WO2024109534A1

WO2024109534A1 - Human serum albumin-insulin conjugate and preparation method therefor

Info

Publication number: WO2024109534A1
Application number: PCT/CN2023/130071
Authority: WO
Inventors: 董亮亮; 杨代常; 夏军; 陈蓉; 徐伟
Original assignee: 武汉禾元生物科技股份有限公司
Priority date: 2022-11-24
Filing date: 2023-11-07
Publication date: 2024-05-30
Also published as: CN115894719A; CN115894719B

Abstract

Provided is a human serum albumin-insulin conjugate, having the following structure: HSA-Linker-Insulin, wherein HSA is recombinant human serum albumin, Linker is a small molecule linker with bifunctional groups, and Insulin is insulin; compared to existing insulin, the half-life and hypoglycemic maintenance time of the insulin-human serum albumin conjugate product are significantly prolonged, such that the frequency of administration can be reduced in the future, increasing patient compliance.

Description

Human serum albumin insulin conjugate and preparation method thereof

Technical Field

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

Background technique

Diabetes is an important chronic disease that currently poses a serious threat to human health, and its incidence is rising at an unprecedented rate. According to WHO statistics, the prevalence of diabetes in my country has risen from 0.67% in 1980 to 12.8% in 2020, and the prevalence continues to rise. By 2020, the number of patients reached 129.8 million, accounting for about 28% of the global number of diabetes patients, ranking first in the world.

Insulin is the only way to treat late-stage diabetes. An expert once commented: "The proportion of type 2 diabetes patients in a country who take insulin reflects the level of diabetes treatment in that country." However, the reality is that the use rate of insulin in my country is only about 2%, far lower than the 30% use rate in the United States. In addition to the high cost of treatment, the fear of injections and the inconvenience of injections make many patients refuse to use insulin treatment. In addition, many details of the patient's use of insulin, such as whether the dosage is accurate, whether the injection timing is appropriate, and whether the blood sugar target adjustment is reasonable, will also affect the effectiveness and safety of insulin treatment. Therefore, the development of longer-acting insulin and the reduction of injection frequency will help increase patients' desire for insulin treatment, control blood sugar steadily for a long time, and ultimately improve patients' prognosis.

Patent WO2016178905A1 "Fusion Protein" describes a method for preparing an Fc-fused insulin analog, which fuses an engineered single-chain insulin analog to the IgG Fc region to achieve a long-term effect once a week. Its related product LY3209590 injection has been accepted for clinical application by the Drug Review Center of the China National Medical Products Administration 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 injection into the human body, Icodec will tightly but reversibly bind to human serum albumin (HSA) to form a "circulating storage reservoir." This result can continuously, slowly and steadily lower blood sugar within a week. Based on its concentrated formula, the dosage of icodec insulin injected once a week is equivalent to that of insulin glargine U100 injected once a day.

HSA is the single component protein with the highest content in human blood, with a blood content of about 50g/L, accounting for 40% to 60% of total plasma protein, and a half-life of up to 19 days. The long half-life of HSA is attributed to the FcRn receptor-mediated HSA recycling mechanism (pH-dependent, preventing lysosomal degradation) and its ability to avoid renal clearance (HSA can be reabsorbed through receptor-mediated endocytosis in the renal proximal tubules, thereby avoiding renal clearance).

HSA plays an irreplaceable role in human blood with its stable inertness. In recent years, the use of HSA to extend the half-life of drugs in clinical therapeutic drug research and development has received increasing attention. For example, both degludec and icodec use endogenous HSA to extend the duration of drug action. 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 drugs.

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

Summary 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 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

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 a bifunctional group is one of the following: 6-(maleimido)hexanoic acid succinimidyl ester (EMCS), 6-(3-bromomaleimido)hexanoic acid succinimidyl ester (Br-EMCS), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), 4-maleimidobenzoic acid succinimidyl ester (SMB), 3-(2-pyridyldithiol)propionic acid N-hydroxysuccinimidyl ester (SPDP) or their derivatives.

The human serum albumin-insulin conjugate of the present invention preferably comprises a recombinant human serum albumin derived from a plant, and in particular, the recombinant human serum albumin is derived from recombinant human serum albumin expressed in rice endosperm cells (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, preferably human insulin.

In the human serum albumin-insulin conjugate of the present invention, the linker is preferably 6-(maleimido)hexanoic 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 of the present invention comprises the following steps:

1) subjecting insulin to a coupling reaction with a bifunctional linker to obtain an insulin-bifunctional linker intermediate coupling product;

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

3) Purifying 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 bicoronavirus linker is EMCS or SPDP, and in the coupling reaction of step 1) and step 2), the reaction molar ratio of insulin, bicoronavirus linker and recombinant human serum albumin is insulin: bicoronavirus linker: recombinant human serum albumin = 1:5:2.

