Efficient Production Of Recombinant Teriparatide Based On CSAT Scheme

There are currently more than 60 peptide drugs approved by the U.S.Food and Drug Administration（FDA）on the market.Relevant market research shows that the global therapeutic peptide market in 2019 was 26.98 billion US$and it is expected to reach 51.24billion US$by 2027.Teriparatide contains 34 amino acids at the N-terminus of parathyroid hormone（PTH）,and it has similar biological activities as intact parathyroid hormone（PTH）.In November 2002,FDA approved teriparatide PTH（1-34）produced by Eli Lilly through recombinant DNA technology.The drug is named Forsteo for the treatment of men or women at high risk of fractures and osteoporosis in postmenopausal women.Compared with organic small molecule drugs,peptide drugs have the advantages of short half-life,stronger efficacy,stronger selectivity and high specificity.However,there are major bottlenecks in the preparation of peptide drugs,such as low production efficiency and high cost.The difficulty of chemical synthesis increases exponentially as the length of the polypeptide increases,and the yield and efficiency are low.Traditional genetic engineering methods have the problems of easy degradation,cumbersome purification and renaturation process,and high cost.These problems increase the cost of preparing peptide drugs and limit the more effective development of new peptide drugs.This research is based on the cleavable self-aggregating tag（c SAT）protein purification and production technology created by our laboratory team to express and purify recombinant teriparatide.c SAT is composed of a fusion protein triplet self-aggregating tag-self-cleaving intein-target protein/peptide.The expression of the fusion protein improves the stability of recombinant teriparatide.This method removed the tag avoiding the use of enzymes.It can simplifie the purification steps and reduces production costs.In this research,the recombinant teriparatide fusion protein expression vector was first constructed,and it could be expressed and cut smoothly,which proved the feasibility of c SAT in the production and purification of recombinant teriparatide.Then the c SAT was optimized,including four MtuΔI-CM mutant strains,cleavage conditions and optimization of self-aggregating tags.The experimental results confirmed that the yield of MtuΔI-CM m2 and MtuΔI-CM m3 is higher,and the purity is better;when the cutting condition is 25℃for 24 h,the yield of recombinant teriparatide is higher;the L₆KD label is more suitable thanα3/EFK8/ELK16/TZ1H tags for the production and purification of recombinant teriparatide.Secondly,fermentation was carried out at the level of shake flask（50 m L fermentation medium）and 2-L fermentor（modified fermentation medium）.The fermentation at shake flask level showed the average yield of recombinant teriparatide after cutting by MtuΔI-CM m2 is 332.9mg/L（purity 82.9%）.The average yield of recombinant teriparatide after cleavage by MtuΔI-CM m3 is 243.3 mg/L（purity 79.0%）.Since teriparatide has higher yield and purity after cutting with MtuΔI-CM m2,we selected strains containing L₆KD-MtuΔI-CM m2-teriparatide fusion protein expression vector for the fermentation test.After fermentation,the fusion protein formed aggregates.After cutting,the yield of recombinant teriparatide in the supernatant was116.9 mg/L（purity 62.3%）.Finally,the recombinant teriparatide was purified and characterized.Recombinant teriparatide was further purified by size exclusion chromatography and ion exchange chromatography,and the recombinant teriparatide was identified by RP-HPLC,mass spectrometry,circular dichroism chromatography and other methods.RP-HPLC showed that the peak time of the recombinant teriparatide produced by the c SAT method and the standard teriparatide Forsteo were the same.Mass spectrometry showed that the molecular weight of teriparatide sample was 4,117.2 Da,which was consistent with the theoretical value.The results of circular dichroism chromatography showed that the secondary structure analysis pattern of the recombinant teriparatide was consistent with the secondary structure analysis pattern of the medical teriparatide standard Forsteo,which proved that the secondary structure of the obtained recombinant teriparatide was correct.With the aging of the population,osteoporosis has increasingly become a global epidemic.Teriparatide can treat osteoporosis,but its price is very high,so reducing the production cost of teriparatide has become an urgent problem to be solved.In this study,the commercial application preparation technology of teriparatide was established through c SAT,which improved the stability of teriparatide and reduced the production cost.This efficient preparation method enabled teriparatide to occupy an advantageous position in commercial competition.