"Hybridization analysis for the quantitative determination of residual DNA in some recombinant proteins expressed in E. Coli cells".

The recent advances in the area of pharmaceutical recombinant DNA products have led to an impressive increase in the number of clinically used therapeutic proteins, for which stringent purity requirements are established by authorities. Nucleic acids are host cell derived contaminants and their allowed level in the final product is in the low pg range, because of the health risk to the patient. Even if fast non-radioactive methods have been recently developed as an alternative to the traditional hybridization assay, the latter is still widely used being a specific and sensitive method. However hybridization quantitative aspects are only partially reviewed in the literature as well as the procedures utilised to cope with the possible interference of the sample protein on the assay results. These topics are described in the present paper by detailing and comparing the methods set up for three recombinant proteins: human pro-Urokinase (r-h-proUK), human basic fibroblast growth factor (r-h-bFGF) and human granulocyte macrophage colony stimulating factor (r-h-GMCSF).

RP-HPLC peptide mapping methods for the analysis of recombinant human pro-urokinase.

Among the techniques available for the detection of protein structure variants such as single point mutations, RP-HPLC peptide mapping plays a key role owing to the high reproducibility of peptide retention times, determined as identity indexes. Because of the possible co-elution of some proteolytic fragments, an improvement of the array of information given by the technique can be achieved by setting up a series of experiments under hydrolytic conditions with different enzymes, followed by appropriate RP-HPLC gradient elutions. Such an experimental approach appears to be particularly useful in the examination of proteins with a high molecular weight, where the resulting RP-HPLC maps are complex. Therefore different RP-HPLC peptide mapping methods have been studied for recombinant human pro-urokinase (r-h-proUK), a thrombolytic agent of apparent molecular weight of 46 kD. The RP-HPLC maps indicate that the methods developed are not only suitable for the qualitative control of the amino acid sequence and arrangement of disulphide bonds but also represent the first demonstration of the identity of the primary structure of the recombinant and of the native species, within the limits of the technique.

Influence of lipophilicity on cytotoxicity of anthracyclines in LoVo and LoVo/Dx human cell lines.

Quantitative structure-activity relationship studies aimed at improving drug activity profiles require the determination of the physicochemical properties possibly involved in biological action. The lipophilic character of selected anthracyclines has been measured by means of reverse-phase high performance liquid chromatography, selecting appropriate experimental conditions. The capacity coefficients at zero percentage of the organic phase (log K0), which are retention indexes, have been used as lipophilicity descriptors in a QSAR study, involving as biological data the cytotoxicity of anthracyclines in a doxorubicin-sensitive (LoVo) and in a doxorubicin-resistant (LoVo/Dx) human cell lines. The results obtained in these in vitro models indicate that lipophilicity plays a role in anthracycline activity, influencing drug availability at the site of action.