CN115894719B - A kind of human serum albumin-insulin conjugate and preparation method thereof - Google Patents

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

A kind of human serum albumin-insulin conjugate and preparation method thereof

Technical field

The invention belongs to the field of biopharmaceuticals. Specifically, the invention relates to a human serum albumin-insulin conjugate and a preparation method thereof.

Background technique

Patent WO2016178905A1 "fusion protein" describes a method for preparing Fc fusion insulin analogues. It fuses an engineered single-chain insulin analogue to the IgG Fc region to achieve long-lasting effects once a week. It is related to The product LY3209590 injection has been accepted for clinical application by the Drug Evaluation Center of the State Food and Drug Administration of China in March 2022. Iocdec is a weekly insulin preparation developed by Novo Nordisk. According to public information, Icodec is a long-acting basal insulin analog with a half-life of 196 hours. After being injected into the human body, Icodec will bind tightly but reversibly to human serum albumin (HSA) to form a "circulating storage bank." The result is a continuous, slow and steady reduction in blood sugar over the course of a week. Based on its concentrated formula, once-weekly injection of icodec insulin is equivalent to once-daily injection of insulin glargine U100.

HSA is the single component protein with the highest content in human blood. The content in blood is about 50g/L, accounting for 40% to 60% of total plasma protein, and its half-life is as long as 19 days. The long half-life of HSA is partly due to the FcRn receptor-mediated HSA recycling mechanism (pH-dependent, preventing degradation in the lysosomal pathway), and on the other hand, it is also related to its ability to avoid renal clearance (HSA can be mediated through receptors in the renal proximal tubule). reabsorbed via endocytosis, thus avoiding renal clearance). HSA plays an irreplaceable function in human blood due to its stable inertness. In recent years, the use of HSA to extend the half-life of drugs has attracted more and more attention in the development of clinical therapeutic drugs. For example, insulin degludec and icodec both use endogenous HSA to extend the drug's action time. Idelvion (recombinant human serum albumin/coagulation factor-IX fusion protein) and albiglutide (recombinant human serum albumin/GLP-1 fusion) use exogenous HSA to extend the half-life of the drug.

However, using endogenous HSA, it is impossible to predict the true binding of drugs to HSA in the body; HSA fusion has problems such as reduced biological activity, uneven expression products, easy degradation, low expression levels, and high cost.

Contents of the invention

An object of the present invention is 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 invention, a human serum albumin-insulin conjugate has the following structure:

HSA-Linker-Insulin

in:

HSA is recombinant human serum albumin (HSA), Linker is a small molecule linker with bifunctional groups, and Insulin is insulin;

The small molecule linker with bifunctional groups is the following 6-(maleimido)hexanoic acid succinimide ester (EMCS), 6-(3-bromomaleimide)hexane. Succinimide acid ester (Br-EMCS), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), 4-maleimide Succinimide benzoate (SMB), N-hydroxysuccinimide 3-(2-pyridinedimercapto)propionate (SPDP) or their derivatives.

In the human serum albumin-insulin conjugate of the present invention, preferably the recombinant human serum albumin is a plant-derived recombinant human serum albumin, especially the recombinant human serum albumin is derived from recombinant human serum albumin expressed in rice endosperm cells. Human serum albumin (OsrHSA).

The insulin of the present invention can be natural or recombinant insulin, and can be from various sources, such as porcine insulin, bovine insulin, and human insulin. Preferred is human insulin.

In the human serum albumin-insulin conjugate of the present invention, preferably the linker is 6-(maleimido)caproate succinimide ester (EMCS) or N-succinimide 3-( 2-Pyridyldithio)propionate (SPDP).

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

1) Perform a coupling reaction between insulin and a bifunctional linker to obtain an insulin-bifunctional linker intermediate coupling product;

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

3) Purify the final coupling product obtained in step 2) to obtain the human serum albumin-insulin conjugate.

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

The method of the present invention, wherein after step 1), it further includes concentrating the insulin-bifunctional group linker reaction solution of step 1) through desalting or ultrafiltration to remove excess bifunctional linker and organic matter in the reaction. Solvent step to obtain the insulin-bifunctional linker intermediate coupling product.

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

3a) Purify the final coupling product obtained in step 2) using a hydrophobic chromatography medium, and the hydrophobic chromatography medium is Phenyl HP;

3b) Use an ultrafiltration membrane to concentrate the purified liquid obtained in step 3a) to obtain human serum albumin-insulin conjugate.

