A novel perfusion system for animal cell cultures by two step sequential sedimentation.

A novel perfusion system was developed for high density culture of animal cells. The system consists of an airlift bioreactor, a setting tank and a flat settler. Both the settling tank and flat settler have two connecting pipes for transporting the cells from and back to the reactor, respectively. Thus, the cell flow in the settlers can be controlled in uni-direction, avoiding the countercurrent flow of the cells. During perfusion cultures, the cells firstly settled in the settling tank, then, unsettled cells in the tank were transferred to the flat settler for re-settling. With the application of the system to hybridoma cell cultures, it was found that the maximum viable cell density, monoclonal antibody concentration and average productivity were 1.31 x 107 cells ml-1, 400 mg l-1 and 461 mg l-1 d-1, respectively, which were much higher than those of a batch culture. Both theoretical analysis and experimental results showed a much higher separation efficiency in such a two-step sedimentation system than that in a conventional one-step sedimentation system. In addition, the volumetric ratio of the sedimentation devices to the culture volume in our developed system is much lower, which may be potentially useful on an industrial-scale.

High-performance liquid chromatographic analysis of mometasone furoate and its degradation products: application to in vitro degradation studies.

A method of analysis of mometasone furoate in pharmaceutical formulations and biological fluids is necessary to study the degradation kinetics and determine its stability. A simple high-performance liquid chromatographic method was developed for simultaneous determination of mometasone furoate and its degradation products in human plasma. Plasma (0.5 ml) was extracted with dichloromethane after addition of the internal standard, dexamethasone 21-acetate. Separation was achieved on a Beckman C(8) column with UV detection at 248 nm. The calibration curve was linear ranging from 0.2 to 100 microg/ml. The mean extraction efficiency was >86%. Precision of the assay was <10% (CV), and was within 10% at the limit of quantitation (0.2 microg/ml). Bias of the assay was lower than 7%. The limit of detection was 50 ng/ml for a 0.5-ml sample. The assay was applied successfully to the in vitro kinetic study of degradation of mometasone furoate in human plasma and simulated biological fluids.

Kinetics of metabolism and degradation of mometasone furoate in rat biological fluids and tissues.

Mometasone furoate (MF) is a potent glucocorticoid developed for the treatment of glucocorticoid-responsive inflammatory disorders. The in-vitro and ex-vivo kinetics of the degradation and metabolism of MF were studied in selected biological fluids of rat and subcellular fractions of different rat tissues. In-vitro, MF was found to degrade slowly into four products in serum and urine, and metabolized rapidly and extensively in rat liver, minimally in extrahepatic tissues, including intestine, stomach, lung and kidney. Further investigation found that the microsomal fraction was the major intracellular site of MF 6 beta-hydroxylation in rat liver. Using chemical inhibitors, CYP3A was found to be the major enzyme involved in the in-vitro MF 6 beta-hydroxylation in rat liver microsomes. Enzyme kinetic studies in rat liver microsomes showed that the overall metabolic process of MF followed biphasic Michaelis-Menten kinetics, while 6 beta-hydroxylation obeyed monophasic Michaelis-Menten kinetics. The kinetic parameters derived from the kinetic models along with the enzyme inhibition studies suggest that MF is mainly metabolized via 6 beta-hydroxylation mediated by CYP3A primarily, and also biotransformed via other pathway(s) catalysed by other enzymes in rat liver in-vitro.