阅读说明：本技术 一种重组人胰岛素或其类似物的前体的制备方法 (Preparation method of precursor of recombinant human insulin or analogue thereof ) 是由 赵亮亮 韩广杰 牛宁宁 李娜 刘衍伟 王宏伟 于 2019-05-23 设计创作，主要内容包括：一种重组人胰岛素或其类似物的前体的制备方法,包括a.菌体发酵,将发酵液进行连续流离心,收集清液；b.将步骤a中的清液经过中空纤维膜过滤,收集滤液；c.将步骤b中的滤液通过层析柱进行纯化。该方法具有步骤精简、易放大生产、无有机溶剂、收率高等优点,胰岛素前体纯度可高于90％,宿主细胞蛋白去除率高于90％,外源性DNA去除率达到89％及以上、低于0.1ng/mg。(A preparation method of precursor of recombinant human insulin or analogues thereof comprises a, fermenting thallus, carrying out continuous flow centrifugation on fermentation liquor, and collecting clear liquid; b. filtering the clear liquid in the step a through a hollow fiber membrane, and collecting filtrate; c. and (c) purifying the filtrate in the step b by a chromatographic column. The method has the advantages of simple steps, easy amplification production, no organic solvent, high yield and the like, the purity of the insulin precursor can be higher than 90%, the removal rate of host cell protein is higher than 90%, and the removal rate of exogenous DNA reaches 89% or more and is lower than 0.1 ng/mg.)

A method for preparing a precursor of recombinant human insulin or an analogue thereof, said method comprising the steps of:

a. fermenting thallus, performing continuous flow centrifugation on the fermentation liquor, and collecting light liquor;

b. b, filtering the light liquid in the step a through a hollow fiber membrane, and collecting filtrate;

c. and (c) purifying the filtrate in the step b by a chromatographic column.

The process of claim 1, wherein the hollow fiber membrane has a preferred pore size of 0.22 μm or 0.45 μm, more preferably 0.22 μm; or the molecular weight cut-off of the hollow fiber membrane is 500-1000KDa, preferably 750 KDa.

The process of claim 1 or 2, the hollow fiber membrane filtration of step b being tangential flow circulation filtration.

The method of claim 1, comprising the steps of:

a. after the thalli are fermented, carrying out continuous flow centrifugation on the fermentation liquor, respectively collecting light liquor and heavy liquor, carrying out at least one-time dilution on the heavy liquor, then carrying out centrifugation to obtain light liquor, and combining the light liquor;

b. b, performing tangential flow circulating filtration on the light liquid in the step a through a hollow fiber membrane, and collecting filtrate;

c. and (c) purifying the filtrate obtained in the step (b) through a chromatographic column, wherein the filler used by the chromatographic column is a composite filler, the composite filler comprises a cation exchange ligand and a hydrophobic ligand, and the preferable cation exchange ligand is a strong cation exchange ligand.

The method of claim 4, wherein in step a, the diluent used for dilution is selected from acidic solution selected from acetic acid-sodium acetate and citrate buffer, or alkaline solution selected from tris-hcl buffer, phosphate buffer, glycine-naoh buffer.

The method of claim 5, wherein the acidic solution has a pH of 2.0 to 6.0 and the basic solution has a pH of 7.0 to 9.0.

The method of claim 5 or 6, wherein the acidic solution has a concentration of 1mM to 100mM, preferably 1mM to 50mM, more preferably 10mM to 20mM, most preferably 10 mM; the concentration of the alkaline solution is 1mM-100mM, preferably 1mM-50mM, more preferably 10mM-30mM, most preferably 20 mM.

The process of any of claims 1 to 7, wherein the fermentation broth in step a is adjusted to a pH of 2.0 to 9.0, preferably to a pH of 3.0 to 8.5, more preferably to a pH of 4.0 to 8.0 before centrifugation.

The method of claim 8, wherein the fermentation broth in step a is adjusted to a pH of about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5 prior to centrifugation.

The process of claims 1-4, wherein the continuous flow centrifugation in step a is performed using a disk centrifuge, preferably with a centrifugal force of 8000- "14000 g.

The method of claim 1, comprising the steps of:

a. after the thalli are fermented, adjusting the pH value of the fermentation liquor to 3.0-8.0, carrying out continuous flow centrifugation, and collecting light liquor;

b. b, filtering the light liquid in the step a by a tangential flow filtering system of a hollow fiber membrane, and collecting filtrate;

c. purifying the filtrate in the step b by a strong cation-hydrophobic exchange ligand chromatographic column;

wherein, the step a comprises the following steps: respectively collecting light liquid and heavy liquid, diluting the heavy liquid at least once, centrifuging to obtain light liquid, and combining the light liquid; the diluent used for dilution is selected from acetic acid-sodium acetate with pH of 3.0-5.0 or trihydroxymethyl aminomethane-hydrochloric acid buffer solution with pH of 7.0-9.0;

the aperture of the hollow fiber membrane in the step b is 0.22 μm.

The method of claims 1-11, wherein the purification of step c comprises the steps of equilibration, loading, washing and elution; the solution used for balancing and impurity washing is acetic acid-sodium acetate buffer solution, and the solution used for elution is tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution.

The method of claim 12, wherein the concentration of the acetic acid-sodium acetate buffer is 1-50mM, preferably 10 mM; the concentration of the tris-hydrochloride buffer is 10mM-150mM, preferably 30mM-100 mM.

The method of claim 12 or 13, the pH of the solution used for equilibration being between 3.0 and 6.0, preferably between 4.0 and 5.0; the pH of the solution used for washing impurities is 5.0 to 7.0, preferably 5.5 to 6.0.

A method of preparing human recombinant insulin or an analog thereof, said method comprising:

1) yeast expresses the precursor of human recombinant insulin or its analogs;

2) purifying a precursor of human recombinant insulin or an analogue thereof according to the method of any one of claims 1 to 14;

3) carrying out enzyme digestion treatment on a precursor of the human recombinant insulin or the analogue thereof to obtain the human recombinant insulin or the analogue thereof;

the human recombinant insulin analogue is preferably B30 deleted human insulin.

A method of preparing an acylated insulin analog, said method comprising:

1) yeast expresses the precursor of human recombinant insulin;

2) purifying a precursor of human recombinant insulin according to the method of any one of claims 1 to 14;

3) carrying out enzyme digestion treatment to obtain human recombinant insulin;

4) the human recombinant insulin is substituted by an acylation group;

the substitution is preferably substituted at the lysine at position B29.

The method of claim 16, the acylated insulin analog being a B30 deleted human recombinant insulin, the preferred substitution product of the substitution being lysine B29 (N29)-(N^α-hexadecane fatty diacid-L-lysine-N-oxobutanoyl)) des (B30) human recombinant insulin.