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Economized large‐scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system
Author(s) -
Negrete Alejandro,
Yang Linda C.,
Mendez Andres F.,
Levy Justin R.,
Kotin Robert M.
Publication year - 2007
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1092
Subject(s) - multiplicity of infection , baculoviridae , genetic enhancement , yield (engineering) , biology , recombinant dna , bioreactor , vector (molecular biology) , cell culture , titer , virology , gene , physics , virus , genetics , botany , spodoptera , thermodynamics
Abstract Background The versatility of recombinant adeno‐associated vector (rAAV) as a gene delivery system is due to the vector's ability to transduce different cell types as well as dividing and non‐dividing cells. Large‐scale production of rAAV remains one of the major challenges for continued development of pre‐clinical and clinical studies, and for its potential commercialization. The baculovirus expression vectors ( BEVS ) and insect cells represent a potential method to produce rAAV economically at large scale. This technology uses three different BEV s (Bac‐Rep, Bac‐GFP, and Bac‐VP) each at a multiplicity of infection (MOI) of 3. We reported previously the production of rAAV at 40 L scale using a stirred‐tank bioreactor (STB). However, production in larger volumes is limited by the stability of the BEV s and amount of BEV s needed to achieve the target MOI of 3 per BEV . Here, the production parameters were optimized and the baculovirus stability was determined. Methods The stability of the three types of baculovirus used to produce rAAV was determined for six expansion passages by protein expression analysis. To economize baculovirus, MOI and cell density at time of infection (TOI) were evaluated initially at small scale and then applied to the 10 L scale. Results An MOI = 0.03 and TOI cell density of 1 × 10 6 cells/mL produced high titer rAAV without comprising yield. To confirm the scalability of the process, rAAV was produced in a 10 L STB using the optimized parameters obtaining a 10× increase in yield (∼1 × 10 14 rAAV DNAse‐resistant particles per liter). Conclusion These findings contribute to the process development for large‐scale production of rAAV for gene therapy applications and its commercialization. Copyright © 2007 John Wiley & Sons, Ltd.