The method of the present invention further comprises, after step 1), a step of removing excess bifunctional linker and organic solvent in the reaction by desalting or ultrafiltration concentration of the insulin-bifunctional linker reaction solution of step 1) to obtain an insulin-bifunctional linker intermediate coupling product.

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

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

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

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

(1) Insulin coupling with EMCS

According to the molar ratio of insulin to EMCS of 1:5, the EMCS solution was added to the insulin solution for coupling reaction. After the reaction was completed, glycine was added to terminate the reaction;

(2) Excess EMCS removal

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

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

The insulin-EMCS desalted in step (2) is added with recombinant human serum albumin at a molar ratio of 1:2 of insulin-EMCS to human serum albumin for coupling reaction, and Cys is added after the reaction is completed to terminate the reaction;

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

The coupling product obtained in step (3) was purified using a Phenyl HP chromatography column under the following chromatography conditions:

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

Eluent: 10 mmol/LPB, 0.025 M ammonium sulfate, pH 7.2;

CIP: _H2O ;

The eluted solution was concentrated using a 50 kDa ultrafiltration membrane, and then concentrated and dialyzed by adding 10 mmol/LPB at pH 7.2. The concentration was repeated to obtain the OsrHSA-EMCS-Insulin conjugate.

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

The present 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 blood sugar lowering effect and a greatly prolonged action time, which can reduce the frequency of administration and improve patient compliance. At the same time, the preparation method has a simple process, good consistency, and is easy to scale up in later production.

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

Figure 3 is the chromatogram of Phenyl HP loading determination (A) and electrophoresis detection results (B);

In (B), L represents the loading solution, and 1-6 represents the flow-through solution when 1-6 column volumes are loaded.

FIG4 is a chromatogram of different sample loading amounts and SDS-PAGE detection results;

A and B, chromatogram of 5 mg/ml filler (A) and SDS-PAGE detection result (B);

C and D, chromatogram of the filler with a sample load of 3 mg/ml (C) and SDS-PAGE detection results (D);

In the figure, M represents the molecular weight marker, L represents the loading solution; FT1-FT4 represent the penetration solutions 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 represents the regeneration solution.

Figure 5 is a process validation of three batches of low-load. A, chromatogram, B, SDS-PAGE test results (1-3 represent different batches, M is the molecular weight marker);

FIG6 is the SDS-PAGE detection result of the high conductivity sample flowthrough; L represents the sample flowthrough, M represents the molecular weight marker, and 17-25 represents the flowthrough when 17-25 column volumes are loaded.

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

Figure 8 shows the SDS-PAGE test results of ultrafiltrates from different membrane packages; M is the molecular weight marker; INS is the Insulin control; "Before" indicates the sample before ultrafiltration and concentration; "Permeate" indicates the permeate from the membrane package during the first concentration; "Together" indicates the permeate from the membrane package after ultrafiltration 1-4 times; 5-7 indicates the permeate from the membrane package after ultrafiltration 5-7 times; and "After" indicates the sample after the final ultrafiltration and concentration.

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

Figure 10 Phenyl HP purification chromatograms and SDS-PAGE results after coupling with different linkers. A, OsrHSA-SMB-Insulin chromatogram; B, OsrHSA-SMB-Insulin chromatographic sample SDS-PAGE test results; C, OsrHSA-Br-EMCS-Insulin chromatographic sample SDS-PAGE test results. M represents molecular weight marker; Load represents loading solution; FT1 represents the penetration solution at the end of loading; FT2 represents the penetration solution at the end of loading and rebalancing; Wash represents washing solution; Elu1 represents elution collection solution; Elu2 represents the tailing part of elution collection solution; CIP represents regeneration solution.

Figure 11 is a glucose tolerance test in rats. A, schematic diagram of blood sampling time; B, blood glucose concentration curve at different times);

Figure 12 is a blood glucose (A) and blood drug concentration (B) curve of rats after a single dose under non-fasting conditions;

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

FIG. 14 is a blood glucose change curve of diabetic mice after subcutaneous injection of OsrHSA-SPDP-Insulin.

Detailed ways

The present invention is further described below in conjunction with specific examples. It should be noted that these examples are provided only to illustrate the content of the present invention, and do not limit the remaining content disclosed by the present invention in any way.

