المؤلفون: Sunderji, R., Piret, J. M., Kennard, M. L.

المصدر: Biotechnology and Bioengineering ; volume 55, issue 1, page 136-147 ; ISSN 0006-3592 1097-0290

الإتاحة: https://doi.org/10.1002Test/(sici)1097-0290(19970705)55:1%3C136::aid-bit14%3E3.0.co%3B2-k

المؤلفون: Kennard, M. L., Piret, J. M.

المصدر: Biotechnology and Bioengineering ; volume 47, issue 5, page 550-556 ; ISSN 0006-3592 1097-0290

الوصف: The influence of the microcarrier type on the performance of a controlled release process used to produce a recombinant glycosyl‐phosphatidylinositol anchored protein was investigated. Chinese hamster ovary (CHO) cells expressing the human melanoma tumor antigen (p97) were cultured in 10% serum on Cultispher‐GH porous microcarriers and then, for protein production, maintained in 2% serum. Cells were harvested every 48 h and p97 was recovered at 90 μg/mL and 40% purity. Harvested p97 concentrations were increased by harvestingfrom spheroid (241 μg/mL) and smaller porous microcarrier, Cultispher‐G (167 μg/mL) cultures. The low total cell specific p97 production of cells cultured on Cultispher‐GH was due to necrosis of cells within the beads, decreased p97 expression of the immobilized cells, dilution by the liquid (up to 40% volume) associated with settled beads, and incomplete recovery of p97 from within the beads. Cells cultured on solid microcarriers, Cytodex‐1, had the highest cell viability and cell specific p97 production, It is recommended that a two‐stage cyclic harvesting process of cells cultured on small Cultispher‐G or on Cytodex‐1 beads would minimize protein loss and maximize cell specific protein recovery. © 1995 John Wiley & Sons Inc.

الإتاحة: https://doi.org/10.1002/bit.260470507Test

المؤلفون: Guarna, M. M., Fann, C. H., Busby, S. J., Walker, K. M., Kilburn, D. G., Piret, J. M.

المصدر: Biotechnology and Bioengineering ; volume 46, issue 1, page 22-27 ; ISSN 0006-3592 1097-0290

الوصف: The secretion rate of activated protein C (APC) by BHK cells was increased 35‐fold by increasing the cDNA copy number per cell from 50 to 240. In this range, the relation between APC secretion and cDNA copy number was not linear and the rate of APC secretion per cDNA copy increased sevenfold. This apparent cooperative effect of multiple cDNA copies could be related to their integration in tandem. For cDNA copy numbers higher than 240, the APC secreation rate per cDNA and per cell decreased dramatically. The γ‐carboxylation of glutamic acid residues, a posttranslational modification required for APC biological activity, was also investigated. The proportion of APC that was fully γ‐carboxylated decreased as the secretion rate of APC increased. © 1995 John Wiley & Sons, Inc.

الإتاحة: https://doi.org/10.1002/bit.260460104Test

المؤلفون: Taylor, D. G., Piret, J. M., Bowen, B. D.

المصدر: AIChE Journal ; volume 40, issue 2, page 321-333 ; ISSN 0001-1541 1547-5905

الوصف: A mathematical model has been developed to predict the coupled hydrodynamics and high‐molecular‐weight protein transport in mammalian‐cell hollow‐fiber bioreactors (HFBRs). The analysis applies to reactors with isotropic ultrafiltration membranes under startup conditions when the extracapillary space (ECS) is essentially unobstructed by cells. The model confirms the experimental finding that secondary ECS flows, engendered by the primary flow in the fiber lumens, can cause significant downstream polarization of ECS proteins at typical mammalian‐cell HFBR operating conditions. It also reveals that the osmotic activity of the proteins, by curtailing transmembrane fluid fluxes, can influence strongly the outcome of the polarization process. In fact, at order‐of‐magnitude higher protein concentrations and/or lower recycle flow rates, the secondary flow velocities can be reduced by as much as six orders‐of‐magnitude throughout the ECS, thereby virtually eliminating the polarization problem. This result has important implications for improved reactor startup procedures.

الإتاحة: https://doi.org/10.1002/aic.690400211Test

المؤلفون: Kennard, M. L., Piret, J. M.

