Establishment of a novel cell line, CHO-MK, derived from Chinese hamster ovary tissues for biologics manufacturing.

Chinese hamster ovary (CHO) cells are widely used as a host for producing recombinant therapeutic proteins due to advantages such as human-like post-translational modification, correct protein folding, higher productivity, and a proven track record in biopharmaceutical development. Much effort has been made to improve the process of recombinant protein production, in terms of its yield and productivity, using conventional CHO cell lines. However, to the best of our knowledge, no attempts have been made to acquire new CHO cell lines from Chinese hamster ovary. In this study, we established and characterized a novel CHO cell line, named CHO-MK, derived from freshly isolated Chinese hamster ovary tissues. Some immortalized cell lines were established via sub-culture derived from primary culture, one of which was selected for further development toward a unique expression system design. After adapting serum-free and suspension culture conditions, the resulting cell line exhibited a considerably shorter doubling time (approximately 10 h) than conventional CHO cell lines (approximately 20 h). Model monoclonal antibody (IgG1)-producing cells were generated, and the IgG1 concentration of fed-batch culture reached approximately 5 g/L on day 8 in a 200-L bioreactor. The cell bank of CHO-MK cells was prepared as a new host and assessed for contamination by adventitious agents, with the results indicating that it was free from any such contaminants, including infectious viruses. Taking these findings together, this study showed the potential of CHO-MK cells with a shorter doubling time/process time and enhanced productivity in biologics manufacturing.

Upacicalcet Is a Novel Secondary Hyperparathyroidism Drug that Targets the Amino Acid Binding Site of Calcium-Sensing Receptor .

The human calcium-sensing receptor (CaSR) is a G protein-coupled receptor that maintains extracellular Ca2+ homeostasis by regulating the secretion of parathyroid hormone. Upacicalcet is a novel positive allosteric modulator of CaSR that is used for the treatment of secondary hyperparathyroidism. In the present study, to clarify the binding site of upacicalcet to CaSR, we conducted binding studies and agonistic activity studies in HEK-293T cells expressing human CaSR (intact and mutant) and an in silico docking-simulation analysis. As a result, upacicalcet competed with L-tryptophan and was thought to affect the amino acid binding site. In addition, the effects of substitutions at the amino acid binding site on the binding abilities to upacicalcet as well as the effects on receptor function as measured using inositol-1 monophosphate accumulation were examined. Upacicalcet interacted with several CaSR residues that constitute the amino acid binding site. Based on these results, we performed an in silico analysis and obtained a binding mode, consistent with the in vitro study results. Our study revealed that upacicalcet is a novel secondary hyperparathyroidism drug that targets the amino acid binding site of CaSR. Upacicalcet is expected to become a new treatment option for secondary hyperparathyroidism because the binding site differs from that of conventional drugs; consequently, it may be effective for patients who are not sensitive to conventional drugs, and it may have a superior safety profile. SIGNIFICANCE STATEMENT: Upacicalcet interacts with several residues that constitute the amino acid binding site of the calcium-sensing receptor (CaSR) and shows a potent positive allosteric activity. This mechanism differs from those of conventional drugs. Therefore, upacicalcet can be regarded as a novel secondary hyperparathyroidism drug that acts on the amino acid binding site of CaSR.

Approaches to Quality Risk Management When Using Single-Use Systems in the Manufacture of Biologics.

Biologics manufacturing technology has made great progress in the last decade. One of the most promising new technologies is the single-use system, which has improved the efficiency of biologics manufacturing processes. To ensure safety of biologics when employing such single-use systems in the manufacturing process, various issues need to be considered including possible extractables/leachables and particles arising from the components used in single-use systems. Japanese pharmaceutical manufacturers, together with single-use suppliers, members of the academia and regulatory authorities have discussed the risks of using single-use systems and established control strategies for the quality assurance of biologics. In this study, we describe approaches for quality risk management when employing single-use systems in the manufacturing of biologics. We consider the potential impact of impurities related to single-use components on drug safety and the potential impact of the single-use system on other critical quality attributes as well as the stable supply of biologics. We also suggest a risk-mitigating strategy combining multiple control methods which includes the selection of appropriate single-use components, their inspections upon receipt and before releasing for use and qualification of single-use systems. Communication between suppliers of single-use systems and the users, as well as change controls in the facilities both of suppliers and users, are also important in risk-mitigating strategies. Implementing these control strategies can mitigate the risks attributed to the use of single-use systems. This study will be useful in promoting the development of biologics as well as in ensuring their safety, quality and stable supply.

Expressions of mass transfer coefficients of bubbles and free surface of culture tanks using the k-epsilon turbulence model.

For mammalian cell culture, getting a continuous supply of oxygen and extracting carbon dioxide are primary challenges even in the most modern biopharmaceutical manufacturing plants, due to the low oxygen solubility and excessive carbon dioxide accumulation. In addition, various independent flow and mass transfer characteristics in the culture tanks vessel make scale-up extremely difficult. One method for overcoming these and providing rational optimization is solving the fluid and mass transport equations by numerical simulation. To develop a simulation program, it is decisively important to know mass transfer coefficients of gaseous species in the culture tank. In this study, oxygen mass transfer coefficients are measured using a beaker with a sparger and impellers. In order to investigate the formulation of the mass transfer coefficients, the turbulent flow statistics is calculated by a CFD code for all cases, and the expressions of the mass transfer coefficients are established as functions of the statistics. Until now, the expression by Kawase is known in this field. This expression becomes a function only of energy dissipation rate epsilon. It does not coincide with the conventional experimental fact that mass transfer coefficient is proportional power 0.5 of impeller rotation speed. The new mass transfer coefficient is dependent on both of energy dissipation rate epsilon and turbulent flow energy k. It satisfies the relation of power of 0.5 of impeller rotation speed.