CN115894719B

CN115894719B - Human serum albumin insulin conjugate and preparation method thereof

Info

Publication number: CN115894719B
Application number: CN202211485710.9A
Authority: CN
Inventors: 董亮亮; 杨代常; 夏军; 陈蓉; 徐伟
Original assignee: Wuhan Healthgen Biotechnology Co Ltd
Current assignee: Wuhan Healthgen Biotechnology Co Ltd
Priority date: 2022-11-24
Filing date: 2022-11-24
Publication date: 2023-10-20
Anticipated expiration: 2042-11-24
Also published as: CN115894719A; WO2024109534A1

Abstract

The invention relates to a human serum albumin insulin conjugate, which has the following structure: HSA-Linker-instrument; wherein: HSA is recombinant human serum albumin, linker is a small molecule Linker with difunctional groups, and Insulin is Insulin; compared with the existing insulin, the half-life period and the blood glucose reduction maintenance time of the insulin human serum albumin coupling product are obviously prolonged, the administration frequency can be reduced in the future, and the compliance of patients is improved.

Description

Human serum albumin insulin conjugate and preparation method thereof

Technical Field

The invention belongs to the field of biological pharmacy, and in particular relates to a human serum albumin insulin conjugate and a preparation method thereof.

Background

Patent WO2016178905A1 "fusion protein" describes a preparation method of an Fc fusion insulin analogue, which fuses an engineered single chain insulin analogue to an IgG Fc region, so as to achieve the aim of long acting once a week, and related product LY3209590 injection has been accepted in 2022 by the clinical application of the drug review center of the national drug administration. Iocdec is a periplasmic insulin formulation developed by norand nod, and according to the disclosure, icodec is a long-acting basal insulin analog with a half-life of 196 hours. After injection into the human body, icotec will bind tightly but reversibly to Human Serum Albumin (HSA) to form a "circulatory reservoir". This result can continuously, slowly and steadily decrease blood glucose over a period of one week. Based on its concentrated formulation, the amount of icodec insulin injected once a week was comparable to insulin glargine U100 injected once a day.

HSA is the highest single component protein in human blood, the content in blood is about 50g/L, accounting for 40% -60% of the total protein in plasma, and the half life is as long as 19 days. HSA half-life is due on the one hand to the FcRn receptor mediated HSA recovery mechanism (pH dependent, preventing lysosomal pathway degradation) and on the other hand to the avoidance of renal clearance (HSA can be reabsorbed by receptor mediated endocytosis in the proximal tubular of the kidney, thereby avoiding clearance by the kidney). HSA plays an irreplaceable function in human blood by its stable inertia, and in recent years, the use of HSA to prolong the half-life of the drug in the development of clinical therapeutic drugs has been increasingly paid attention. For example, insulin digluc and icodec are both used to prolong the action time of the medicine by utilizing endogenous HSA. Idelvion (recombinant human serum albumin/coagulation factor-IX fusion protein) and Abirudin (recombinant human serum albumin/GLP-1 fusion) utilize exogenous HSA to prolong the half-life of the drug.

However, with endogenous HSA, the actual binding of the drug to HSA in vivo cannot be predicted; HSA fusion has the problems of reduced biological activity, non-uniform expression products, easy degradation, low expression level, high cost and the like.

Disclosure of Invention

It is an object of the present invention to provide a human serum albumin insulin conjugate.

Another object of the present invention is to provide a method for preparing the above-mentioned human serum albumin insulin conjugate.

According to one aspect of the present invention, a human serum albumin insulin conjugate has the following structure:

HSA-Linker-Insulin

wherein:

HSA is recombinant Human Serum Albumin (HSA), linker is small molecule Linker with difunctional group, and Insulin is Insulin;

the small molecule linker with double functional groups is one of 6- (maleimide group) caproic acid succinimidyl Ester (EMCS), 6- (3-bromomaleimide group) caproic acid succinimidyl ester (Br-EMCS), 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), 4-maleimidobenzoic acid succinimidyl ester (SMB), 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester (SPDP) or derivatives thereof.

The human serum albumin insulin conjugate of the invention is preferably a plant-derived recombinant human serum albumin, in particular a recombinant human serum albumin (OsrHSA) expressed by rice endosperm cells.

The insulin of the present invention may be natural or recombinant insulin and may be of various origins, such as porcine insulin, bovine insulin, human insulin. Human insulin is preferred.

The human serum albumin insulin conjugate of the invention preferably the linker is 6- (maleimido) caproic acid succinimidyl Ester (EMCS) or N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP).

According to another aspect of the present invention, a method for preparing the human serum albumin insulin conjugate according to the present invention comprises the steps of:

1) Coupling reaction is carried out on insulin and the bifunctional linker to obtain an intermediate coupling product of the insulin-bifunctional linker;

2) Reacting the intermediate coupling product of the insulin-bifunctional linker obtained in the step 1) with recombinant human serum albumin to obtain a final coupling product of the recombinant human serum albumin and insulin;

3) Purifying the final coupled product obtained in the step 2) to obtain the human serum albumin insulin conjugate.