More preferably, the method of the present invention includes the following steps:

(1) Insulin coupled to EMCS

According to the molar ratio of insulin to EMCS of 1:5, add the EMCS solution to the insulin solution to perform the coupling reaction. After the reaction is completed, add glycine to terminate the reaction;

(2) Excess EMCS removal

Use G-25 desalting column to remove excess EMCS after the reaction in step (1);

(3) Insulin-EMCS coupled with recombinant human serum albumin

Add recombinant human serum albumin to the desalted insulin-EMCS in step (2) according to the molar ratio of insulin-EMCS: human serum albumin of 1:2 to perform a coupling reaction. After the reaction is completed, Cys is added to terminate the reaction;

(4) Purification of OsrHSA-EMCS-Insulin coupling product

The coupling product obtained in step (3) is purified using a Phenyl HP chromatography column. The chromatography conditions are:

Balance solution: 10mmol/LPB, 0.5M ammonium sulfate, pH 6.5;

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

CIP: H ₂ O;

After concentrating the elution collection liquid with a 50kDa ultrafiltration membrane bag, add 10mmol/LPB at pH 7.2 for concentration and dialysis, and repeat to obtain the OsrHSA-EMCS-Insulin conjugate.

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

The invention provides a conjugate based on human serum albumin and insulin and a preparation method thereof. The human serum albumin-insulin conjugate has an obvious hypoglycemic effect, has a greatly prolonged action time, and can reduce the frequency of administration. , improving patient compliance; at the same time, this preparation method has simple process, good consistency, and is easy to scale up in later production.

Description of the drawings

Figure 1 shows the SDS-PAGE detection of the coupling products of Insulin and EMCS under different coupling conditions;

Figure 2 shows the SDS-PAGE detection results of OsrHSA and Insulin-EMCS coupling solution (based on the OsrHSA spotting amount); the left picture shows: the coupling ratio of Insulin and EMCS is 1:1; the right picture shows: the coupling ratio of Insulin and EMCS 1 :5.

Figure 3 shows the chromatogram (A) and electrophoresis detection results (B) of Phenyl HP capacity measurement;

In (B), L represents the loading liquid, and 1-6 represents the penetration liquid when loading 1-6 column volumes.

Figure 4 shows the chromatograms and SDS-PAGE detection results of different loading amounts;

A and B, 5mg/ml packing volume chromatogram (A) and SDS-PAGE detection results (B);

C and D, chromatogram (C) and SDS-PAGE detection results (D) of packing material loading 3 mg/ml;

In the figure, M represents the molecular weight marker, and L represents the loading solution; FT1-FT4 represent the penetration fluid at the positions shown in Figures A and C respectively; Elu1 represents the main peak of the elution collection solution, and Elu2 represents the tailing part of the elution collection peak; CIP Indicates regeneration fluid.

Figure 5 shows the low-load 3-batch process verification. A chromatogram, B, SDS-PAGE detection results (1-3 represent different batches, M is the molecular weight marker);

Figure 6 shows the SDS-PAGE detection results of high conductivity loading and penetration fluid; L represents the loading fluid, M is the molecular weight marker, and 17-25 represents the penetration fluid when loading 17-25 column volumes.

Figure 7 shows the chromatograms and SDS-PAGE detection results of three batches of high-capacity process verification; A, chromatogram. B, SDS-PAGE test results (01-03 respectively represent 3 different batches)

Figure 8 shows the SDS-PAGE test results of ultrafiltrate from different membrane packages; M is the molecular weight makrer; INS is the Insulin control; “pre” represents the sample before ultrafiltration concentration; “penetration” represents the penetration of the membrane package during the first concentration. liquid; "He" represents the membrane packet permeate obtained by ultrafiltration for 1-4 times; 5-7 represents the membrane packet permeate obtained by ultrafiltration for 5-7 times; and "he" represents the sample after final ultrafiltration and concentration.

Figure 9 shows the chromatograms and SDS-PAGE detection results of loading ammonium sulfate with different concentrations; A, the ammonium sulfate concentration of the loading solution is 0.35mol/L; B, the ammonium sulfate concentration of the loading solution is 0.4mol/L; C, the loading solution The concentration of liquid ammonium sulfate is 0.45mol/L; D, SDS-PAGE detection result. M represents the molecular weight marker; L represents the loading solution; Re represents the re-equilibration penetration liquid; W represents the washing liquid; Elu represents the elution collection liquid; CIP represents the regeneration liquid.

Figure 10 Different Linker coupling, Phenyl HP purification chromatogram and SDS-PAGE detection results. A, OsrHSA-SMB-Insulin chromatogram; B, SDS-PAGE detection results of OsrHSA-SMB-Insulin chromatography sample; C, OsrHSA-Br-EMCS-Insulin chromatogram; D, OsrHSA-Br-EMCS-Insulin layer Analyze the SDS-PAGE test results of the samples. M represents the molecular weight marker; Load represents the loading liquid; FT1 represents the penetration liquid at the end of loading; FT2 represents the penetration liquid when re-equilibrating at the end of loading; Wash represents the washing liquid; Elu1 represents the elution collection liquid; Elu2 represents wash Remove the tailing part of the collection liquid; CIP represents the regeneration liquid.

Figure 11 shows the glucose tolerance test in rats. A, Schematic diagram of blood collection time; B, Blood glucose concentration curve at different times);

Figure 12 shows the blood glucose (A) and blood drug concentration (B) curves of rats after a single administration under non-fasting conditions;

Figure 13 shows the purification chromatogram (A) and electrophoresis detection results (B) of the OsrHSA-SPDP-Insulin coupling product; M represents the molecular weight marker; Load represents the loading solution; FT represents the loading penetration solution; Wash represents the washing solution. ; Elu represents the elution collection solution; CIP represents the regeneration solution; non-reducing means that no reducing agent is added during the Elu sample preparation process; reduction means that the reducing agent is added during the Elu sample preparation process.

