GMP Production of Biopharmaceuticals Using High Density, Fluidized-Bed Cell Culture Technology

Runstadler, Peter W.; Young, Michael W.

doi:10.1007/978-94-011-3550-4_13

Cited by 3 publications

References 2 publications

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“…First, reduced metabolism of producer cells may increase vims production per cell; cells grow slower at 32°C than at 37°C. Reports have been published on enhanced protein production from genetically engineered mammalian cells under low metabolism using reduced temperatures or limiting nutrients (Runstadler and Young, 1991;Zhang et at., 1993). This may also apply to rettoviral vectors.…”

Section: Discussionmentioning

confidence: 99%

Improved Methods of Retroviral Vector Transduction and Production for Gene Therapy

Kotani¹,

Newton²,

Zhang³

et al. 1994

Human Gene Therapy

360

280

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To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detectable replication-competent retroviruses. The purpose of this study was to optimize methods of retroviral vector production and transduction. Studies were conducted using 22 retroviral vector producer cell lines. Inactivation of retroviral vectors was greater at 37 degrees C than at 32 degrees C. A 5- to 15-fold increase of vectors was produced at 32 degrees C compared to 37 degrees C; the vector increase at 34 degrees C was intermediate. For example, PA317/G1Na.40 grew to a titer of 1.8 x 10(7) cfu/ml at 32 degrees C, compared to 5.0 x 10(5) cfu/ml at 37 degrees C. The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor. Retroviral vectors were concentrated 15-24 times with vector recovery ranging from 91 to 96% in a Pellicon tangential flow filtration system. Retroviral supernatants were successfully lyophilized. The combination of glucose or sorbitol with gelatin resulted in recovery rates of 64-83%. In studies on transduction by retroviral vectors, centrifugation of vector supernatants onto target cells significantly increased transduction efficiency as measured by vector titration for G418 resistance, fluorescence-activated cell sorting (FACS), and polymerase chain reaction (PCR) analyses. The combination of the above methods has significantly increased the growth and transduction by this vector system.

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Section: Discussionmentioning

confidence: 99%