In the method of the present invention, preferably, the double crown linker is EMCS or SPDP, and in the coupling reaction of step 1) and step 2), the reaction molar ratio of insulin, the double crown linker and recombinant human serum albumin is: double crown energy group linker: recombinant human serum albumin = 1:5:2.

The method of the invention, wherein the step 1) is followed by a step of concentrating the insulin-double crown energy linker reaction solution of the step 1) by desalting or ultrafiltration to remove excessive double-functional group linker and organic solvent in the reaction, thereby obtaining an insulin-double-functional group linker intermediate coupling product.

The method of the present invention, wherein the step 3) comprises:

3a) Purifying the final coupled product obtained in the step 2) by using a hydrophobic chromatography medium, wherein the hydrophobic chromatography medium is Phenyl HP;

3b) Concentrating the purified solution obtained in the step 3 a) by using an ultrafiltration membrane to obtain the human serum albumin insulin conjugate.

More preferably, the method of the present invention comprises the steps of:

(1) Coupling of insulin to EMCS

Adding the insulin into the EMCS solution according to the molar ratio of insulin to EMCS of 1:5 for coupling reaction, and adding glycine to terminate the reaction after the reaction is finished;

(2) Excess EMCS removal

Removing excessive EMCS after the reaction in the step (1) by adopting a G-25 desalting column;

(3) Coupling of insulin-EMCS to recombinant human serum albumin

Desalting the insulin-EMCS obtained in the step (2), and according to the insulin-EMCS: human serum albumin molar ratio 1:2, adding recombinant human serum albumin for coupling reaction, and adding Cys for stopping reaction after the reaction is finished;

(4) Purification of OsrHSA-EMCS-Injulin coupled products

Purifying the coupling product obtained in the step (3) by adopting a Phenyl HP chromatographic column under the following chromatographic conditions:

balancing solution: 10mmol/LPB,0.5M ammonium sulfate, pH6.5;

eluent: 10mmol/LPB,0.025M ammonium sulfate, pH7.2;

CIP：H ₂ O；

after concentrating the eluate collection with 50kDa ultrafiltration membrane, 10mmol/LPB concentration dialysis at pH7.2 was added, and the reaction was repeated to obtain OsrHSA-EMCS-instrument conjugate.

In a third aspect, the invention provides a pharmaceutical composition comprising the human serum albumin and insulin conjugate of the invention, wherein the conjugate of the invention can be added with pharmaceutically or physiologically acceptable auxiliary materials or excipients and the like to form the pharmaceutical composition, and the pharmaceutical composition has application value in treating diabetes as long-acting insulin.

The invention provides a conjugate based on human serum albumin and insulin and a preparation method thereof, wherein the human serum albumin insulin conjugate has obvious blood sugar reducing effect, greatly prolongs the action time, can reduce the administration frequency and provides compliance for patients; meanwhile, the preparation method is simple in process, good in consistency and easy to produce and amplify in the later period.

Drawings

FIG. 1 shows SDS-PAGE detection of coupled products under different coupling conditions of Insulin and EMCS;

FIG. 2 shows the SDS-PAGE detection result of the coupling solution of OsrHSA and Insulin-EMCS (consistent sample application amount of OsrHSA); the left graph is: the right graph of the coupling ratio of Insulin to EMCS of 1:1 is: the coupling ratio of the Insulin to the EMCS is 1:5.

FIG. 3 shows a Phenyl HP loading assay chromatogram (A) and an electrophoresis detection result (B);

(B) L represents loading solution, and 1-6 represents penetrating solution when loading 1-6 column volumes.

FIG. 4 shows the chromatographic patterns of different loading amounts and the detection results of SDS-PAGE;

a and B, 5mg/ml loading of filler chromatographic patterns (A) and SDS-PAGE detection results (B);

c and D, loading 3mg/ml filler chromatographic patterns (C) and SDS-PAGE detection results (D);

in the figure, M represents a molecular weight Marker, and L represents a sample loading liquid; FT1 to FT4 represent the penetration liquid at the positions shown in the A and C diagrams, respectively; elu1 represents the main peak of the elution collection liquid, elu represents the trailing portion of the elution collection peak; CIP represents the regeneration liquid.

Figure 5 is a low load 3 batch process validation. A chromatographic patterns, B, SDS-PAGE detection results (1-3 represents different batches, M is a molecular weight Marker);

FIG. 6 shows the result of SDS-PAGE detection of high conductivity loading penetrating fluid; l represents loading liquid, M represents molecular weight Marker, and 17-25 represents penetrating liquid when 17-25 column volumes are loaded.

FIG. 7 shows high-load 3-batch process validation chromatography and SDS-PAGE detection; a, chromatographic profile. B, SDS-PAGE detection (01-03 shows 3 different batches respectively)

FIG. 8 shows the SDS-PAGE detection result of ultrafiltrates of different membrane packages; m is the molecular weight makrer; INS is an instrin control; "front" means the sample before ultrafiltration concentration; "permeate" means the permeate of the membrane envelope at the time of first concentration; "combined" means that the membrane-packed permeate is ultrafiltered 1-4 times; 5-7 represents membrane-packed permeate obtained by ultrafiltration for 5-7 times; the post-indicated samples after final ultrafiltration concentration.

