Refolding and purification method of recombinant human granulocyte stimulating factor

Abstract

A method for renaturation and purification of recombinant human granulocyte-stimulating factor, which improves the controllability and renaturation rate of the renaturation process by removing the reducing agent by buffer replacement before renaturation, and optimizes the concentration of the renaturation buffer composition. The above-mentioned method is suitable for large-scale industrial scale-up production, has low requirements on equipment, simple operation, high purity and good stability of the obtained protein, can reduce the side effects of the final product in clinical practice, and improve the safety and effectiveness of medicines.

Description

technical field [0001] The present invention relates to the field of protein purification downstream of biomedicine and bioengineering, in particular to a renaturation and purification method for recombinant human granulocyte colony stimulating factor (rhG-CSF). The renaturation rate of the present invention is High, simple operation, suitable for industrial scale-up production. Background technique [0002] Human granulocyte colony stimulating factor (hG-CSF) belongs to the hematopoietic growth factor family. hG-CSF is a protein synthesized by mononuclear macrophages, vascular endothelial cells and fibroblasts. It consists of 174 amino acids and has a molecular weight of 19KD. Its sequence is known, as shown in sequence 1. The main mechanisms of action of hG-CSF are: (1) specifically acting on the precursor cells of bone marrow granulocytes and macrophages, promoting their differentiation and proliferation into mature granulocytes; (2) acting on bone marrow mature neutroph...

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Problems solved by technology

The disadvantage of this method is that there is a risk of trace metal ions remaining, which in turn affects drug-related testing and drug safety. It is rarely used in industrial production at present.

[0009] Chinese patent CN1167150A reports the renaturation of rhG-CSF by using hollow fiber ultrafiltration. Although it can be used in large-scale production, it has the following disadvantages: ① Affected by the phenomenon of concentration polarization, the renaturation protein solution in the hollow fiber column The internal concentration is not uniform, and protein flocculation, precipitation and even clogging of hollow fiber columns are prone to occur; ②In large-scale production, hollow fiber columns with a length of more than 90cm are often required to provide sufficient membrane area, and the length of hollow fiber columns in the research and development stage is In the range of 30-60cm, the operation of hollow fiber ultrafiltration cannot be scaled up linearly, and it is difficult to carry out detailed process research on the complex renaturation process to ensure the stability of the process

[0010] Chinese patent CN102344931A provides a method for renaturation on a nickel affinity chromatography column, which is cumbersome to operate and difficult to scale up

Chinese patent CN 104120159 A provides a Sephadex G-25 column chromatographic refolding method, the chromatographic column diameter is 5-20cm, the column bed height is 60-100cm, and the chromatographic loading protein concentration is 1.0-2.0mg / ml, The loading volume is less than 20% of the column bed volume, and the loading and elution flow rate is 5-10cm / h; calculated according to its reported scale, the batch size is about 10-20g, which is difficult to meet the needs of commercial production

[0011] Compared with other methods, dilution renaturation has the advantages of simple equipment requirements, convenient operation, low cost, and easy scale-up, but there are also a large number of wrong folding and polymerization, and even protein precipitation, which greatly reduces the renaturation yield. around 20%

[0012] The renaturation method of rhG-CSF reported in Chinese patent CN1718739 A is: the first dilution and renaturation for more than 20 hours → ultrafiltration concentration → the second dilution and renaturation for more than 96 hours → ultrafiltration concentration, that is, twice dilution and renaturation operation and two ultrafiltration treatments, although this method combines dilution renaturation and ultrafiltration treatment, the renaturation rate is high, but there are many renaturation steps, too long renaturation time, and low efficiency, which will inevitably affect the overall Yield and increase the difficulty of process scale-up

[0013] In view of the above problems, considering the whole process of inclusion body denaturation, dissolution and renaturation, a reducing agent should be added to reduce it during the denaturation and dissolution process of inclusion body protein, usually using dithiothreitol (DL-Dithiotheitol, DTT), however , because DTT is easy to be oxidized, has poor stability, and is easy to volatilize, its effect is easily affected by the environment, and it is difficult to control. Therefore, it is safe to increase the step of removing DTT after the dissolution of inclusion bodies and then dilute and refold.

[0014] Chinese patent CN101045742A provides a renaturation and purification method for recombinant human granulocyte stimulating factor. Before renaturation, the step of separating the lysate with Sephadex G-25 and removing the redundant reducing agent DTT is added, and the renaturation quality yield is high. At 50%, the RP-HPLC purity of rhG-CSF after purification is higher than 97%. Although this method can achieve the purpose of removing excess reducing agent in the lysate by Sephadex G-25 chromatography, the lysate containing Urea has a very high salt concentration. High, there is a large back pressure when performing Sephadex G-25 chromatography, there are shortcomings such as slow chromatography flow rate, long processing time, and difficult industrial scale-up

[0015] At the same time, it is necessary to provide an appropriate redox environment for the correct formation of disulfide bonds in the protein molecule during the renaturation process. The redox agent usually used is GSH / GSSG, and the ratio of GSH and GSSG will also affect the renaturation efficiency of the protein.

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