Sei Murakami

Automated monitoring of cell concentration and viability using an image analysis system

In order to automate measurements of cell concentration and viability in a suspended animal cell culture, we have developed an in situ microscopic image analysis system with an effective cell recognition algorithm. With a small amount of sample, this system can measure the cell density rapidly and aseptically. In addition, it can measure a cell size histogram including cell debris small particle distribution. These small particles have been found to be related to the viability of the mouse-mouse hybridoma STK1 cell line. By using cell debris small particle density as an indicator of cell viability, the developed system provides non-destructive viability monitoring without trypan blue staining.

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

ABSTRACTBiologics 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–ɛ 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 ɛ. 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 ɛ and turbulent flow energy k. It satisfies the relation of power of 0.5 of impeller rotation speed.

High-Density Animal Cell Culture by Gas Sparging

In order to enhance the oxygen supply for an animal cell culture, several foam-suppressing methods which enable direct gas sparging into culture medium were investigated. When an anti-foaming reagent was added to the medium to suppress foaming, the amount of reagent needed to be controlled in accordance with the aeration rate. With sparger optimization, increasing the sparger pore size reduced the remaining foam time. However, with the sparger pore size larger than certain value, the remaining foam time became constant, and this pore size can be regarded as the optimum. A defoaming device with a hydrophobic net has also been developed, and a culture system equipped with this defoaming net was constructed. A series of culture experiments was performed to confirm the efficacy of the defoaming net, and enhanced cell density and productivity were confirmed.