FIG. 9 shows sample chromatographic patterns of ammonium sulfate with different concentrations and SDS-PAGE detection results; a, the concentration of ammonium sulfate in the sample solution is 0.35mol/L; b, the concentration of ammonium sulfate in the sample loading liquid is 0.4mol/L; c, the concentration of ammonium sulfate in the sample loading liquid is 0.45mol/L; d, SDS-PAGE detection result. M represents a molecular weight Marker; l represents loading liquid; re represents the rebalancing penetration fluid; w represents a cleaning solution; elu the elution collection liquid; CIP represents the regeneration liquid.

FIG. 10 shows the results of SDS-PAGE detection by different Linker couplings, phenyl HP purification chromatograms. A, osrHSA-SMB-instrument chromatographic profile; b, an OsrHSA-SMB-ins chromatographic sample SDS-PAGE detection result; c, osrHSA-Br-EMCS-ins chromatographic profile; and D, SDS-PAGE detection results of the OsrHSA-Br-EMCS-ins chromatographic sample. M represents a molecular weight Marker; load represents loading solution; FT1 represents the penetration fluid at the end of loading; FT2 is penetrating fluid when loading and rebalancing are finished; wash represents a Wash; elu1 represents an elution collection liquid; elu2 the eluting tail portion of the collection liquid; CIP represents the regeneration liquid.

FIG. 11 is a glucose tolerance test in rats. A, a blood sampling time schematic diagram; b, different time blood glucose concentration curves);

FIG. 12 is a graph of blood glucose (A) and blood concentration (B) of rats administered a single time under non-fasting conditions;

FIG. 13 shows a purification chromatographic profile (A) of OsrHSA-SPDP-instrument coupled product and an electrophoresis detection result (B); m represents a molecular weight Marker; load represents loading solution; FT represents the loading penetration liquid; wash represents a Wash; elu the elution collection liquid; CIP represents the regeneration liquid; non-reducing means Elu no reducing agent is added during sample preparation; reduction means Elu addition of reducing agent during sample preparation.

FIG. 14 shows the blood glucose change profile of OsrHSA-SPDP-instrument injected subcutaneously in diabetic mice.

Detailed Description

The invention will be further elucidated with reference to specific examples. It should be noted that these examples are provided merely to illustrate the present disclosure and are not intended to limit the remainder of the disclosure in any way whatsoever.

The experimental procedures not specifically described in the following examples were either conventional or performed according to the instructions provided by the manufacturer. Recombinant human serum albumin (osrsa) expressed by rice seeds used in the present invention: prepared according to patent CN100540667C and CN103880947 a; GMP grade recombinant human insulin was purchased from east Biocide (cat# GMP-045); the remaining reagents, unless otherwise specified, are commercially available or conventional products.

EXAMPLE 1 Process for coupling Insulin (Insulin) to OsrHSA

(1) Coupling of Insulin with EMCS

7ml of an Insulin solution (0.4 mmol/L) was taken for 3 branches, according to a molar ratio of Insulin to EMCS molecule of 1: 1. 1:2.5, 1:5, 28. Mu.l, 70. Mu.l, 140. Mu.l EMCS solution (100 mmol/L in DMSO) were slowly added dropwise under stirring, and the reaction was stirred at 25 ℃. At the time of 0.5h,1h and 2h, 2ml of each sample was taken, and 10 times of EMCS molar amount of Glycine was added to terminate the reaction. Excess EMCS was removed using a G-25 desalting column (column volume 25ml, flow rate 5 ml/min). The desalted Insulin-EMCS was added to 0.186ml of an OsrHSA injection (3 mmol/L) at a molar ratio of OsrHSA to Insulin-EMCS of 1:1, and reacted at room temperature for 1h. After the reaction, a proper amount of reaction solution was taken for SDS-PAGE detection. As a result, as shown in FIG. 1, the higher the coupling ratio of EMCS to Insulin, the higher the yield of the target product (red (lower) arrow) was, and when the coupling ratio of both was 5:1, the coupling efficiency of OsrHSA was nearly 50%. Whereas the coupling time of EMCS with ins has no significant effect on the yield of the protein of interest within 0.5-2 h. It is further noted that as the coupling ratio of EMCS to ins increases, so does the non-target components such as dimers (yellow (upper) arrow) and the like.

