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Scalable production of adeno‐associated virus type 2 vectors via suspension transfection
Author(s) -
Park Joon Young,
Lim ByungPil,
Lee Kyuhyun,
Kim YoungGun,
Jo EuiCheol
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20776
Subject(s) - transfection , hek 293 cells , polyethylenimine , cell culture , bioreactor , virus , adeno associated virus , suspension (topology) , gene delivery , recombinant dna , chemistry , biology , virology , gene , vector (molecular biology) , genetics , mathematics , organic chemistry , homotopy , pure mathematics
Vectors derived from adeno‐associated virus type 2 (AAV2) are promising gene delivery vehicles, but it is still challenging to get the large number of recombinant adeno‐associated virus (rAAV) particles required for large animal and clinical studies. Current transfection technology requires adherent cultures of HEK 293 cells that can only be expanded by preparing multiple culture plates. A single large‐scale suspension culture could replace these multiple culture preparations, but there is currently no effective co‐transfection scheme for generating rAAV from cells in suspension culture. Here, we weaned HEK 293 cells to suspension culture using hydrogel‐coated six‐well culture plates and established an efficient transfection strategy suitable for these cells. Then the cultures were gradually scaled up. We used linear polyethylenimine (PEI) to mediate transfection and obtained high transfection efficiencies ranging from 54% to 99%, thereby allowing efficient generation of rAAV vectors. Up to 10 13 rAAV particles and, more importantly, up to 10 11 infectious particles were generated from a 2‐L bioreactor culture. The suspension‐transfection strategy of this study facilitates the homogeneous preparation of rAAV at a large scale, and holds further potential as the basis for establishing a manufacturing process in a larger bioreactor. © 2006 Wiley Periodicals, Inc.