Figure 14 shows the blood glucose change curve of diabetic mice injected subcutaneously with OsrHSA-SPDP-Insulin.

Detailed ways

The present invention will be further elucidated below with reference to specific examples. It should be noted that these provided embodiments are only illustrative of the content of the present invention and do not limit the remaining content disclosed in the present invention in any way.

Experimental methods not specifically described in the following examples are all routine operations or performed in accordance with the operating instructions provided by the manufacturer. The recombinant human serum albumin (OsrHSA) expressed from rice seeds used in the present invention: prepared according to patents CN100540667C and CN103880947A; GMP grade recombinant human insulin was purchased from Dongkang Biotechnology (Product No.: GMP-045); the remaining reagents, unless otherwise specified, are For commercially available or conventional products.

Example 1 Coupling process of insulin (Insulin) and OsrHSA

(1) Insulin and EMCS coupling

Take 3 bottles of 7ml Insulin solution (0.4mmol/L), and slowly add 28μl, 70μl, 140μl EMCS solution (100mmol) under stirring conditions according to the molar ratio of Insulin to EMCS molecules: 1:1, 1:2.5, 1:5. /L, prepared in DMSO), stir and react at 25°C. At 0.5h, 1h, and 2h of reaction, 2 ml samples were taken respectively, and 10 times the molar amount of Glycine of EMCS was added to terminate the reaction. Use a G-25 desalting column (column volume 25 ml, flow rate 5 ml/min) to remove excess EMCS. Add 0.186ml OsrHSA injection (3mmol/L) to the desalted Insulin-EMCS at a molar ratio of OsrHSA to Insulin-EMCS molecules of 1:1, and react at room temperature for 1 hour. After the reaction is completed, take an appropriate amount of the reaction solution for SDS-PAGE detection. The results are shown in Figure 1. The higher the coupling ratio of EMCS to Insulin, the higher the yield of the target product (shown by the red (lower) arrow). When the coupling ratio of the two is 5:1, the coupling ratio of OsrHSA The connection efficiency is close to 50%. The coupling time of EMCS and Insulin has no significant impact on the yield of the target protein within 0.5-2h. It should also be noted that as the coupling ratio of EMCS to Insulin increases, non-target components such as dimers (shown by the yellow (upper) arrow) also increase.

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

Take 2 bottles of 11ml Insulin solution (0.4mmol/L) respectively. According to the molar ratio of Insulin to EMCS is 1:1 and 1:5, slowly add 44μl and 220μl EMCS solution (100mmol/L) under stirring conditions, and stir at 25°C. Reaction 0.5h. The reaction was terminated by adding 10 times the molar amount of Glycine to EMCS. Use a G-25 desalting column (column volume 52 ml, flow rate 4 ml/min) to remove excess EMCS. Add 3 tubes of each component of the desalted Insulin-EMCS sample, 5 ml each, and add 0.103 ml, 0.205 ml, and 0.411 ml respectively according to the molar ratio of OsrHSA to Insulin-EMCS: 1:2, 1:1, and 2:1. OsrHSA (3mmol/L), react at 25°C, take 2ml samples each at 1h and 2h, and add 10 times the molar amount of L-cysteine of OsrHSA to terminate the reaction. All reaction solutions were diluted to the same concentration (based on OsrHSA), and SDS-PAGE detection was performed at the same spotting volume. The results are shown in Figure 2. The reaction time between OsrHSA and Insulin-EMCS has little effect on the coupling efficiency; under the same OsrHSA coupling ratio, the coupling ratio of Insulin to EMCS is 1:1, and the yield of the coupling product is lower than 1:1. 5 coupling; when the coupling ratio of Insulin to EMCS is 1:5, the ratio of the coupling products in electrophoresis is relatively consistent when the coupling ratio of OsrHSA to Insulin-EMCS is 0.5:1 and 1:1, but due to The reaction volume of the 1:1 coupling product is twice that of 0.5:1, so the final amount of OsrHSA-EMCS-Insulin obtained from the 1:1 reaction is more than that of 0.5:1; similarly, from the electrophoresis point of view, OsrHSA and Insulin-EMCS When the coupling ratio is 2:1, OsrHSA-EMCS-Insulin is slightly lower than 1:1, but since its volume is 2 times that of 1:1, the amount of the final product is higher than that of 1:1 coupling.

In summary, the optimal reaction ratio of Insulin, EMCS and OsrHSA is 1:5:2, in which the reaction time of Insulin and EMCS is 0.5h, and the reaction time of Insulin-EMCS is 1h.