(2) Determination of the coupling Process of Insulin-EMCS and OsrHSA

Respectively taking 2 pieces of 11ml of Insulin solution (0.4 mmol/L), slowly dripping 44 μl and 220 μl of EMCS solution (100 mmol/L) under stirring according to the molar ratio of Insulin to EMCS of 1:1 and 1:5, and stirring at 25 ℃ for reaction for 0.5h. The reaction was stopped by adding 10 times the molar amount of Glycine to EMCS. Excess EMCS was removed using a G-25 desalting column (column volume 52ml, flow rate 4 ml/min). 3 pieces of desalted Insulin-EMCS sample are respectively added into 5ml of desalted Insulin-EMCS sample according to the molar ratio of OsrHSA to Insulin-EMCS of 1:2, 1:1 and 2:1, 0.103ml, 0.205ml and 0.411ml of OsrHSA (3 mmol/L) are respectively added for reaction at 25 ℃, 2ml of sample is respectively taken when the reaction is carried out for 1h and 2h, and the reaction is stopped by adding L-cysteine with 10 times of the molar amount of OsrHSA. All reactions were diluted to the same concentration (as OsrHSA) and subjected to SDS-PAGE detection at the same spot volume. The results are shown in FIG. 2, wherein the reaction time of OsrHSA and Insulin-EMCS has less influence on the coupling efficiency; under the same OsrHSA coupling ratio, the coupling ratio of Insulin to EMCS is 1:1, and the coupling product yield is lower than 1:5; when the coupling ratio of Insulin to EMCS is 1:5, the ratio of coupled products in electrophoresis is consistent with that of OsrHSA to Insulin-EMCS being 0.5:1 and that of coupled products in electrophoresis being 1:1, but the reaction volume of the coupled products in 1:1 is 2 times of that of the coupled products in 0.5:1, so that the reaction in 1:1 finally obtains more OsrHSA-EMCS-Insulin in ratio of 0.5:1; also, when the coupling ratio of OsrHSA to Insulin-EMCS was 2:1, the ratio was slightly lower than 1:1, but the amount of the final product was higher than 1:1, since the volume was 2 times 1:1, from the viewpoint of electrophoresis.

In summary, the optimal reaction ratio of Insulin, EMCS and OsrHSA was 1:5:2, with a reaction time of 0.5h for Insulin and EMCS and 1h for Insulin-EMCS.

EXAMPLE 2OsrHSA-EMCS-Insulin purification Process

9ml of an Insulin solution (4 mg/ml,0.689 mmol/L) was taken, 310. Mu.l of an EMCS solution (100 mmol/L) was slowly added dropwise in a molar ratio of Insulin to EMCS of 1:5 (converted to a mass ratio of 1:0.265) under stirring, the reaction was stirred at room temperature for 30min, and after the reaction was completed, 5 times of the molar amount of EMCS Glycine was added to terminate the reaction. Excess EMCS was removed using a G-25 desalting column (column volume 170ml, flow20 ml/min).

Desalted Insulin-EMCS is prepared according to Insulin-EMCS: osrHSA molecular mole ratio 1:2, 4ml of OsrHSA injection (3 mmol/L) was added and reacted at room temperature for 30-60min. After 45min of reaction, 5 molar times of OsrHSA was added to terminate the reaction.

(1) Phenyl HP loading determination

The reaction solution of Insulin-EMCS and OsrHSA is adjusted to be 36mS/cm by using 3mol/L ammonium sulfate, then diluted to be about 2mg/ml by using balance solution (10 mmol/LPB,0.2M ammonium sulfate and pH 6.5) based on the protein content of OsrHSA, and filtered by using a microporous filter membrane with pH adjusted to 6.5,0.22 mu M by using dilute hydrochloric acid, thus obtaining the sample loading solution. The loading was applied to a Phenyl HP chromatography column (C10/40, column height 20 cm) of 15ml in equilibrated Column Volume (CV) at a flow rate of 3ml/min and collected using a distribution collector at 15 ml/tube (i.e., 1 CV/tube). The total loading was about 12CV. Elution and H with 10mmol/LPB ₂ And O is regenerated. As shown in FIG. 3, when the protein content of the loading solution is about 2mg/ml, the conductance is about 36mS/cm (corresponding to about 0.2mol/L ammonium sulfate), and the loading of Phenyl HP is only about 3CV, i.e., corresponding to about 6mg protein/ml filler.

(2) Load confirmation

To further determine the loading of Phenyl HP, the loading was diluted to about 1mg/ml and confirmed by 5mg/ml of packing using an XK16/40 column (column volume 40 ml). As shown in fig. 4-a-B, in the process of ending the re-equilibration in the loading, the penetrating peak (FT 2) is significantly raised, and the equilibration is continued, so that the target protein is significantly exfoliated (FT 3), and as the equilibration volume increases, the penetrating peak is substantially maintained at about 20mAu (FT 4), and the target protein begins to exfoliate substantially. The purity of the eluent (Elu) is relatively high. The results are shown in FIGS. 4-C-D, considering that the loading was reduced to 3mg/ml of filler at 5mg/ml of filler overload. When the loading is reduced to 3mg/ml of filler, no obvious target protein penetrates in the rebalancing process. The purity of the target protein of the main peak (Elu 1) of the eluent is relatively high. Calculated, the yield of Insulin was 17.7% as 5mg/ml filler loading, and 32.3% as 3mg/ml filler loading.

(3) Low load process verification

According to the established coupling and chromatographic purification process, the Insulin is subjected to 3 batches of verification according to the feeding amount of 70mg, and the results are shown in figure 5, and the consistency of the three batches of processes is good.

Example 3 OsrHSA-EMCS-Injulin high-load purification Process

The OsrHSA-EMCS-instrument preparation process of example 2 has good consistency, but the Phenyl HP loading is only 3mg/ml filler, which is not beneficial to the amplification of the later process. The loading of Phenyl HP was increased by increasing the conductance of the loading solution.

