scholarly journals Isolation and analysis of human parathyrin in parathyroid tissue and plasma Use of reversed-phase liquid chromatography

1981 ◽  
Vol 197 (2) ◽  
pp. 391-400 ◽  
Author(s):  
H P J Bennett ◽  
S Solomon ◽  
D Goltzman
Keyword(s):  
Liquid Chromatography ◽  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Parathyroid Tissue ◽  
Reversed Phase ◽  
Resolving Power ◽  
Molecular Forms ◽  
Reversed Phase Liquid Chromatography ◽  
Phase Liquid

Reversed-phase liquid chromatography techniques have been used to extract and purify human parathyrin from parathyroid adenomas and to analyse the circulating forms of human parathyrin in plasma. Both the supernatant from tissue homogenates, and plasma were extracted with octadecylsilyl-silica (ODS-silica) in a batch procedure. Extracts were subjected to reversed-phase high-pressure liquid chromatography (h.p.l.c.) employing solvent systems composed of aqueous acetonitrile containing trifluoroacetic acid or heptafluorobutyric acid as hydrophobic ion-pairing reagents. The volatile solvents facilitated the radioimmunoassay, bioassay in vitro and amino acid analysis of column fractions and permitted monitoring for u.v. absorbance at 210nm. Isolated glandular parathyrin was found to be homogeneous by sodium dodecyl sulphate/urea/polyacrylamide-gel electrophoresis, to have an amino acid composition conforming to that of human parathyrin-(1-84)-tetraoctacontapeptide and to be bioactive in both renal adenylate cyclase and cytochemical bioassays. ODS-silica extraction permitted examination of large plasms samples by reversed-phase h.p.l.c., facilitating the resolution of the various circulating molecular forms of parathyrin according to their hydrophobic character. Because of its rapidity, excellent recovery and high resolving power, the methodology utilized is uniquely suited to the purification and analysis of parathyrin in tissues and body fluids.

scholarly journals At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization

2021 ◽  
Vol 8 (6) ◽  
pp. 78
Author(s):  
Julian Ebner ◽  
Diana Humer ◽  
Robert Klausser ◽  
Viktor Rubus ◽  
Reinhard Pell ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Inclusion Body ◽  
Sodium Dodecyl ◽  
Reversed Phase ◽  
Reversed Phase Liquid Chromatography ◽  
Chromatography Method ◽  
Sds Page ◽  
Phase Liquid ◽  
Liquid Chromatography Method

Refolding is known as the bottleneck in inclusion body (IB) downstream processing in the pharmaceutical industry: high dilutions leading to large operating volumes, slow refolding kinetics and low refolding yields are only a few of the problems that impede industrial application. Solubilization prior to refolding is often carried out empirically and the effects of the solubilizate on the subsequent refolding step are rarely investigated. The results obtained in this study, however, indicate that the quality of the IB solubilizate has a severe effect on subsequent refolding. As the solubilizate contains chaotropic reagents in high molarities, it is commonly analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE, however, suffers from a long analysis time, making at-line analytical implementation difficult. In this study, we established an at-line reversed phase liquid chromatography method to investigate the time-dependent quality of the solubilizate. To verify the necessity of at-line solubilization monitoring, we varied the essential solubilization conditions for horseradish peroxidase IBs. The solubilization time was found to have a major influence on subsequent refolding, underlining the high need for an at-line analysis of solubilization. Furthermore, we used the developed reversed phase liquid chromatography method for an in-process control (IPC). In conclusion, the presented reversed phase liquid chromatography method allows a proper control of IB solubilization applicable for tailored refolding.

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