Example 2 OsrHSA-EMCS-Insulin purification process

Take 9ml Insulin solution (4mg/ml, 0.689mmol/L), according to the molar ratio of Insulin to EMCS 1:5 (converted to mass ratio 1:0.265), slowly add 310μl EMCS solution (100mmol/L) under stirring conditions. , stir the reaction at room temperature for 30 minutes. After the reaction is completed, add 5 times the molar amount of Glycine of EMCS to terminate the reaction. Use a G-25 desalting column (column volume 170 ml, flow rate 20 ml/min) to remove excess EMCS.

Add 4 ml of OsrHSA injection (3 mmol/L) to the desalted Insulin-EMCS at a molar ratio of Insulin-EMCS: OsrHSA molecules of 1:2, and react at room temperature for 30-60 minutes. After 45 min of reaction, 5 times the molar amount of Cys of OsrHSA was added to terminate the reaction.

(1)Phenyl HP load measurement

Use 3mol/L ammonium sulfate to adjust the conductivity of the reaction solution of Insulin-EMCS and OsrHSA to 36mS/cm, and then use equilibrium solution (10mmol/LPB, 0.2M ammonium sulfate, pH 6.5) to dilute it to about 2mg/ml (based on OsrHSA protein). (content meter), use dilute hydrochloric acid to adjust the pH to 6.5, and filter it with a 0.22 μm microporous membrane to obtain the loading solution. Pour the sample solution onto a Phenyl HP chromatography column (C10/40, column height 20cm) with a balanced column volume (CV) of 15ml at a flow rate of 3ml/min, and use a distribution collector to follow the flow rate of 15ml/tube (i.e. 1CV/tube). ) to collect. A total of approximately 12CV of sample was loaded. Use 10 mmol/LPB for elution and H ₂ O for regeneration. As shown in Figure 3, when the protein content of the sample solution is approximately 2 mg/ml and the conductivity is approximately 36 mS/cm (approximately equivalent to 0.2 mol/L ammonium sulfate), the loading capacity of Phenyl HP is only approximately 3CV, which is approximately equivalent to 6 mg. Protein/ml filler.

(2) Loading capacity confirmation

In order to further determine the loading capacity of Phenyl HP, the loading solution was diluted to approximately 1 mg/ml, and an XK16/40 chromatography column (column volume 40 ml) was used to confirm the loading capacity of 5 mg/ml packing material. The results are shown in Figure 4-A-B. During the re-equilibration process after sample loading, the penetration peak (FT2) increased significantly. As the equilibrium continued, obvious target protein shedding (FT3) was visible. As the equilibrium volume increased, the penetration peak The peak is basically maintained at around 20mAu (FT4), and the target protein begins to fall off in large quantities. The purity of (Elu) in the eluent is relatively high. Considering that the sample loading based on 5 mg/ml packing was overloaded, the loading amount was reduced to 3 mg/ml packing, and the results are shown in Figure 4-C-D. When the loading amount was reduced to 3 mg/ml packing, there was no obvious target protein penetration during the reequilibration process. The purity of the target protein in the main peak of the eluent (Elu1) is relatively high. After calculation, according to the loading of 5 mg/ml packing, the yield of Insulin is 17.7%, and according to the loading of 3 mg/ml packing, the yield of Insulin is 32.3%.

(3) Low-load process verification

According to the determined coupling and chromatography purification process, Insulin was verified in three batches according to the input amount of 70 mg. The results are shown in Figure 5. The consistency of the three batches of process is good.

Example 3 OsrHSA-EMCS-Insulin high-capacity purification process

The preparation process of OsrHSA-EMCS-Insulin in Example 2 has good consistency, but the loading capacity of Phenyl HP is only 3 mg/ml filler, which is not conducive to the amplification of the later process. Therefore, the loading capacity of Phenyl HP can be increased by increasing the conductance of the loading solution.

The reaction solution of OsrHSA and Insulin-EMCS was prepared according to Example 2, and then the conductivity of the reaction solution was adjusted with ammonium sulfate to be consistent with the equilibrium solution (10mmol/LPB, 0.5M ammonium sulfate, pH 6.5), and a 16ml Phenyl HP chromatography column was used. (C10/40) for capacity determination. The results are shown in Figure 6. The obvious OsrHSA-EMCS-Insulin coupling product can be seen in the 23rd tube (15ml/tube). The sample collected in each tube is 15ml, that is, the loading capacity is (23×15ml)/16ml=21.56, that is The maximum loading volume is 20CV (approximately 1CV remains in the pipeline), that is, 20 mg (calculated as OsrHSA)/ml filler, which is significantly increased compared to Example 2.

Example 4 OsrHSA-EMCS-Insulin high-load preparation process verification

(1) Insulin and EMCS coupling

Weigh 400mg of Insulin dry powder, add about 20ml of 4mmol/L dilute hydrochloric acid (pH2.0) and fully dissolve it. Under stirring conditions, use 0.5mol/L NaOH solution to slowly adjust the pH to 7.2 (the pH cannot exceed 8.0), and then add even Add coupling buffer (20mmol/LPB, 2mmol/LEDTA, pH7.2) to 100ml. Then according to the ratio of Insulin to EMCS mass ratio 1:0.266 (molar ratio 1:5), slowly add 3.43ml EMCS solution (31mg/ml) under stirring conditions, stir and react at 25°C for 30 minutes, after the reaction is completed, add 5 times EMCS molar amounts of Glycine terminate the reaction.