OsrHSA and Insulin-EMCS reaction solutions were prepared according to example 2, and the conductance of the reaction solutions was adjusted with ammonium sulfate to match the equilibrium solution (10 mmol/LPB,0.5M ammonium sulfate, pH 6.5), and the load was measured using a 16ml Phenyl HP column (C10/40). As a result, as shown in FIG. 6, the more remarkable OsrHSA-EMCS-Insulin coupled product was seen in the 23 rd tube (15 ml/tube), and the sample collected per tube was 15ml, i.e., the loading amount was (23X 15 ml)/16 ml=21.56, i.e., the maximum loading amount was 20CV (about 1CV of tube residue), i.e., 20mg (based on OsrHSA)/ml of the filler, which was significantly improved as compared with example 2.

Example 4OsrHSA-EMCS-Insulin high load preparation Process validation

(1) Coupling of Insulin with EMCS

400mg of Insulin dry powder are weighed, after adding about 20ml of 4mmol/L dilute hydrochloric acid (pH 2.0) for complete dissolution, the pH is slowly adjusted to 7.2 (pH cannot exceed 8.0) with 0.5mol/L NaOH solution under stirring, and then coupling buffer (20 mmol/LPB,2mmol/LEDTA, pH 7.2) is added to 100ml. Then according to the mass ratio of Insulin to EMCS of 1:0.266 (molar ratio 1:5), 3.43ml of EMCS solution (31 mg/ml) was slowly added dropwise under stirring, the reaction was stirred at 25℃for 30 minutes, and after the completion of the reaction, 5 times the molar amount of EMCS Glycine was added to terminate the reaction.

(2) Excess EMCS removal

Excess EMCS was removed using a G-25 desalting column (column volume 490ml, flow rate 25-40 ml/min). The buffer used is a coupling buffer.

(3) Coupling of Insulin-EMCS with OsrHSA

The desalted Insulin-EMCS was diluted to about 1600ml with coupling buffer and then following Insulin-EMCS: osrHSA was added in a ratio of 1:22.9 (molar ratio 1:2) by mass to 50.9ml of OsrHSA (153 mg/ml) (the final concentration of OsrHSA in the system after the addition was 5 mg/ml). After 50min of reaction at 25 ℃,5 times of the molar amount of OsrHSA was added to terminate the reaction.

(4) Purification of OsrHSA-EMCS-Injulin coupled products

The reaction solution of the coupled product was diluted to about 1mg/ml (calculated as OsrHSA) and purified by using Phenyl HP column. The chromatographic conditions were as follows: chromatography column: GCC-50-400; the height of the column is 20cm; column Volume (CV) 390ml; the flow rate was 35ml/min. Sample loading volume: about 7.8L,20CV; balancing solution: 10mmol/LPB,0.5M ammonium sulphate, pH6.5 (rebalancing volume: 3CV,1170 ml); eluent: 10mmol/LPB,0.025M ammonium sulfate, pH7.2 (collected to UV 30 mAu); CIP: h ₂ O; concentrating the eluted collection liquid to a smaller volume by using a 50kDa ultrafiltration membrane bag, then adding 10mmol/LPB (pH 7.2) with at least 2 times of volume, concentrating and dialyzing, and repeating for 7 times to obtain the OsrHSA-EMCS-instrument stock solution.

The verification result shows that the chromatographic spectrum and the electrophoresis detection result have good consistency; the yields (Table 1) and purities were confirmed to be consistent in 3 batches (Table 2 and FIG. 7).

Table 1 high load 3 batch process to verify chromatographic yield

TABLE 2 summary of SEC-HPLC detection results for three stock solutions of OsrHSA-EMCS-Insulin

Example 5Linker removal Process

After coupling of the instrin with the Linker, the excess Linker needs to be removed to avoid direct reaction of the excess Linker with OsrHSA. Since Linker molecular weight is generally small, it can be removed by means of desalination, dialysis, ultrafiltration, etc., and since dialysis is unfavorable for industrial amplification, desalination and ultrafiltration means are selected for comparative study. The method comprises the following steps: 320mg of Insulin dry powder is weighed, after about 20ml of 4mmol/L diluted hydrochloric acid (pH 2.0) is added for full dissolution, the pH is slowly adjusted to 7.2 (pH cannot exceed 8.0) by adopting 0.5mol/L NaOH solution under stirring, then coupling buffer solution (20 mmol/LPB,2mmol/LEDTA, pH 7.2) is added to 80ml, and the mixture is stirred and mixed uniformly, wherein the protein content is 4mg/ml. Then the Insulin solution is equally divided into 2 parts, each part is 40ml, 1.38ml of EMCS solution (31 mg/ml) is slowly added dropwise under the stirring condition according to the mass ratio of the Insulin to the EMCS of 1:5, the reaction is stirred for 30min at 25 ℃, and after the reaction is finished, the reaction is stopped by adding Glycine with the molar quantity of 5 times of the EMCS. 40ml of the Injulin-EMCS reaction product was initially concentrated to a minimum volume (tubing residual-15 ml) using 10kDa and 5kDa ultrafiltration membrane bags, respectively. After the initial concentration, 50ml of coupling buffer solution is added, the concentration is continued to the minimum volume, after the exchange of the solution, the membrane is rinsed with a proper amount of dialysis solution, and the volume is restored to 80ml (2 times of the initial volume). The results are shown in FIG. 8, which shows an increase in aggregates after ultrafiltration dialysis, whether it is a 5kDa membrane packet or a 10kDa membrane packet; the 5kDa membrane covered yield was approximately 80% while the 10kDa membrane covered yield was only 64% and both were lower than the 90% yield of the G-25 desalting column; the low membrane package yield of one side is related to the molecular weight cut-off and the membrane package material of the selected membrane package, and an ultrafiltration membrane package of 1-3kDa can be selected for improving the yield; but the ultrafiltration time of the membrane package with smaller molecular weight cut-off is longer; the selection of a desalting column such as G-25 is more suitable from the viewpoint of yield and operation time.

