Bioreactor production of recombinant herpes simplex virus vectors.
Serotypical application of herpes simplex virus (HSV) vectors to gene therapy (type 1) and prophylactic vaccines (types 1 and 2) has garnered substantial clinical interest recently. HSV vectors and amplicons have also been employed as helper virus constructs for manufacture of the dependovirus adeno-associated virus (AAV). Large quantities of infectious HSV stocks are requisite for these therapeutic applications, requiring a scalable vector manufacturing and processing platform comprised of unit operations which accommodate the fragility of HSV. In this study, production of a replication deficient rHSV-1 vector bearing the rep and cap genes of AAV-2 (denoted rHSV-rep2/cap2) was investigated. Adaptation of rHSV production from T225 flasks to a packed bed, fed-batch bioreactor permitted an 1100-fold increment in total vector production without a decrease in specific vector yield (pfu/cell). The fed-batch bioreactor system afforded a rHSV-rep2/cap2 vector recovery of 2.8 x 10(12) pfu. The recovered vector was concentrated by tangential flow filtration (TFF), permitting vector stocks to be formulated at greater than 1.5 x 10(9) pfu/mL.
Knop
David R
DR
Applied Genetic Technologies Corporation (AGTC), Alachua, Florida 32615, USA. dknop@agtc.com
Harrell
Heather
H
D000818:Animals; D019149:Bioreactors / Q000821:virology*; D018929:Cell Culture Techniques / Q000379:methods; D002522:Chlorocebus aethiops; D004274:DNA, Recombinant / Q000235:genetics; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D018139:Simplexvirus / Q000235:genetics / Q000254:growth & development*; D013696:Temperature; D014357:Trypsin / Q000378:metabolism; D014709:Vero Cells
D004274:DNA, Recombinant; D014357:Trypsin