(2) Excess EMCS removal

Use a G-25 desalting column (column volume 490 ml, flow rate 25-40 ml/min) to remove excess EMCS. The buffer used is coupling buffer.

(3) Insulin-EMCS coupled with OsrHSA

Dilute the desalted Insulin-EMCS to about 1600ml with coupling buffer, then add 50.9ml of OsrHSA (153mg/ml) according to the ratio of Insulin-EMCS: OsrHSA mass ratio 1:22.9 (molar ratio 1:2) (After OsrHSA is added, the final concentration in the system is 5 mg/ml). After 50 min of reaction at 25°C, Cys with 5 times the molar amount of OsrHSA was added to terminate the reaction.

(4) Purification of OsrHSA-EMCS-Insulin coupling product

The reaction solution of the coupling product was diluted to approximately 1 mg/ml (calculated as OsrHSA), and purified using a Phenyl HP chromatography column. The chromatography conditions are as follows: chromatography column: GCC-50-400; column height 20cm; column volume (CV) 390ml; flow rate 35ml/min. Loading volume: about 7.8L, 20CV; equilibrium solution: 10mmol/LPB, 0.5M ammonium sulfate, pH6.5 (reequilibration volume: 3CV, 1170ml); eluent: 10mmol/LPB, 0.025M ammonium sulfate, pH7. 2 (Collect to UV 30mAu); CIP: H ₂ O; Concentrate the elution collection liquid to a smaller volume with a 50kDa ultrafiltration membrane bag, then add at least 2 times the volume of 10mmol/LPB (pH7.2), concentrate and dialyze, Repeat 7 times to obtain OsrHSA-EMCS-Insulin stock solution.

The verification results showed that the chromatography and electrophoresis detection results were in good consistency; the verification yields (Table 1) and purity of the three batches were consistent (Table 2 and Figure 7).

Table 1 High-loading 3-batch process validation chromatography yields

Table 2 Summary of SEC-HPLC test results of three batches of OsrHSA-EMCS-Insulin stock solution

Example 5 Linker removal process

After conjugation of Insulin and Linker, excess Linker needs to be removed to avoid direct reaction between excess Linker and OsrHSA. Since the molecular weight of Linker is generally small, it can be removed by desalting, dialysis, ultrafiltration, etc. Since dialysis is not conducive to industrial scale-up, desalting and ultrafiltration were chosen for comparative research. The details are as follows: Weigh 320mg of Insulin dry powder, add about 20ml of 4mmol/L dilute hydrochloric acid (pH2.0) to fully dissolve it, then slowly adjust the pH to 7.2 with 0.5mol/L NaOH solution under stirring conditions (the pH cannot exceed 8.0), and then Add coupling buffer (20mmol/LPB, 2mmol/LEDTA, pH7.2) to 80ml, stir and mix, and the protein content is 4mg/ml. Then divide the above Insulin solution into 2 equal parts, each part is 40 ml. According to the mass molar ratio of Insulin to EMCS 1:5, under stirring conditions, slowly add 1.38 ml EMCS solution (31 mg/ml) respectively, and stir the reaction at 25°C. After 30 minutes, after the reaction is completed, 5 times the molar amount of Glycine of EMCS is added to terminate the reaction. Using 10kDa and 5kDa ultrafiltration membrane packages respectively, 40ml of Insulin-EMCS reaction product was initially concentrated to the minimum volume (pipeline residual - 15ml). After the initial concentration, add 50 ml of coupling buffer and continue to concentrate to the minimum volume. After changing the medium, use an appropriate amount of dialysate to rinse the membrane and restore the volume to 80 ml (twice the initial volume). The results are shown in Figure 8. Whether it is a 5kDa membrane cassette or a 10kDa membrane cassette, the aggregates increase after ultrafiltration dialysis; the 5kDa membrane cassette recovery rate is about 80%, while the 10kDa membrane cassette recovery rate is only 64%, both lower than G The yield of -25 desalting column is 90%; on the one hand, the low yield of the membrane bag is related to the molecular weight cutoff of the selected membrane bag and the material of the membrane bag. In order to increase the yield, an ultrafiltration membrane bag of 1-3kDa can be selected; but the molecular weight cutoff is higher Small membrane cassette ultrafiltration takes longer; considering the yield and operating time, it is more appropriate to choose a desalting column such as G-25.

Example 6 OsrHSA-EMCS-Insulin multimer removal process

In Example 2, Phenyl HP is loaded with 0.25M ammonium sulfate. The dimer content of the collection liquid is only about 2-2.5%, but its maximum loading amount is only 3 mg/ml. In Example 4, the ammonium sulfate in the loading liquid is Increasing the concentration to 0.5M, the loading capacity increased to 20 mg/ml (5-6 times increase), but the dimer increased to 5-6%.