EXAMPLE 6 OsrHSA-EMCS-Injulin Polymer removal Process

In example 2, phenyl HP was loaded with 0.25M ammonium sulfate, the dimer content of the collected solution was only about 2-2.5%, but the maximum loading was only 3mg/ml, and in example 4, the ammonium sulfate concentration in the loaded solution was increased to 0.5M, the loading was increased to 20mg/ml (5-6 fold increase), but the dimer was increased to 5-6%.

Based on the results, the concentration of ammonium sulfate in the sample loading solution is optimized to obtain the process with higher loading capacity and lower polymer and dimer content. The OsrHSA and instrin-EMCS reaction solution was prepared according to the previous examples, and purification conditions were optimized according to the following conditions: (1) Loading 0.45mol/L ammonium sulfate, and washing impurities with 0.4mol/L ammonium sulfate; (2) Loading 0.4mol/L ammonium sulfate, and washing impurities with 0.35mol/L ammonium sulfate; (3) Loading 0.35mol/L ammonium sulfate and washing impurities with 0.3mol/L ammonium sulfate. As a result, as shown in FIG. 9, the maximum loading was reduced due to the reduced concentration of ammonium sulfate in the loading solution, wherein 0.35mol/L and 0.4mol/L of ammonium sulfate was loaded with about 12mg/ml of the filler, and 0.45mol/L of ammonium sulfate was loaded with about 20mg/ml of the filler; performing SEC-HPLC detection on the impurity-washing liquid and the elution collection liquid under each condition, wherein 0.45mol/L ammonium sulfate is loaded, the aggregate in the impurity-washing liquid is 11.6%, and the polymer content of the elution liquid is 4.0%; loading 0.4mol/L ammonium sulfate, wherein the content of polymer in the washing liquid is 7.6%, and the content of polymer in the eluent is 3.6%; the sample was loaded with 0.35mol/L ammonium sulfate, the polymer content of the washing liquid was 7.6%, and the polymer content of the eluent was 3.6%. The results show that the lower the concentration of ammonium sulfate in the sample loading solution is, the lower the content of dimer and multimer in the eluent is; although the electrophoretic band of the wash solution showed that it was predominantly the target protein, the SEC-HPLC results showed that the aggregate content phase was significantly higher than the eluate. The yield of the Insulin is reduced by about 25% due to the addition of the impurity washing step; as can be seen from the above optimization, the dimer and multimer are mainly removed by sample penetration and washing, and the content thereof is inversely related to the chromatographic yield and the sample loading amount.

Example 7Insulin different Linker coupling Process

In the above examples, EMCS was used as a Linker, and in order to demonstrate that other linkers also have the same or better coupling effect, the analogue Br-EMCS of EMCS and the analogue SMB of SMCC were selected as test subjects.

Weighing 340mg of Insulin dry powder, adding 34ml of 4mmol/L dilute hydrochloric acid (pH 2.0), fully dissolving, slowly adjusting the pH to 7.2 by adopting 0.5mol/L NaOH solution under the stirring condition, then adding PB-1 to 85ml (4 mg/ml of Insulin final concentration), adjusting the pH to 7.10-7.20, and stirring and uniformly mixing; dividing the Insulin solution into 2 parts, respectively according to the molar ratio of Insulin to SMB or Br-EMCS of 1:5, slowly dropwise adding 1.242ml of SMB solution (100 mmol/L) or Br-EMCS solution (100 mmol/L) under the stirring condition, stirring at room temperature for 30min, and adding Glycine to terminate the reaction after the reaction is finished. Excess SMB or Br-EMCS was removed using a G-25 desalting column (XK 26/40, column volume 175ml; flow rate 15 ml/in).

