METHOD FOR PREPARING PRECURSOR OF RECOMBINANT HUMAN INSULIN OR ANALOGUE THEREOF

Abstract

Disclosed is a method for preparing a precursor of a recombinant human insulin or an analogue thereof, comprising: a. bacterial fermentation, centrifuging a fermentation broth in a continuous flow to collect a supernatant; b. filtering the supernatant in step (a) by means of a hollow fiber membrane and collecting the filtrate; and c. purifying the filtrate in the step (b) by means of a chromatography column. The method has the advantages of streamlined steps, scalability, no use of organic solvents, high yield, etc. The purity of the insulin precursor can exceed 90%, the host cell protein removal rate exceeds 90%, and the exogenous DNA removal rate is 89% or greater, achieving less than 0.1 ng/mg.

Claims

1. A method for preparing a precursor of recombinant human insulin or an analogue thereof comprising the following steps: a. performing continuous flow centrifugation on obtained fermentation broth after the fermentation of bacteria, and collecting a light liquid; b. filtering the light liquid in step a by hollow fiber membrane filtration and collecting obtained filtrate; c. purifying the filtrate in the step b by chromatography column.

2. The method of claim 1, wherein the hollow fiber membrane has a pore size of 0.22 μm or 0.45 μm; or, the hollow fiber membrane has a molecular weight cut-off of 500-1000 KDa.

3. The method of claim 1, wherein the hollow fiber membrane filtration in step b is a cyclical tangential flow filtration.

4. The method of claim 1, comprising the following steps: a. performing continuous flow centrifugation on obtained fermentation broth after the fermentation of bacteria, and collecting a light liquid and a heavy liquid respectively; diluting the heavy liquid at least once and then performing centrifugation again to obtain another light liquid, and pooling two obtained light liquids into one; b. filtering the light liquid obtained in step a by a cyclical tangential flow filtration using a hollow fiber membrane and collecting the filtrate; c. purifying the filtrate in step b by a chromatography column using a composite filler as filler, which comprises a cation exchange ligand and a hydrophobic ligand, and the preferred cation exchange ligand is a strong cation exchange ligand.

5. The method of claim 4, wherein the diluting in step a adopts a diluent that is selected from an acidic solution or an alkaline solution, wherein the acidic solution is selected from an acetic acid-sodium acetate buffer and a citrate buffer, and the alkaline solution is selected from a trihydroxymethyl-aminomethane-hydrochloric acid buffer, a phosphate buffer and a glycine sodium hydroxide buffer.

6. The method of claim 5, wherein the acidic solution has a pH of 2.0-6.0, and the alkaline solution has a pH of 7.0-9.0.

7. The method of claim 5 or 6, wherein the acidic solution has a concentration of 1 mM-100 mM; the alkaline solution has a concentration of 1 mM-100 mM.

8. The method of claim 1, wherein the fermentation broth in step a is adjusted to a pH of 2.0-9.0 before centrifugation.

9. The method of claim 8, wherein the pH of the fermentation broth in step a is adjusted to 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 before centrifugation.

10. The method of claim 1, wherein the continuous flow centrifugation in step a is performed by a disc centrifuge.

11. The method of claim 1, comprising following steps: a. adjusting the fermentation broth to a pH of 3.0-8.0 after the fermentation of bacteria, performing continuous flow centrifugation and collecting a light liquid; b. filtering the light liquid in step a by a cyclical tangential flow filtration system using a hollow fiber membrane and collecting the filtrate; c. purifying the filtrate in step b by chromatography column using a strong cation-hydrophobic exchange ligand; wherein, step a comprises following steps: collecting light liquid and heavy liquid, respectively, diluting the heavy liquid at least once and then performing centrifugation to obtain another light liquid, and pooling two light liquids into one; the diluting adopts a diluent that is selected from an acetic acid-sodium acetate buffer with a pH of 3.0-5.0 or a trihydroxymethyl-aminomethane-hydrochloric acid buffer with a pH of 7.0-9.0; the hollow fiber membrane in step b has a pore size of 0.22 μm.