Based on the above-mentioned results, the ammonium sulfate concentration in the loading solution was optimized in order to obtain a process with higher loading capacity and lower polymer and dimer content. The reaction solution of OsrHSA and Insulin-EMCS was prepared according to the above examples, and the purification conditions were optimized according to the following conditions: (1) 0.45mol/L ammonium sulfate was loaded, and 0.4mol/L ammonium sulfate was used to wash impurities; (2) 0.4mol/L sulfate Ammonium was loaded into the sample, and 0.35mol/L ammonium sulfate was used to wash out the impurities; (3) 0.35mol/L ammonium sulfate was loaded into the sample, and 0.3mol/L ammonium sulfate was used to wash out the impurities. The results are shown in Figure 9. As the concentration of ammonium sulfate in the loading solution decreases, the maximum loading volume also decreases. The maximum loading volume for 0.35mol/L and 0.4mol/L ammonium sulfate is about 12mg/ml. Filling, and the maximum loading amount of 0.45mol/L ammonium sulfate is about 20mg/ml filling; the impurities and elution collection liquids under various conditions were tested by SEC-HPLC, among which 0.45mol/L ammonium sulfate was used. When the sample was loaded with 0.4 mol/L ammonium sulfate, the aggregate content in the eluate was 11.6%, and the polymer content in the eluate was 7.6%, and the polymer content in the eluate was 7.6%. The solid content is 3.6%; when 0.35mol/L ammonium sulfate is loaded, the polymer content of the eluate is 7.6% and the polymer content of the eluate is 3.6%. The above results show that the lower the concentration of ammonium sulfate in the loading solution, the lower the content of dimers and polymers in the eluate; although the electrophoresis band of the eluate shows that it is mainly the target protein, the SEC-HPLC results It shows that the content of aggregates is significantly higher than that of the eluent. Due to the addition of impurity washing steps, the yield of Insulin has decreased to about 25%. From the above optimization, it can be seen that dimers and multimers are mainly removed through sample loading and impurity washing, and their content is related to the chromatography yield. There is a negative correlation with the loading amount.

Example 7 Insulin Different Linker Coupling Processes

The above embodiments all use EMCS as the linker. In order to prove that other linkers also have the same or better coupling effect, Br-EMCS, an analog of EMCS, and SMB, an analog of SMCC, were selected as test objects.

Weigh 340mg of Insulin dry powder, add 34ml of 4mmol/L dilute hydrochloric acid (pH2.0) to fully dissolve it, slowly adjust the pH to 7.2 with 0.5mol/L NaOH solution under stirring conditions, and then add PB-1 to 85ml (Insulin final Concentration 4mg/ml), adjust the pH to 7.10-7.20, stir and mix; divide the above Insulin solution into 2 parts, respectively according to the molar ratio of Insulin to SMB or Br-EMCS 1:5, slowly add dropwise under stirring conditions 1. 242ml SMB solution (100mmol/L) or Br-EMCS solution (100mmol/L), stir and react for 30 minutes at room temperature. After the reaction is completed, add Glycine to terminate the reaction. Use G-25 desalting column (XK26/40, column volume 175ml; flow rate 15ml/in) to remove excess SMB or Br-EMCS.

Use coupling buffer to dilute the desalted Insulin-SMB or Insulin-Br-EMCS to about 715 ml, then add 15 ml of OsrHSA (240 mg/ml) according to the mass ratio of Insulin: OsrHSA 1:21 (after adding OsrHSA, add The final concentration in the system is 4-5 mg/ml), and the reaction is stirred for 2 hours at room temperature. Use sodium sulfate to adjust the conductance of the reaction solution to 77±2mS/cm, then dilute it with balancing solution to about 1mg/ml (calculated as OsrHSA, a total of about 3570ml), adjust the pH to 6.0-6.1 with dilute hydrochloric acid, and filter with 0.22μm micropores After membrane filtration, it is the Phenyl HP chromatography loading solution. Phenyl HP chromatography (GCC-40-200; column height 14cm; column volume 175ml; flow rate 30ml/min) steps are as follows: Equilibration: use 3-5CV equilibrium solution (10mmol/PB, 0.5mol/L ammonium sulfate, conductivity 75- 79 mS/cm, pH 6.0) to flush the equilibrium chromatography column until the baseline, elution pH and conductivity are stable; load the sample: load the sample to the chromatography column with a loading volume of about 3500ml (20CV); rebalance: use 5CV equilibrium solution Rinse the chromatography column until the baseline, elution pH and conductivity are stable; wash impurities: wash the chromatography column with 3CV of impurity washing solution (10mM PB, 0.45mol/L ammonium sulfate, conductivity 62-66mS/cm, pH 6.0) ; Elution: Rinse the chromatography column with eluent (10mmol/L, 50mmol/L ammonium sulfate, conductivity 10-12mS/cm, pH 6.0), reduce to 20-30mAu and stop collection;

Use a 50kDa ultrafiltration membrane bag (Sartorius, vivaflow200, PES material) to concentrate the collection liquid (-700ml) to about 20ml, then add at least 2 times the volume of dialysate, mix evenly and concentrate again to 20ml, repeat 7 Second-rate. After dialysis, concentrate the concentrate to a smaller volume (about 15 ml), then empty the membrane bag, collect the concentrate, and store it at -80°C for later use. The coupling product and purified electrophoresis pattern of OsrHSA and Insulin using Br-EMCS or SMB as the linker are shown in Figure 10.