The desalted Insulin-SMB or Insulin-Br-EMCS was diluted to about 715ml with coupling buffer, and then following Insulin: 15ml of OsrHSA (240 mg/ml) was added in a mass ratio of OsrHSA of 1:21 (the final concentration of OsrHSA in the system after the addition was 4-5 mg/ml), and the reaction was stirred at room temperature for 2 hours. The conductance of the reaction solution was adjusted to 77.+ -.2 mS/cm by sodium sulfate, then diluted to about 1mg/ml (calculated as OsrHSA, total about 3570 ml) by equilibration solution, and pH was adjusted to 6.0-6.1,0.22 μm by dilute hydrochloric acid, and the reaction solution was filtered through a microporous filter membrane to obtain Phenyl HP chromatography loading solution. Phenyl HP chromatography (GCC-40-200; column height 14cm; column volume 175ml; flow rate 30 ml/min) was performed as follows: balance: the equilibrium chromatographic column was rinsed with 3-5CV of equilibration solution (10 mmol/PB,0.5mol/L ammonium sulfate, conductance 75-79mS/cm, pH 6.0) until baseline, elution pH and conductance were stable; loading: the sample was loaded onto the chromatography column in a volume of about 3500ml (20 CV); rebalancing: flushing the chromatographic column with 5CV balance liquid until the baseline, the eluting pH and the electric conductance are stable; washing: the column was washed with 3CV of wash solution (10 mM PB,0.45mol/L ammonium sulfate, conductance 62-66mS/cm, pH 6.0); eluting: washing the chromatographic column with eluent (10 mmol/L,50mmol/L ammonium sulfate, conductance 10-12mS/cm, pH 6.0), reducing to 20-30mAu, and stopping collecting;

the collection solution (-700 ml) was concentrated to about 20ml using a 50kDa ultrafiltration membrane pack (Sidoris, vivaflow200, PES material), then at least 2 volumes of dialysate was added, and after mixing well, again concentrated to 20ml, and repeated 7 times. After the dialysis is finished, the concentrated solution is concentrated to a small volume (about 15 ml), then the membrane bag is drained, the concentrated solution is collected and is preserved at the temperature of minus 80 ℃ for standby. The coupled product of OsrHSA and ins using Br-EMCS or SMB as Linker and the purification electrophoresis pattern are shown in FIG. 10.

Example 8 glucose tolerance test (IPGTT)

A rat glucose tolerance test (IPGTT) was performed on OsrHSA-EMCS-Insulin (prepared according to example 4). The experiments were divided into 3 groups, control group and experimental group, 6 SD rats per group. The experimental group was given by subcutaneous injection once at a dose of 125nmol/kg, and the control group was given the same volume of placebo saline. 16 hours before the glucose tolerance test, no water is forbidden after fasting. Taking blood from the orbital venous plexus of the rat, wherein the blood taking time points are respectively-0.5 h,0h,0.5h,1h,2h,4h and 6h; wherein-0.5 h is the fasting blood glucose concentration before administration, 0h is the blood glucose concentration before intraperitoneal injection of 20g/kg glucose after administration for 0.5h, and blood glucose concentrations are measured at 0.5h,1h,2h,4h, and 6h after glucose injection. The results showed (FIG. 11) that the efficacy of OsrHSA-EMCS-Insulin could last for 45-49 hours, suggesting that OsrHSA-EMCS-Insulin had longer efficacy compared to the Insulin group and the negative control group; after 5 rounds of IPGTT, the blood glucose concentration of OsrHSA-EMCS-Insulin was substantially the same as that of the control group, indicating that the duration of action of OsrHSA-EMCS-Insulin in rats was about 24 hours.

Example 9 traditional Chinese medicine efficacy and pharmacokinetic study of OsrHSA-EMCS-Insulin in SD rats.

To demonstrate the long-term efficacy and effectiveness of OsrHSA-EMCS-instrument, SD rats were selected for testing as subjects. The specific operation is as follows: SD male rats of 200g-250g of 7-week-old body weight were randomly grouped, different doses of OsrHSA-EMCS-Insulin (prepared according to example 4) and physiological saline (negative control) of the same volume were subcutaneously injected under non-fasted condition for a single time, blood was taken from the orbital venous plexus of the rats at 4h, 8h, 24h, 32h and 48h after administration, treated at 37℃for 30min, and centrifuged at 4000rpm for 15min, and the supernatants were kept at-80 ℃. Blood sugar and Insulin content were determined using a blood sugar measurement test strip (Roche) and an ins ELISA kit (R & D, DY 8056-05). The results are shown in FIG. 12, wherein OsrHSA-EMCS-instrument shows obvious dose effect, and the larger the administration dose, the better the hypoglycemic effect and the longer the duration; the blood glucose reducing effect of SD rats can be maintained for 32-48h under the highest dosage of 1000 nmol/kg. The measurement result of the concentration of the blood medicine (Insulin) shows that the concentration of the Insulin in the blood is positively correlated with the dosage of the medicine within 0-32h, and after 48h, the content of the Insulin in three groups of blood tends to the background level; the variation of the ins content in the blood is basically consistent with the variation trend of the blood sugar concentration.

EXAMPLE 10 preparation of recombinant human serum Albumin recombinant human Insulin conjugate (OsrHSA-SPDP-Insulin)

Recombinant human Insulin (ins) coupling to SPDP

42ml of an Insulin solution (0.4 mmol/L, coupling solution is dissolved) was taken, then 8ml of an N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) solution (100 mmol/L, DMSO solution) was slowly added dropwise in a molar ratio of Insulin to SPDP of 1:5, and the reaction was stirred at room temperature for 1 hour, followed by 5-fold molar amount of Glycine solution.