12. The method of claim 1, wherein the purifying in step c comprises steps of equilibration, loading, washing impurities and elution; wherein the equilibration and washing use a solution of acetic acid-sodium acetate buffer, and the elution uses a solution of trihydroxymethyl-aminomethane-hydrochloric acid buffer.

13. The method of claim 12, wherein the acetic acid-sodium acetate buffer has a concentration of 1-50 mM; the trihydroxymethyl-aminomethane-hydrochloric acid buffer has a concentration of 10 mM-150 mM.

14. The method of claim 12 or 13, wherein the solution used for the equilibration has a pH of 3.0-6.0; the solution used for washing impurities has a pH of 5.0-7.0.

15. A method for preparing a human recombinant insulin or an analogue thereof comprising: 1) expressing a precursor of human recombinant insulin or an analogue thereof by yeast; 2) purifying the precursor of human recombinant insulin or the analogue thereof according to the method of claim 1; 3) performing enzymatic digestion on the precursor of human recombinant insulin or the analogue thereof to obtain the human recombinant insulin or the analogue thereof.

16. A method for preparing an acylated insulin analogue, wherein the method comprising: 1) expressing a precursor of human recombinant insulin by yeast; 2) purifying the precursor of human recombinant insulin according to the method of claim 1; 3) performing enzymatic digestion on obtained human recombinant insulin; 4) conducting a substitution of an acylation group on the human recombinant insulin.

17. The method of claim 16, wherein the acylated insulin analogue is a human recombinant insulin with B30 deletion.

18. The method of claim 15, wherein the human recombinant insulin analogue is a human insulin with B30 deletion.

19. The method of claim 16, wherein the substitution is a substitution of lysine at position B29.

20. The method of claim 17, wherein the substitution has a product of Lysine B29 (N.sup.ε—(N.sup.α-hexadecanedioic acid-L-lysine-N.sup.ε-oxobutanoyl)) des(B30) human recombinant insulin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1: HPLC detection chart of captured sample of insulin precursor sample prepared by the method of Example 9.

[0049] FIG. 2: HPLC detection chart of captured sample of insulin precursor sample prepared by the method of Example 10.

[0050] FIG. 3: HPLC detection chart of captured sample of insulin precursor sample prepared by the method of Example 11.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0051] The following examples are used to further describe the present invention, but the scope of present invention is not limited thereto.

[0052] Materials: MaxCell hollow fiber column and tangential flow filtration system were purchased from General Electric (China) Medical Group; Eshmuno®HCX was purchased from Merck Millipore China.

[0053] Pichia pastoris: All of the conventional Pichia pastoris for fermentation in the art can be used herein, for example, the GS115 type Pichia pastoris (purchased from Invitrogen, USA) in CN106282274A can be used to ferment and express recombinant human insulin precursor.

[0054] Recombinant human insulin and precursors thereof: The recombinant human insulin and precursors thereof herein can be any type of recombinant human insulin and precursors thereof, such as recombinant human insulin Lysine B29 (N.sup.ε—(N.sup.α-hexadecanedioic acid-L-lysine-N.sup.ε-oxobutanoyl)) des(B30) human insulin and precursors thereof.

[0055] Confirmation of the structure of human insulin: After purification of the recombinant human insulin precursors in the present invention, the target product can be digested with, for example, V8 protease, and the digested product can be analyzed by LC-MS. Recombinant human insulin: the recombinant human insulin herein can be any kind of recombinant human insulin, such as the recombinant human insulin Lysine B29 (N.sup.ε—(N.sup.α-hexadecanedioic acid-L-lysine-N.sup.ε-oxobutanoyl)) des(B30) human insulin in PCT/CN2019/074146.

Example 1 Centrifugal Separation of Fermentation Broth

[0056] Pichia pastoris was used to ferment and express insulin precursor. A 50 L of fermentation broth with 40% bacteria content was adjusted to pH 4.0 with acetic acid, and treated with an Alfa Laval flow centrifuge T20 with a centrifugal force of 10,000 g, a flow rate of 60 L/h and a frequency of slag discharge once every 2 min. A 25 L of light liquid and a 25 L of heavy liquid were collected. The heavy liquid was diluted to 50 L with 10 mM acetic acid-sodium acetate of pH 4.0 and centrifuged again, and another 25 L of light liquid was collected, thus collecting a total of 50 L light liquid. The total yield of insulin precursor was 90% and the bacterial content was less than 1%.

