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Effects of heat shock on the production of human erythropoietin from recombinant CHO cells (1992)

Tsao, Eric I. ; Bohn, Martie A. ; Omstead, Daniel R. ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 40 (1992), S. 1190-1196

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ISSN: 0006-3592

Keywords: erythropoietin ; recombinant protein ; heat shock ; protein synthesis ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: The production of recombinant proteins by mammalian cells demands a highly controlled environment for cell cultivation. Temperature stress represents a commonly encountered disturbance in both research and process environments. In this study, we examined the effects of heat shock on the expression of recombinant human erythropoietin (EPO) in a Chinese hamster ovary (CHO) cell line. Biosynthetic radiolabeling experiments indicated that the cells exposed to a 42°C/1-hour heat shock exhibit a transient reprogramming of transcription and translation characterized by the inhibition of protein synthesis and induction of heat shock proteins. The rate of protein synthesis decreased by 50% after the heat shock, while the rate of RNA synthesis increased by 50% initially and then quickly reduced to 80% of the control level. The protein and RNA synthesis rates were fully recovered in approximately 48 hours after the heat shock. However, we found that the expression of EPO was not arrested by the heat shock. The glycosylation patterns, as examined by isoelectric focusing, of either the culture supernatant or the purified EPO were not affected by the heat shock. In contrast, a 45°C/1-hour heat shock terminated RNA and protein synthesis immediately and caused culture death in 12 hours. Cellular responses to temperature stress were affected by the metabolic state of the cells; cells maintained in serum-free medium were more sensitive than cells growing exponentially in the presence of serum. We have also examined the kinetics of metabolic responses of the cells to heat shock with respect to nutrient uptake and metabolite accumulation. © 1992 John Wiley & Sons, Inc.

Additional Material: 9 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/bit.260401008

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Production of parvovirus B19 vaccine in insect cells co-infected with double baculoviruses (1996)

Tsao, Eric I. ; Mason, Michael R. ; Cacciuttolo, Marco A. ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 49 (1996), S. 130-138

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ISSN: 0006-3592

Keywords: recombinant baculovirus ; virus-like particles ; parvovirus B19 ; multiplicity of infection ; co-infection ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Recombinant human parvovirus B19 virus-like particles (VLPs), a candidate vaccine, were produced using the insect cell (Sf-9)-baculovirus (AcNPV) expression system. The synthesis and assembly of the particles in Sf-9 cells are directed by double infections with one recombinant virus (bacVP1) expressing the parvovirus minor viral protein VP1 and a second virus (bacVP2) expressing the major viral protein VP2. Previous animal studies demonstrated that the polypeptide composition of the VLPs strongly affects the elicitation of virus neutralizing antibodies. The key factor controlling the production of an immunologically potent product in bioreactors was identified to be the multiplicity of infection (MOI) of bacVP1 and bacVP2 used for infection. A probabilistic model, which correlates well with the experimental results, was employed to facilitate the selection of MOIs and to provide a better understanding of the baculovirus co-infection process. A novel production process based on secondary infections was developed to ensure product consistency and to simplify large-scale logistics. The effects of other critical process parameters, such as temperature, dissolved oxygen concentration, lactate concentration, cell concentration at infection, and harvest time, were also investigated. © 1996 John Wiley & Sons, Inc.

Additional Material: 11 Ill.

Type of Medium: Electronic Resource

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