Method for producing fusion protein having IgG Fc domain
Inventors
Park, Soon Jae • Chung, Hye Shin • YOU, Sun Ah • CHO, Jeong Soo
Assignees
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Publication Number
US-11414476-B2
Publication Date
2022-08-16
Expiration Date
Abstract
The present invention relates to a method for preparing a fusion protein having an IgG Fc domain and, specifically, to a method for preparing a fusion protein having an IgG Fc domain, the method additionally comprising a step of culturing cells, which produce the fusion protein, at a decreased culture temperature, thereby increasing cell growth and cell viability so as to increase fusion protein productivity and inhibiting aggregate generation so as to improve quality and production yield.
Core Innovation
The invention relates to a method for producing a fusion protein in a cell culture by culturing cells capable of producing the fusion protein at a first temperature range of 33°C to 38°C and then culturing the cells at a second temperature range of 28°C or greater and less than or equal to 35°C. The lower temperature in the second culturing step is lower than the temperature in the first culturing step, and the sum of a culture period of the first temperature step and the second temperature step is 3 days or more.
The fusion protein is a soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor fused to a human immunoglobulin G (IgG) Fc domain. The decreased-temperature culture is stated to increase cell growth/viability and fusion protein expression while inhibiting aggregate formation.
The method is supported by experimental results in flask and bioreactor studies, reporting increased expression/production metrics including integrated viable cell number (IVC), PV, and specific production rate, and reduced aggregates and monomer impurity measured by size exclusion-HPLC (SE-HPLC). The described culturing approach is also linked to downstream recovery and use in pharmaceutical compositions for therapeutic protein applications.
Claims Coverage
The partial content includes one independent claim (clm-00001). It recites a temperature-shift cell culture workflow with quantitative temperature ranges, a cell-density trigger for switching, and a total culture-period requirement to increase fusion protein expression for a VEGF receptor–IgG Fc fusion protein.
Temperature-shift fusion protein production with first and second culture temperature ranges
Culturing cells capable of producing a fusion protein at a temperature range of 33°C to 38°C, and culturing the cells at a temperature range of 28°C or greater and less than or equal to 35°C when a cell density reaches 4×10^6 cells/ml to 1×10^8 cells/ml, wherein the temperature in b) is lower than the temperature in a).
Increased fusion protein expression with total culture period of 3 days or more
Increasing an expression level of the fusion protein compared to culturing the cells without the temperature range of b) being lower than the temperature the cells are cultured in a), wherein the sum of a culture period of a) and b) is 3 days or more.
VEGF receptor soluble extracellular domain fused to human IgG Fc domain
Producing a fusion protein comprising a soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor and a human immunoglobulin G (IgG) Fc domain.
The core claim coverage is the combination of a two-step temperature regime tied to a cell-density window, a total culture period of 3 days or more, and the specific fusion protein composition to increase expression and reduce aggregate formation.
Stated Advantages
Increase an expression level of the fusion protein compared to culturing the cells without the temperature range of b) being lower than the temperature the cells are cultured in a).
Increase cell growth/viability and fusion protein expression while inhibiting aggregate formation.
Reduce aggregate formation and monomer impurity, as measured by size exclusion-HPLC (SE-HPLC).
Improve productivity and product quality, supported by increased expression/production metrics (IVC, PV, specific production rate).
Documented Applications
Downstream recovery and use in pharmaceutical compositions for therapeutic protein applications.
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