Example 2 Centrifugal Separation of Fermentation Broth

[0057] Pichia pastoris was used to ferment and express insulin precursor. A 50 L of fermentation broth with 40% bacteria content was adjusted to pH 5.0 with Tris, and treated with an Alfa Laval flow centrifuge T20 with a centrifugal force of 10,000 g, a flow rate of 60 L/h and a frequency of slag discharge once every 2 min. A 25 L of light liquid and a 25 L of heavy liquid were collected. The heavy liquid was diluted to 50 L with 10 mM acetic acid-sodium acetate of pH 5.0 and centrifuged again, and another 25 L of light liquid was collected, thus collecting a total of 50 L light liquid. The total yield of the insulin precursor was 95% and the bacterial content was less than 1%.

Example 3 Centrifugal Separation of Fermentation Broth

[0058] Pichia pastoris was used to ferment and express insulin precursor. A 500 L of fermentation broth with 34% bacteria content was adjusted to pH 5.0 with acetic acid, and treated with a continuous flow centrifuge with a centrifugal force of 10,000 g, a feeding speed of 1000 L/h and a light liquid outlet pressure of 5 bar. A 210 L of light liquid and a 290 L of heavy liquid were collected. The heavy liquid was diluted to 400 L with 10 mM acetic acid-sodium acetate of pH 5.0 and centrifuged again, and another 120 L of light liquid was collected, thus collecting a total of 330 L light liquid. The total yield of insulin precursor was 88% and the bacterial content was less than 1%.

Example 4 Centrifugal Separation of Fermentation Broth

[0059] Pichia pastoris was used to ferment and express insulin precursor. A 350 L of fermentation broth with 41% bacteria content was adjusted to pH 3.0 with acetic acid, and treated with a continuous flow centrifuge with a centrifugal force of 10,000 g, a feeding speed of 800 L/h and a light liquid outlet pressure of 5 bar. A 110 L of light liquid and a 240 L of heavy liquid were collected. The heavy liquid was diluted to 350 L with 10 mM acetic acid-sodium acetate of pH 3.0 and centrifuged again, and another 120 L of light liquid was collected, thus collecting a total of 230 L light liquid. The total yield of insulin precursor was 85% and the bacterial content was less than 1%.

Example 5 Centrifugal Separation of Fermentation Broth

[0060] Pichia pastoris was used to ferment and express insulin precursor. A 350 L of fermentation broth with 40% bacteria content was adjusted to pH 8.0 with 5 M NaOH solution, and treated with an Alfa Laval flow centrifuge T20 with a centrifugal force of 10,000 g, a flow rate of 60 L/h, a feeding speed of 800 L/h and a light liquid outlet pressure of 5 bar. A 130 L of light liquid and a 220 L of heavy liquid were collected. The heavy liquid was diluted to 350 L with 20 mM Tris-HCl of pH 8.0 and centrifuged again, and another 140 L of light liquid was collected, thus collecting a total of 270 L light liquid. The total yield of insulin precursor was 95% and the bacterial content was less than 1%.

Example 6 Hollow Fiber Microfiltration

[0061] A 0.22 μm of hollow fiber column was washed cyclically with an equilibration solution (10 mM acetic acid-sodium acetate, pH 4.0), until the pH in the reflux end and the feed inlet are equal. 50 L of the light liquid obtained in Example 1 was subjected to hollow fiber microfiltration, with a transmembrane pressure of 15 psi. About 48 L of filtrate was collected from the permeate outlet, and the yield of insulin precursor was 95%.

Example 7 Hollow Fiber Microfiltration

[0062] A 0.22 μm of hollow fiber column was washed cyclically with an equilibration solution (10 mM acetic acid-sodium acetate, pH 5.0), until the pH in the reflux end and the feed inlet are equal. 50 L of the light liquid obtained in Example 2 was subjected to hollow fiber microfiltration, with a transmembrane pressure of 15 psi. About 48 L of filtrate was collected from the permeate outlet, and the yield of insulin precursor was 90%.

