Cell Growth Arrest by Nucleotides, Nucleosides and Bases as a Tool for Improved Production of Recombinant Proteins

Abstract

Arresting cell growth and thus decreasing cell division potentially lessens the chance for genetic drift in the cell population; this would be of utmost importance for the consistent production of biopharmaceuticals during long periods. The drawback of the addition of well-known synchronizing agents, such as chemotherapeutics, is that they cause a disproportionate accumulation of cellular constituents, leading to cell death. The use of compounds that are naturally synthesized by the cell, as is the case of nucleotides, nucleosides, and bases (Nt/Ns/B), is shown in this work to be a promising tool. The addition of purines and pyrimidines was tested using a CHO cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP). From the chemical alternatives tested, AMP was the most promising compound for protein production improvement; it reduced cell growth and maintained the culture with high cell viability for long periods, while increasing SEAP specific productivity 3-fold. The use of CHO and BHK mammalian cells producing Factor VII and the use of a insect cell line (Sf9) showed that the effect of AMP addition seems to be independent of the r-protein and cell line. With the addition of AMP, accumulation of cells at the S phase was accompanied by an increase of the protein specific productivity. Addition of known synchronizing drugs (aphidicolin and doxorubicin) and application of environmental cell growth arrest strategies (depletion of nutrients and byproduct accumulation) showed also to effectively arrest CHO cell growth. A careful look onto cell cycle distribution in the different scenarios created, shows whether it is important to consider r-protein expression dependency upon cell cycle in process optimization and operation strategies.

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium

“…2,4,5,23 Moreover, it has been reported that the cell cycles phase, specifically the percentage of cells in the G0/G1 phase, significantly influences the specific productivity and production of recombinant proteins in CHO cells. [16][17][18][19] Many researchers have focused on screening for chemicals that pause the cells in the G0/ G1 phase to enhance protein specific productivity in CHO cells. 16,24 Several tested chemicals had toxic effect on the cell growth and led to the decline of viable cell density.…”

“…[13][14][15] In addition, SFM formulae have been optimized to better support the physiology and metabolism of the cell and enhance target protein expression. [16][17][18] The aim of this study was to enhance the expression of rFVII by screening signal peptides and optimizing fed-batch media for efficient and long-term production. Five signal peptides from different sources were fused to the N-terminus of rFVII and its expression level during suspension culture was analyzed.…”

Effects of hydroxyurea on monoclonal antibody production induced by anti-mIgG and LPS stimulation on murine B cell hybridomas

“…These strategies have been divided by some authors (Carvalhal et al 2003) into four categories: (1) genetic, (2) environmental, (3) chemical, and (4) physical strategies. Although all these strategies may improve the protein production rate, addition of stimulatory agents is considered as a simple and low cost method, which could quickly impact on protein productivity.…”

293 cell cycle synchronisation adenovirus vector production

“…Several methods for obtaining synchronised populations of mammalian cells in vitro have been reported; genetic, chemical, environmental, and physical strategies have been used. 16,17 For reversible chemical synchronisation several compounds are available, as thymidine 18 for G1 phase synchronisation, mimosine 19 for late G1 phase synchronisation, aphidicolin, 20 rapamycine. 18 and hydroxyurea 21 for synchronisation at the G1/S transition phase, staurosporine 22 for G2 phase synchronisation and nocodazole 23 for synchronisation at the G2/M transition phase.…”