Example 8 Glucose Tolerance Test (IPGTT)

Rat glucose tolerance test (IPGTT) was performed on OsrHSA-EMCS-Insulin (prepared according to Example 4). The experiment was divided into 3 groups, control group and experimental group, with 6 SD rats in each group. The experimental group was given a subcutaneous injection at a dose of 125 nmol/kg, and the control group was given the same volume of placebo saline. Do not eat or drink anything 16 hours before the glucose tolerance test. Blood was collected from the orbital venous plexus of rats, and the blood collection time points were -0.5h, 0h, 0.5h, 1h, 2h, 4h, and 6h respectively; -0.5h was the fasting blood glucose concentration before administration, and 0h was 0.5h after administration. Afterwards, the blood glucose concentration was measured before intraperitoneal injection of 20g/kg glucose, and the blood glucose concentration was measured at 0.5h, 1h, 2h, 4h, and 6h after glucose injection. The results show (Figure 11) that compared with the Insulin group and the negative control group, the efficacy of OsrHSA-EMCS-Insulin can last until 45-49h, suggesting that OsrHSA-EMCS-Insulin has a longer efficacy; after 5 rounds of IPGTT, The blood glucose concentration of OsrHSA-EMCS-Insulin was basically consistent with that of the control group, indicating that the action time of OsrHSA-EMCS-Insulin in rats was approximately 24 hours.

Example 9 Study on the efficacy and pharmacokinetics of OsrHSA-EMCS-Insulin in SD rats.

In order to prove the long-term effect and effectiveness of OsrHSA-EMCS-Insulin, SD rats were selected as the research subjects for testing. The specific operation is as follows: 7-week-old SD male rats weighing 200g-250g are randomly divided into groups, and under non-fasting conditions, a single subcutaneous injection of different doses of OsrHSA-EMCS-Insulin (prepared according to Example 4) and the same volume Normal saline (negative control), then take blood from the rat orbital venous plexus at 4h, 8h, 24h, 32h and 48h after administration, treat it at 37℃ for 30min, then centrifuge at 4000rpm for 15min, take the supernatant and store it at -80℃ save. Blood glucose and insulin content were measured using blood glucose test strips (Roche) and Insulin ELISA kit (R&D, DY8056-05). The results are shown in Figure 12. OsrHSA-EMCS-Insulin showed an obvious dose effect. The greater the dose, the better the hypoglycemic effect and the longer the duration. At the highest dose of 1000 nmol/kg, the hypoglycemic effect in SD rats can be Maintain for 32-48h. The blood drug (Insulin) concentration measurement results showed that the Insulin concentration in the blood was positively correlated with the dosage within 0-32 hours. After 48 hours, the Insulin content in the blood of the three groups tended to the background level; changes in the Insulin content in the blood were related to changes in blood glucose concentration. The trend remains basically the same.

[Example 10] Preparation of recombinant human serum albumin and recombinant human insulin conjugate (OsrHSA-SPDP-Insulin)

Recombinant human insulin (Insulin) conjugated to SPDP

Take 42ml of Insulin solution (Hefei Tianmai Biotechnology Development Co., Ltd., 0.4mmol/L, dissolved in coupling solution), and then slowly add 8ml of N-succinimide dropwise according to the molar ratio of Insulin to SPDP molecules of 1:5. 3-(2-Pyridyldithio)propionate (SPDP) solution (100mmol/L, DMSO dissolved), stir and react at room temperature for 1 hour, then add 5 times the molar amount of Glycine solution of SPDP to terminate the reaction.

Insulin-SPDP conjugated with OsrHSA

After the reaction, use Bestdex G-25M desalting column to remove excess SPDP and DMSO. Add 3.88 ml of OsrHSA solution (3 mmol/L) to the Insulin-SPDP obtained after desalting at a molar ratio of 1:1 to OsrHSA molecules, mix thoroughly, and let stand for 18 hours at 4°C.

Purification of OsrHSA-SPDP-Insulin coupling product

Use 3M ammonium sulfate to adjust the conductivity of the above reaction solution to be consistent with the balance solution (10mM PB, 0.2M ammonium sulfate, pH 6.5), then dilute it with the balance solution to about 1mg/ml (based on OsrHSA), and adjust the conductivity to 15ml/min. Load the sample at the flow rate to a chromatography column filled with 176ml Phenyl Bestrose HP chromatography medium and a column height of 25cm. After loading the sample, balance the column again with balancing solution until the UV is basically consistent with the baseline. Use 10mM PB, 0.18M ammonium sulfate, pH 6.5 eluent to remove dimers, and finally use 10mM PB, pH 7.2 eluent to elute the target protein. The elution collection liquid was concentrated using a 50kDa ultrafiltration membrane bag and the liquid was changed to obtain the OsrHSA-SPDP-Insulin conjugate stock solution. The purification chromatogram (A) and electrophoresis detection results (B) of the OsrHSA-SPDP-Insulin coupling product are shown in Figure 13.

