Stability of the Third International Standard for Corticotrophin: Accelerated Degradation Study Using Different Bioassays and Isoelectric Focusing

Abstract

The corticotrophin activity of ampoules of the Third International Standard for Corticotrophin (IS) kept at 20 and 37 \ s=deg\ Cfor 15 years was compared with that of ampoules of the IS stored under normal conditions ( \ m=-\ 20 \s=deg\Cin the dark), using adrenal ascorbate depletion assays after subcutaneous administration of the hormone, assays of an increase in plasma corticosterone after intravenous administration of the hormone and in-vitro adrenal cell corticosterone production assays. Estimates of activity by all three methods are homogeneous and give combined weighted geometric means (with 95% confidence limits), as per cent of the activity in the IS stored at \m=-\20 \ s=deg\ C, of 92\m=.\1(84\m=.\8\p=n-\100) and 77\m=.\8(71\ m=. \ 6\ p=n-\ 84\ m=. \ 5) for the 20 and 37 \ s=deg\ C degradation samples respectively. Isoelectric focusing studies of the ampoule contents of the three preparations showed that ampoules of the IS stored at 20 and 37 \ s=deg\ Ccontained 90 and 79% respectively, of the component representing native corticotrophin found in the IS. These estimates of corticotrophin content are comparable to the estimates of biological activity of these preparations.The stability of the IS was calculated from the combined bioassay data assuming that degradation follows first order kinetics. The predicted half-life for the activity of the IS is 2800 years with an approximate lower 95% confidence limit of 500 years; the predicted activity of the IS remaining now, after 20 years at \m=-\20\ s=deg\ C, is 99\m=.\5% of the original activity with an approximate lower 95% confidence limit of 97\m=.\3%

CIEF with hydrodynamic and chemical mobilization for the separation of forms of α‐1‐acid glycoprotein

“…Different profiles observed can be attributed to the different purification methods used for both samples. It is known that purification methods have an influence on what forms of a glycoprotein are isolated [38]. It is also possible that the different health conditions would influence changes in the AGP forms between standard and ovary cancer samples.…”

Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time‐of‐flight mass spectrometry

“…Different pharmaceuticals of rHuEPO have been developed for clinical use. Epoetin-a and -b are obtained from Chinese hamster ovary (CHO) cells, whereas epoetin-o is obtained from baby hamster kidney (BHK) cells [3][4]. These rHuEPOs present differences in the carbohydrate structure.…”

Estimation of the composition of recombinant human erythropoietin mixtures using capillary electrophoresis and multivariate calibration methods

“…Figures 1a-c clearly show that the separated peaks and their quantitative amounts from the obtained electrophoretic profiles represent a characteristic fingerprint of each type of rHuEPO. Thus, epoetin beta and also rHuEPO BRP, which is a mixture of epoetin beta and epoetin alpha, show a wider variety of glycoforms with two more basic electrophoretic peaks than epoetin alpha, owing to their lower sialic acid residue content [8]. Since there are no differences among the UV spectra of the separated glycoforms, the identification of rHuEPO products using CE-UV has been based on their characteristic single-wavelength electrophoretic profiles, and rHuEPO BRP has been extensively used as a standard reference.…”

“…Consequently, EPO exists as a mixture of glycoforms which differ in composition, structure and charge of their carbohydrate moieties. The glycosylation of rHuEPO has been demonstrated to be critical for its biological activity and it is influenced during the manufacturing process by the specific cell line used for gene expression, the incubation culture conditions and the purification procedure [8][9][10]. This is the case of epoetin beta, which contains a greater proportion of more basic glycoforms than epoetin alpha, despite being both obtained in Chinese hamster ovary cells (CHO).…”