Study And Application Of A CSAT Scheme-based Method For Preparation Of Pharmaceutical Proteins Containing Disulfide Bonds

Recombinant pharmaceutical protein is a protein with certain function and activity obtained by using gene recombination technology.It has been widely used in the research,development and production of biological drugs,cellular immunotherapy and diagnostic reagents.It has strong specificity,low toxicity,small side effects,clear biological function and other advantages.It is the pillar of the modern biopharmaceutical field,occupying more than one third of the market share of biopharmaceutical neighborhood.Many of these medicinal proteins are rich in disulfide bonds and have complex structures.When expressed in Escherichia coli,insoluble inclusion bodies are easily formed,which requires repeated refolding.Due to the complex structure,difficult preparation,multiple purification steps,long R&D and production cycle,and high cost of pharmaceutical proteins containing disulfide bonds,their sustainable development is ultimately affected.In this rapidly growing,highly competitive market environment for protein drugs,it will be a trend to develop a more economical and efficient production and purification process.In this paper,the recombinant human growth hormone(hGH)containing disulfide bond as a breakthrough point,based on the cleavable self aggregation tag(cSAT),the validation of different self-aggregation peptides and contained peptides,the optimization and evaluation of the expression conditions and the cutting conditions were carried out to obtain more efficient cSAT tag.In this method,specific aggregation of hGH is induced by cSAT label during expression to form aggregate precipitation with retained activity,and then hGH is released into solution by simple change of buffer p H to induce intein self-cleavage in vitro.At the level of shake flask,hGH with yield of 89 mg/L and purity of more than 80% can be obtained.Then,a standardized and efficient preparation method of pharmaceutical protein containing disulfide bond was constructed by combining the two-step standard column purification(ion exchange and molecular sieve).Through two-step column purification,hGH with yield of 73 mg/L and purity of>99% could be obtained,greatly shortening the production cycle.Furthermore,the structure and biological activity of hGH were characterized by reverse phase liquid chromatography(RP-HPLC),Native PAGE,mass spectrometry(MS),disulfide bond identification,N-terminal and C-terminal sequencing,circular dichroism(CD)and Nb2-11 cell proliferation test.It was found that the purified hGH sample contained two pairs of disulfide bonds and natural N-terminal/C-terminal,and the molecular weight,secondary structure and biological activity were the same as the commercial hGH standard.Furthermore,the aggregation mechanism of hGH fusion protein was preliminarily explored by scanning electron microscopy(SEM),transmission electron microscopy(TEM),dynamic light scattering(DLS),X-ray diffraction(XRD),thioflavine T staining(Th-T)and biofilm interference(BLI).The results show that the cSAT tag can induce the fusion protein to form an ordered amyloid fibrous structure based on cross-β,and the formation of this structure provides a relatively isolated space for the folding of the target protein,which is conducive to the expression of the protein into an active conformation.Subsequently,the method was scaled up on 2-L and 30-L fermenters.Among them,on the level of 30-L fermenter,the hGH yield obtained by cSAT method reached 2.6 g/L of culture medium,with purity>80%,which is the highest production level of hGH prepared by biological method reported at present.It is confirmed that this method has the potential of large-scale and efficient preparation of hGH.Finally,three other medicinal proteins containing disulfide bonds were selected: such as cytokine protein human interferon-α2a(IFN-α2a),nano antibody protein caplacizumab and hormone protein leptin,were used as cases to test the efficient preparation method of medical proteins containing disulfide bonds based on cSAT.The results show that the proposed method(cSAT+2 step column purification)can successfully express and purify these three medicinal proteins containing disulfide bonds,and the purity is > 99% after RP-HPLC identification,and after disulfide bond analysis,it is found that they all contain disulfide bonds,which confirms that the efficient preparation method of cSAT tag-based pharmaceutical proteins containing disulfide bonds constructed in this paper has good versatility.The innovation point of this paper is mainly lies in the fact that cSAT tag can facilitate the correct folding of the target protein containing disulfide bonds through ordered self-aggregation,and obtain the expression of active proteins.On the other hand,the formation of specific aggregation and precipitation separation,so that it only takes two steps of standardized column purification to achieve a purity of >99%,thereby simplifying the downstream process of pharmaceutical protein preparation containing disulfide bonds,shortening the research and development and production cycle,and saving costs.It provides a new generation of preparation technology platform for the production of pharmaceutical proteins containing disulfide bonds,and has a wide range of commercial application prospects.