The use of a two-way analysis of variance to compare the methods for the assay of anhydrous dibasic calcium phosphate in the European, Japanese and United States Pharmacopoeias.

Tri-PEC (IPEC-Americas, IPEC-Europe and JPEC) carried out a two-way analysis of variance (Round Robin testing) for the assay of Anhydrous Dibasic Calcium Phosphate (CaHPO4) at the request of the Pharmacopoeial Discussion Group (PDG). On the basis of the result obtained, the difference in the assay results for anhydrous CaHPO4 using the different methods of the 3 Pharmacopoeias was not significant, while the difference in the results for the 3 different batch samples tested by 3 different methods was significant (p < 0.05). On the basis of these results, the methods from the 3 Pharmacopoeias, did not give different results, and thus the methods themselves should be considered equivalent.

Quantitative in vitro bioassay for recombinant human interleukin-11.

A cell culture-based in vitro bioassay was developed to measure the biological activity of recombinant human interleukin-11 (rhIL-11). The bioassay measures induced proliferation of T10 cells, derived from the T1165 murine plasmacytoma line. A colorimetrically detectable formazan product, obtained by cellular reduction of the tetrazolium compound, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1), was used as an endpoint for response of clone T10 to added rhIL-11. Positions of the samples and the standards in 96-well microplates affected the precision of this bioassay, which was improved by using 2 microplates where serially diluted sample and standard lines were interleaved and their positions were alternated. The coefficient of variation for this bioassay was less than 8%. This method is suitable for quality control of rhIL-11 because of its simplicity, reproducibility, and accuracy.

Adsorption of endotoxin on glass in the presence of rhIL-11.

Poor recovery of spiked endotoxin in the Limulus amebocyte lysate assay (LAL assay) was observed in the presence of recombinant human interleukin-11 (rhIL-11), a cationic, hydrophobic protein. Detection of endotoxin activity remaining in the empty glass tubes in which endotoxin and rhIL-11 mixtures were incubated indicated adsorption of endotoxin on glass. At low concentrations of rhIL-11, a correlation between endotoxin adsorbed on glass and a decrease of endotoxin in solution was observed. Adsorption of rhIL-11 on glass correlated with adsorption of endotoxin, which indicates that rhIL-11 mediates adsorption of endotoxin on glass. Consequently, adsorption of endotoxin on glass may occur in the presence of other substances which bind to both of endotoxin and glass.

Monosaccharide composition analysis of pamiteplase by anion exchange chromatography with pulsed amperometric detection.

The monosaccharides (neutral and amino sugars) of palmiteplase (recombinant modified human tissue plasminogen activator) were analyzed by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Since the palmiteplase formulation contains sucrose, it was removed by reverse-phase high-performance liquid chromatography (RP-HPLC) prior to analysis. Acid hydrolysis with TFA was performed at 100 degrees C for 4 h. Fucose, glucosamine, galactose and mannose were detected by HPAEC-PAD analysis after hydrolysis. The linearity range of HPAEC-PAD analysis was 20-200 pmol/ml(-1) (r > 0.999) and the RSD value for repeatability was less than 7%. The recovery of each monosaccharide spiked into samples was more than 90%. The monosaccharide composition of palmiteplase suggests that it has complex-type oligosaccharides lacking in high-mannose-type oligosaccharides.