Coupling of Insulin-SPDP with OsrHSA

After the reaction was completed, the excess SPDP and DMSO were removed using Bestdex G-25M desalting column. The desalted and obtained Insulin-SPDP was added to 3.88ml of OsrHSA solution (3 mmol/L) in a molar ratio of OsrHSA molecule of 1:1, and after thoroughly mixing, the mixture was allowed to stand at 4℃for 18 hours.

Purification of OsrHSA-SPDP-Injulin coupled products

The reaction solution was adjusted to have a conductivity consistent with that of a equilibration solution (10 mM PB,0.2M ammonium sulfate, pH 6.5) by using 3M ammonium sulfate, and then diluted to about 1mg/ml (based on OsrHSA) with the equilibration solution, and the reaction solution was applied to a chromatography column packed with 176ml Phenyl Bestrose HP chromatography medium at a flow rate of 15ml/min, and a column height of 25 cm. After loading was completed, the column was again equilibrated with equilibration solution until UV was substantially consistent with baseline. The dimer was removed with 10mM PB,0.18M ammonium sulfate, pH6.5, and finally the target protein was eluted with 10mM PB, pH 7.2. Concentrating the elution collecting solution by adopting a 50kDa ultrafiltration membrane bag to change the solution to obtain the OsrHSA-SPDP-Insulin conjugate stock solution. The purification chromatographic profile (A) and the electrophoresis detection result (B) of the OsrHSA-SPDP-Insulin coupling product are shown in figure 13.

EXAMPLE 11 blood glucose reducing Effect study of OsrHSA-SPDP-Insulin in diabetic mouse model

To demonstrate the long-term efficacy of OsrHSA-SPDP-Insulin, a male BSK-DB diabetic mouse model was selected for study. OsrHSA-SPDP-instrument was prepared according to example 7. Mice were fasted overnight without water control, 5 mice per test group. The administration was carried out in a single injection mode, wherein the administration dose of OsrHSA-SPDP-Insulin was set to 25IU/kg,50IU/kg (assuming that the activity remained unchanged after Insulin was coupled with OsrHSA, the Insulin activity was 28 IU/mg), the administration dose of positive control Insulin was 10IU/kg, and the negative control group was given an equivalent volume of physiological saline. Mouse tail venous blood was taken before and 8h after dosing, respectively, and blood glucose was measured using a Roche glucometer and test strip. The blood glucose change curve is plotted according to the change value (Tn/T0) of the blood glucose value (Tn) relative to the initial blood glucose value (T0) at different time points. As shown in fig. 14, the blood glucose of the instrin control group was reduced to the minimum 2 hours after administration, and the blood glucose was restored to be consistent with the physiological saline group for about 4 hours; while OsrHSA-SPDP-Insulin (OsrHSA-Insulin in the legend) shows a certain dosage effect, and has more obvious hypoglycemic effect, and the acting time is obviously prolonged (more than 8 hours). This example demonstrates that HSA conjugates can significantly extend the duration of action of the conjugated drug.

The present invention is described with reference to the above examples to illustrate a detailed preparation method of insulin analogues, but the present invention is not limited to the above detailed method, i.e., it does not mean that the present invention must be carried out depending on the above detailed preparation method. It should be apparent to those skilled in the art that any modifications to the present invention, substitutions of the linker, insulin, etc. are within the scope of the invention as disclosed herein.

Claims

1. A human serum albumin insulin conjugate having the structure:

HSA-Linker-Insulin

wherein:

HSA is recombinant human serum albumin expressed by rice seeds, linker is a small molecule Linker with difunctional groups, and Insulin is Insulin;

the small molecule linker with double functional groups is 6- (maleimide) caproic acid succinimidyl ester EMCS.

2. A method of preparing the human serum albumin insulin conjugate of claim 1, comprising the steps of:

3. The method according to claim 2, wherein in the coupling reaction of step 1) and step 2), the molar ratio of insulin, bifunctional linker and recombinant human serum albumin is: difunctional linkers: recombinant human serum albumin = 1:5:2.

4. The method according to claim 2, wherein the step 1) is followed by a step of concentrating the insulin-double crown-linker reaction solution of step 1) by desalting or ultrafiltration to remove excess double-functional-linker and organic solvent in the reaction, thereby obtaining an insulin-double-functional-linker intermediate coupling product.

5. The method of claim 2, wherein said step 3) comprises:

6. The method according to claim 2, comprising the steps of:

(1) Coupling insulin to EMCS:

(2) Excess EMCS removal:

(3) insulin-EMCS coupled to recombinant human serum albumin:

(4) Purification of the coupled product:

balancing solution: 10mmol/LPB,0.5M ammonium sulfate, pH6.5;

eluent: 10mmol/LPB,0.025M ammonium sulfate, pH7.2;

CIP：H ₂ O；

after the elution collection liquid is concentrated by a 50kDa ultrafiltration membrane bag, 10mmol/L PB of pH7.2 is added for concentration and dialysis, and the process is repeated to obtain the human serum albumin insulin conjugate.

7. A pharmaceutical composition comprising the human serum albumin insulin conjugate of claim 1.