MTS dye based colorimetric CTLL-2 cell proliferation assay for product release and stability monitoring of interleukin-15: assay qualification, standardization and statistical analysis.

A colorimetric cell proliferation assay using soluble tetrazolium salt [(CellTiter 96(R) Aqueous One Solution) cell proliferation reagent, containing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent phenazine ethosulfate], was optimized and qualified for quantitative determination of IL-15 dependent CTLL-2 cell proliferation activity. An in-house recombinant Human (rHu)IL-15 reference lot was standardized (IU/mg) against an international reference standard. Specificity of the assay for IL-15 was documented by illustrating the ability of neutralizing anti-IL-15 antibodies to block the product specific CTLL-2 cell proliferation and the lack of blocking effect with anti-IL-2 antibodies. Under the defined assay conditions, the linear dose-response concentration range was between 0.04 and 0.17ng/ml of the rHuIL-15 produced in-house and 0.5-3.0IU/ml for the international standard. Statistical analysis of the data was performed with the use of scripts written in the R Statistical Language and Environment utilizing a four-parameter logistic regression fit analysis procedure. The overall variation in the ED(50) values for the in-house reference standard from 55 independent estimates performed over the period of 1year was 12.3% of the average. Excellent intra-plate and within-day/inter-plate consistency was observed for all four parameter estimates in the model. Different preparations of rHuIL-15 showed excellent intra-plate consistency in the parameter estimates corresponding to the lower and upper asymptotes as well as to the 'slope' factor at the mid-point. The ED(50) values showed statistically significant differences for different lots and for control versus stressed samples. Three R-scripts improve data analysis capabilities allowing one to describe assay variations, to draw inferences between data sets from formal statistical tests, and to set up improved assay acceptance criteria based on comparability and consistency in the four parameters of the model. The assay is precise, accurate and robust and can be fully validated. Applications of the assay were established including process development support, release of the rHuIL-15 product for pre-clinical and clinical studies, and for monitoring storage stability.

Recombinant human albumin as a stabilizer for biological materials and for the preparation of international reference reagents.

Recombinant human albumin expressed in Saccharomyces cerevisiae was compared with native human serum albumin in its physicochemical properties and in its use as a stabilizer in lyophilized preparations of thyroid-stimulating hormone (TSH), interleukin 15 (IL-15) and granulocyte colony-stimulating factor (G-CSF). Advantages of recombinant albumin include its lack of potential human contaminants and infectious agents. When used at concentrations of 0.1-0.2% (w/v), recombinant albumin was equivalent to native serum albumin in its capacity to protect immunological, biological and biochemical properties of TSH, IL-15 and G-CSF. Physicochemical characteristics of the two forms of albumin including their binding to fatty acids were also similar. The recombinant form of albumin used in this study should be considered as a suitable stabilizer in the preparation of lyophilized products and reference reagents.