Example 8 Hollow Fiber Microfiltration

[0063] A 0.22 μm of hollow fiber column was washed cyclically with an equilibration solution (20 mM Tris-HCl pH 8.0), until the pH in the reflux end and the feed inlet are equal. 270 L of the light liquid obtained in Example 5 was subjected to hollow fiber microfiltration, with a transmembrane pressure of 15 psi. About 255 L of filtrate was collected from the permeate outlet, and the yield of insulin precursor was 93%.

Example 9 Capture of Insulin Precursor

[0064] The filtrate obtained in Example 6 was subjected to cation chromatography, Eshmuno®HCX was used as composite chromatography medium, and the chromatography column was washed 3 times the column volume with an equilibration solution (10 mM acetic acid-sodium acetate buffer, pH 4.0). The filtrate was directly loaded to the column with a sample volume of 60 g/L, then the column was washed 5 times the column volume with a washing solution (10 mM acetic acid-sodium acetate buffer, pH 5.8) to remove portion of the pigment and HCP. Finally, an eluent (100 mM Tris-HCl, pH 8.0) was used for elution and the elution peak was collected to obtain insulin precursor with a purity of 93.1% and a yield of 81%. The results are shown in FIG. 1. When detecting the host cell protein and exogenous DNA in the elution peak, it was found that the removal rate of the host cell protein and the exogenous DNA reached 95.6% and 89.6% respectively after one step of chromatography of the filtrate. The results are shown in Table 1.

Example 10 Capture of Insulin Precursor

[0065] The filtrate obtained in Example 7 was subjected to cation chromatography, Eshmuno®HCX was used as composite chromatography medium, the chromatography column was washed 3 times the column volume with an equilibration solution (10 mM acetic acid-sodium acetate buffer, pH 5.0). The filtrate was directly loaded to the column with a sample volume of 80 g/L, then the column was washed by 5 times the column volume with a washing solution (10 mM acetic acid-sodium acetate buffer, pH 6.0) to remove portion of the pigment and HCP. Finally, an eluent (30 mM Tris-HCl, pH 8.5) was used for elution and the elution peak was collected to obtain insulin precursor with a purity of 93.5% and a yield of 85%. The results are shown in FIG. 2. When detecting the host cell protein and exogenous DNA in the elution peak, it was found that the removal rate of the host cell protein and the exogenous DNA reached 96.8% and 89.8% respectively after one step of chromatography of the filtrate. The results are shown in Table 1.

Example 11 Capture of Insulin Precursor

[0066] The filtrate obtained in Example 8 was adjusted to pH 5.0, followed by cation chromatography, Eshmuno®HCX was used as composite chromatography medium, the chromatography column was washed 3 times the column volume with an equilibration solution (10 mM acetic acid-sodium acetate buffer, pH 5.0). The filtrate was directly loaded to the column with a sample volume of 80 g/L, then the column was washed 5 times the column volume with a washing solution (10 mM acetic acid-sodium acetate buffer, pH 6.0) to remove portion of the pigment and HCP. Finally, an eluent (30 mM Tris-HCl, pH 8.5) was used for elution and the elution peak was collected to obtain insulin precursor with a purity of 90.1% and a yield of 93%. The results are shown in FIG. 3. When detecting the host cell protein and exogenous DNA in the elution peak, it was found that the removal rate of the host cell protein and the exogenous DNA reached 91.0% and 89.0% respectively after one step of chromatography of the filtrate. The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Detection results and removal rate of HCP and HCD in precursor capture sample Corresponding HCP removal HCD removal example Sample name rate rate Example 9 Capture liquid 95.6% 89.6% Example 10 Capture liquid 96.8% 89.8% Example 11 Capture liquid 91.0% 89.0% Note: The detection method of HCP is General Rule 3414 of Volume VI of the Chinese Pharmacopoeia, 2015 edition, and commercial kits are used for detection. HCD was detected by real-time fluorescent quantitative PCR method (q-PCR method) based on SYBR Green fluorescent dye.

[0067] It is to be understood for those skilled in the art that the foregoing description of specific examples is purely illustrative, and that various changes and modifications can be made to these examples without departing from the principle and essence of the present invention. Hence the protection scope of the present invention is defined by the appended claims.