Semi-works Production Study Of Recombinant Human Parathyroid Hormone(1-34) Purification

Parathyroid Hormone (PTH) is a kinds of hormone which can stimulate the normal bonetissue update and metabolic. It is an effective drug to be treat osteoporosis of osteoporosisclinical.The object of this paper is PTH (1-34) which is active center of PTH. The article studiesthe injection with recombinant human parathyroid hormone (1-34) which is according to thecDNA PTH sequence, through PET32constructing fusion protein, then cloning it to E. coli.Express carrier, finally expressing in E. coli. to get genetic engineering drugs. According toexisting drugs to declare the purification process materials as the prototype, firstly gettingfusion protein fermentation clear liquid, then through the nickel ions chelating affinitychromatography, fusion protein ultra filtration and enzyme cut, anion exchangechromatography, inverse column chromatography, cationic SP exchange chromatography,eventually obtaining purity for more than98%of PTH (1-34).To explore the optimal load of each step of column chromatography, eluting processoptimization, affecting the purity of factor analysis and economic benefit analysis to obtainthe following conclusions:1. The nickel ions chelating with the sample50%saturated sample more appropriate,fillers combined with about27ml/mg, According to the kind of saturation in the simplifiedformula, can easily calculate the required sample volume. The process: After loading theBuffer A solution is leaching7CV directly eluted with Buffer B, electrophoresis purity can beobtained about80%of the fusion protein containing the target protein.2. Anion exchange chromatography in Q column binding capacity of the hybrid proteinin about40%, no significant impact on the effect of purification. Through the collection ofprotein samples after wear clothing with liquid column chromatography, to obtain a purity of75%target protein. The target protein pure reason preliminary guess is that the fusion proteinin the molecular weight of about14KDa and the hybrid protein.3. Reversed phase chromatography packing contained less than10mg/ml packing.Mobile phase A10%ethanol was added to help improve the separation and purification effect.The optimized elution gradient of40%to60%of B, elution length20CV, collected the elutionpeak purity of about95%of the target protein.4. Cation exchange chromatography packing load should be less than10mg/ml packing. Change the pH on the system will help improve sample purity but greater losses, not suitablefor pilot production. Purification accord the original process, the purity of more than98%ofthe target protein.According to the above results as well as the original small batch production experience,in accordance with the filler loading and calculation of recovery rate for each step columnchromatography column volume, and then developed to enlarge the production of pilotoperation procedure and set the work plan, and then conducted a series of three batch ofparathyroid hormone (1-34) amplification in trial production. From the existing data, reachand exceed the original expectations, the main performance is: each batch output of about1100mg; the purity of the product is99%; the examination indicators consistent with thestandards; fusion protein from start to calculate the total recovery rate is65.9%, than itsproduction increased by32%.The article takes PTH (1-34) pilot amplification research as the medium; provide someideas for protein purification of amplification, and also provide some reference ideas for theother production of protein separation and purification.