[Example 11] Study on the hypoglycemic effect of OsrHSA-SPDP-Insulin in diabetic mouse model

In order to prove the long-term effect of OsrHSA-SPDP-Insulin, the male BSK-DB diabetic mouse model was selected for study. OsrHSA-SPDP-Insulin was prepared according to Example 7. The mice were fasted overnight without water, and there were 5 mice in each experimental group. A single dose was administered by subcutaneous injection, in which the dosage of OsrHSA-SPDP-Insulin was set at 25IU/kg and 50IU/kg (assuming that the activity of Insulin remains unchanged after conjugation with OsrHSA, the activity of Insulin is 28IU/mg), positive The dosage of control Insulin was 10IU/kg, and the negative control group was given the same volume of normal saline. The tail vein blood of mice was collected before administration and 8 hours after administration, and blood glucose was measured using Roche blood glucose meter and test strips. Draw a blood sugar change curve based on the change value (Tn/T0) of blood sugar value (Tn) relative to the initial blood sugar value (T0) at different time points. As shown in Figure 14, the blood sugar of the Insulin control group dropped to the lowest level 2 hours after administration, and returned to the same level as the normal saline group around 4 hours; while OsrHSA-SPDP-Insulin (OsrHSA-Insulin in the legend) showed a certain dose effect and lowered blood sugar. The effect is more obvious and the action time is significantly prolonged (more than 8h). This example proves that the HSA conjugate can significantly extend the action time of the conjugated drug.

The present invention illustrates detailed preparation methods of insulin analogs through the above examples, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must rely on the above detailed preparation methods to be implemented. Those skilled in the art should understand that any improvements to the present invention, replacement of linkers and insulin in the present invention, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims (7)

1. A human serum albumin-insulin conjugate having the following structure: HSA-Linker-Insulin in: HSA is recombinant human serum albumin expressed from rice seeds, Linker is a small molecule linker with bifunctional groups, and Insulin is insulin; The small molecule linker with bifunctional groups is 6-(maleimido)hexanoic acid succinimide ester EMCS. 2. The method for preparing the human serum albumin-insulin conjugate according to claim 1, comprising the following steps: 1) Perform a coupling reaction between insulin and a bifunctional linker to obtain an insulin-bifunctional linker intermediate coupling product; 2) React the insulin-bifunctional linker intermediate coupling product obtained in step 1) with recombinant human serum albumin to obtain the final coupling product of recombinant human serum albumin and insulin; 3) Purify the final coupling product obtained in step 2) to obtain the human serum albumin-insulin conjugate. 3. The method according to claim 2, characterized in that in the coupling reaction of steps 1) and 2), the reaction molar ratio of insulin, bifunctional linker and recombinant human serum albumin is insulin:bifunctional linker. : Recombinant human serum albumin=1:5:2. 4. The method according to claim 2, wherein after the step 1), it further includes concentrating the insulin-bifunctional group linker reaction solution of step 1) by desalting or ultrafiltration to remove excess bifunctional linker and In the step of using an organic solvent in the reaction, an intermediate coupling product of insulin-bifunctional linker is obtained. 5. The method of claim 2, wherein step 3) includes: 3a) Purify the final coupling product obtained in step 2) using a hydrophobic chromatography medium, and the hydrophobic chromatography medium is Phenyl HP; 3b) Use an ultrafiltration membrane to concentrate the purified liquid obtained in step 3a) to obtain human serum albumin-insulin conjugate. 6. The method according to claim 2, comprising the steps: (1) Coupling of insulin and EMCS: According to the molar ratio of insulin to EMCS of 1:5, add the EMCS solution to the insulin solution to perform the coupling reaction. After the reaction is completed, add glycine to terminate the reaction; (2) Excess EMCS removal: Use G-25 desalting column to remove excess EMCS after the reaction in step (1); (3) Insulin-EMCS coupled with recombinant human serum albumin: Add recombinant human serum albumin to the desalted insulin-EMCS in step (2) according to the molar ratio of insulin-EMCS: human serum albumin of 1:2 to perform a coupling reaction. After the reaction is completed, Cys is added to terminate the reaction; (4) Purification of coupling products: The coupling product obtained in step (3) is purified using a Phenyl HP chromatography column. The chromatography conditions are: Balance solution: 10mmol/LPB, 0.5M ammonium sulfate, pH 6.5; Eluent: 10mmol/LPB, 0.025M ammonium sulfate, pH7.2; CIP: H ₂ O; After concentrating the elution collection liquid with a 50kDa ultrafiltration membrane bag, add 10mmol/L PB at pH 7.2 for concentration and dialysis, and repeat to obtain the human serum albumin-insulin conjugate. 7. A pharmaceutical composition containing the human serum albumin-insulin conjugate of claim 1.