Effect of cold stress on physicochemical characteristics and biological activity of equine chorionic gonadotropin

The purpose of this study was to evaluate iffreezing-thawing and cooling processes affect thestructural properties and biological activity ofcommercial equine chorionic gonadotropin (eCG). First,the structure profile of diluted eCG underwent none,one or three cycles of freezing-thawing was analysed byreverse phase high-performance liquid chromatography(RP-HPLC). In a second experiment, groups ofprepuberal rats were treated with sterile water forinjection USP or eCG that underwent none, one or threecycles of freezing-thawing to assess the increase ofovarian weigh. Finally, groups of prepubertal gilts weretreated with diluted eCG immediately afterreconstitution (T1), after refrigeration for six months(T2) and after freezing and subsequently thawing for one(T3) or three (T4) cycles. The control group (T5) receivedsterile water for injection USP without eCG. Ovulationwas induced with human chorionic gonadotropin (hCG),administered 72 h after the eCG. Gilts were slaughteredfive days after the hCG injection and ovaries wererecovered and analysed for the presence of corporalutea. Data were analysed by ANOVA and Fisher’sexact tests. In the analyses by RP-HPLC, the retentiontimes of cold stressed eCG were similar to unstressedcontrol. The mean ovarian weight of rats treated with coldstressed and unstressed eCG was statistically higher thanwater control (P < 0.05). Lastly, significantly more giltsovulated in groups T1, T2, T3 and T4 than in thecontrol T5 (P < 0.05). It was concluded that freezingthawing,as well as cooling over a period of up to sixmonths, did not significantly change the structuralproperties or biological activity of eCG.

Effect of cold stress on physicochemical characteristics and biological activity of equine chorionic gonadotropin

The purpose of this study was to evaluate iffreezing-thawing and cooling processes affect thestructural properties and biological activity ofcommercial equine chorionic gonadotropin (eCG). First,the structure profile of diluted eCG underwent none,one or three cycles of freezing-thawing was analysed byreverse phase high-performance liquid chromatography(RP-HPLC). In a second experiment, groups ofprepuberal rats were treated with sterile water forinjection USP or eCG that underwent none, one or threecycles of freezing-thawing to assess the increase ofovarian weigh. Finally, groups of prepubertal gilts weretreated with diluted eCG immediately afterreconstitution (T1), after refrigeration for six months(T2) and after freezing and subsequently thawing for one(T3) or three (T4) cycles. The control group (T5) receivedsterile water for injection USP without eCG. Ovulationwas induced with human chorionic gonadotropin (hCG),administered 72 h after the eCG. Gilts were slaughteredfive days after the hCG injection and ovaries wererecovered and analysed for the presence of corporalutea. Data were analysed by ANOVA and Fishersexact tests. In the analyses by RP-HPLC, the retentiontimes of cold stressed eCG were similar to unstressedcontrol. The mean ovarian weight of rats treated with coldstressed and unstressed eCG was statistically higher thanwater control (P < 0.05). Lastly, significantly more giltsovulated in groups T1, T2, T3 and T4 than in thecontrol T5 (P < 0.05). It was concluded that freezingthawing,as well as cooling over a period of up to sixmonths, did not significantly change the structuralproperties or biological activity of eCG.(AU)

Qualitative and quantitative reversed-phase high performance liquid chromatographic analysis of glycoprotein hormones in the presence of a large excess of human serum albumin.

The present work describes reversed-phase high performance liquid chromatographic methodologies (RP-HPLC) for the qualitative and quantitative analysis of the human glycoprotein hormones thyrotropin (hTSH), follitropin (hFSH), choriogonadotropin (hCG) and lutropin (hLH) in the presence of a large excess (up to 250:1) of human serum albumin (HSA). Chromatographic profiles with a good separation between the hormone and HSA were obtained by using a C4 column and specific gradient elution conditions for each hormone. Parameters such as resolution factor, tailing factor and relative retention time, were determined, and are useful for the evaluation of the quality of the separation obtained between the active pharmaceutical ingredient and the excipient present in the final formulation. The potential of each method for quantification of both HSA and the hormone was also demonstrated. Besides furnishing chromatographic quantifications that can substitute for in vivo bioassays and animal use, the chromatograms also provide a direct panorama of the quality and heterogeneity of the protein of interest.

A pilot study on potency determination of human follicle-stimulating hormone: a comparison between reversed-phase high-performance liquid chromatography method and the in vivo bioassay.

Reversed-phase high-performance liquid chromatography (RP-HPLC) was compared with the classical Steelman-Pohley bioassay (BA), based on animal use, for the determination of human follicle-stimulating hormone (hFSH) biological activity. A linear relationship (BA(IU)=0.9925 RP-HPLC(IU)-1.3165) with a highly significant correlation (r=0.9371; p<0.0001; n=24) was found for these two methods for six hFSH preparations of different origins. The mean difference between the bioactivity predicted from RP-HPLC data via this equation and the mean of the bioactivities obtained with the two methods for six other hFSH preparations was -1.4%, with a 95% confidence interval of -9.3 to +6.6%. The precision of these parameters was 1.63% and 2.82%, respectively. These results demonstrate that RP-HPLC is a viable physical-chemical alternative to the use of an in vivo bioassay for hFSH potency determination, applicable also to hFSH Standards containing large amounts of human serum albumin.

Analysis of human luteinizing hormone and human chorionic gonadotropin preparations of different origins by reversed-phase high-performance liquid chromatography.

Specific reversed-phase high-performance liquid chromatography conditions are reported for the analysis of recombinant and native human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) preparations. Heterodimeric hLH, hCG and their alpha- and beta-subunits migrated with significantly different retention times (t(R)) in the following order of increasing hydrophobicity: alpha-hCG

Efficient isolation of the subunits of recombinant and pituitary glycoprotein hormones.

Complete dissociation into subunits was attained by incubating Chinese hamster ovary (CHO)-derived or native human thyrotropin, follitropin and lutropin overnight at 37 degrees C in acetic acid. The alpha-and beta-subunits of the pituitary glycoprotein hormones were rapidly and quantitatively isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). A dissociation efficiency of > 98% was obtained on the basis of mass determinations of the heterodimers and subunits carried out via mass spectrometry. CHO-derived or native subunits were isolated on a C4 column (80-90% total recovery) and characterized comparatively for purity, hydrophobicity, molecular mass and charge distribution by HPLC, mass spectrometry, sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Thyrotropin was used as a model for showing that, after subunit reassociation, the in vivo bioactivity of the hormone was completely restored. The method described is mild, practical, flexible, and can be adapted to dissociate microgram amounts of native or recombinant glycoprotein hormones, allowing characterization of each subunit.