المصدر: Biotechnology and Bioengineering ; volume 44, issue 1, page 45-54 ; ISSN 0006-3592 1097-0290

الوصف: Recombinant proteins were harvested from Chinese hamster ovary (CHO) cells by a controlled release process, which increased the purity and concentration of the harvested protein. Recombinant human melano‐transferrin (p97) was expressed linked to the outer surface of CHO cells by a glycosyl‐phosphatidylinositol (GPI) membrane anchor. Cells were grown to confluence in T‐flask culture, and the p97 harvested by replacing the growth medium for 30 min with phosphate‐buffered saline (PBS) containing 10 mU/mL phosphatidylinositol‐phospholipase C (PI‐PLC). The GPI anchor was selectively cleaved by PI‐PLC. In fresh medium, the CHO cells regained over 95% of their p97 expression within 40 h. The process was repeated for eight harvests. Harvested protein concentrations varied from 1.5 to 3.8 μg/mL due to difficulties in maintaining stable confluent T‐flask cultures. Harvesting from cells growing on porous microcarriers was investigated to increase p97 product concentrations and to overcome culture stability problems. Semicontinuous cultures were maintained in spinners for up to 76 days with average bioreactor cell densities of over 10 7 cell/mL. The p97 was harvested at up to 100 μg/mL and 30% purity with protein production remaining stable for 4 harvest cycles. Production of high levels of p97 from CHO cells was maintained at 0.5% serum. © 1994 John Wiley & Sons, Inc.

الإتاحة: https://doi.org/10.1002/bit.260440108Test

المؤلفون: DEMAIN, A. L., PIRET, J. M., YU, H., COQUE, J.‐J., LIRAS, P., MARTIN, J. F.

المصدر: Annals of the New York Academy of Sciences ; volume 721, issue 1, page 117-122 ; ISSN 0077-8923 1749-6632

الإتاحة: https://doi.org/10.1111Test/j.1749-6632.1994.tb47383.x

المؤلفون: Kennard, M. L., Food, M. R., Jefferies, W. A., Piret, J. M.

المصدر: Biotechnology and Bioengineering ; volume 42, issue 4, page 480-486 ; ISSN 0006-3592 1097-0290

الوصف: A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol–phospholipase C (PI–PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI–PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re‐expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44‐day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins. © 1993 John Wiley & Sons, Inc.

الإتاحة: https://doi.org/10.1002/bit.260420411Test

المؤلفون: LEE, D. W., PIRET, J. M., GREGORY, D., HADDOW, D. J., KILBURN, D. G.

المصدر: Annals of the New York Academy of Sciences ; volume 665, issue 1, page 137-145 ; ISSN 0077-8923 1749-6632

الإتاحة: https://doi.org/10.1111Test/j.1749-6632.1992.tb42581.x

المؤلفون: Piret, J. M., Devens, D. A., Cooney, C. L.

المصدر: The Canadian Journal of Chemical Engineering ; volume 69, issue 2, page 421-428 ; ISSN 0008-4034 1939-019X

الوصف: This work has investigated high density CRL‐1606 hybridoma cell culture in the shell‐side of ultrafiltration hollow fiber bioreactors. Oxygen depletion was measured in the recycle stream, and has been identified as the critical, axial scale‐limiting factor in these reactors. The composition of the extracapillary space has been studied by rapidly freezing these reactors in liquid nitrogen to recover samples of the shell‐side environment. The accumulation of inhibitory metabolites should have only a marginal impact on growth and product formation of this hybridoma cell line. Oxygen depletion is expected to be the major limiting factor for both the axial and radial scale‐up of hollow fiber bioreactors.

الإتاحة: https://doi.org/10.1002/cjce.5450690204Test

المؤلفون: Bentzen, G., Piret, J. M., Daher, E., Demain, A. L.

المصدر: Applied Microbiology and Biotechnology ; volume 32, issue 6, page 708-710 ; ISSN 0175-7598 1432-0614

مصطلحات موضوعية: Applied Microbiology and Biotechnology, General Medicine, Biotechnology

الإتاحة: https://doi.org/10.1007/bf00164745Test