full particles), provides postpurification yields (1 × 10(13) vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered into patients. In addition, we report a minimum of a fivefold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection. |
| Databáze: | OpenAIRE |
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