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

Example 1 Coupling process of insulin and OsrHSA

(1) Insulin-EMCS coupling

Take 3 tubes of 7ml Insulin solution (0.4mmol/L), and slowly add 28μl, 70μl, and 140μl EMCS solution (100mmol/L, prepared in DMSO) under stirring conditions according to the ratio of Insulin to EMCS molecular molar ratio of 1:1, 1:2.5, and 1:5, and react at 25℃. Take 2ml of sample at 0.5h, 1h, and 2h of reaction, and add 10 times the molar amount of Glycine of EMCS to terminate the reaction. Use G-25 desalting column (column volume 25ml, flow rate 5ml/min) to remove excess EMCS. Add 0.186ml OsrHSA injection solution (3mmol/L) to the desalted Insulin-EMCS according to the ratio of OsrHSA to Insulin-EMCS molecular molar ratio of 1:1, and react at room temperature for 1h. After the reaction, take an appropriate amount of 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 (indicated by the red (lower) arrow). When the coupling ratio of the two is 5:1, the coupling efficiency of OsrHSA is close to 50%. The coupling time of EMCS to Insulin has no significant effect 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 (indicated by the yellow (upper) arrow) also increase.

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

Take 2 tubes of 11 ml Insulin solution (0.4 mmol/L) respectively, and slowly add 44 μl and 220 μl EMCS solution (100 mmol/L) under stirring conditions according to the molar ratio of Insulin to EMCS of 1:1 and 1:5, and react at 25℃ for 0.5 h. Add 10 times the molar amount of Glycine to terminate the reaction. Use G-25 desalting column (column volume 52 ml, flow rate 4 ml/min) to remove excess EMCS. The desalted Insulin-EMCS sample is divided into 3 tubes per group, each with 5 ml. According to the molar molecular ratio of OsrHSA to Insulin-EMCS of 1:2, 1:1, and 2:1, add 0.103 ml, 0.205 ml, and 0.411 ml of OsrHSA (3 mmol/L) respectively, react at 25℃, take 2 ml of sample at 1h and 2h of reaction, and add 10 times the molar amount of L-cysteine to terminate the reaction. All reaction solutions were diluted to the same concentration (based on OsrHSA) and subjected to SDS-PAGE detection at the same spotting volume. The results are shown in Figure 2. The reaction time of OsrHSA and Insulin-EMCS has little effect on the coupling efficiency. At 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 that of 1:5 coupling. When the coupling ratio of Insulin to EMCS is 1:5, the ratio of the coupling product in electrophoresis is consistent with that of OsrHSA to Insulin-EMCS when the coupling ratios are 0.5:1 and 1:1, but because the reaction volume of the 1:1 coupling product is twice that of 0.5:1, the amount of OsrHSA-EMCS-Insulin obtained in the 1:1 reaction is more than that of 0.5:1. Similarly, from the electrophoresis, when the coupling ratio of OsrHSA to Insulin-EMCS is 2:1, OsrHSA-EMCS-Insulin is slightly lower than 1:1, but because its volume is twice that of 1:1, the amount of its 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 9 ml of Insulin solution (4 mg/ml, 0.689 mmol/L), and slowly add 310 μl of EMCS solution (100 mmol/L) under stirring conditions according to the molar ratio of Insulin to EMCS of 1:5 (converted to a mass ratio of 1:0.265). Stir and react at room temperature for 30 minutes. After the reaction is completed, add 5 times the molar amount 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.

The desalted Insulin-EMCS was added with 4 ml of OsrHSA injection solution (3 mmol/L) at a molecular molar ratio of 1:2 to Insulin-EMCS: OsrHSA, and reacted at room temperature for 30-60 min. After reacting for 45 min, Cys with a molar amount of 5 times that of OsrHSA was added to terminate the reaction.

(1) Phenyl HP loading determination

The reaction solution of Insulin-EMCS and OsrHSA was adjusted to 36 mS/cm with 3 mol/L ammonium sulfate, then diluted to about 2 mg/ml (based on OsrHSA protein content) with a balance solution (10 mmol/LPB, 0.2 M ammonium sulfate, pH 6.5), adjusted to pH 6.5 with dilute hydrochloric acid, and filtered with a 0.22 μm microporous filter membrane to obtain the loading solution. The loading solution was loaded onto a Phenyl HP chromatography column (C10/40, column height 20 cm) with a balanced column volume (CV) of 15 ml at a flow rate of 3 ml/min, and collected with a distribution collector at 15 ml/tube (i.e., 1 CV/tube). A total of about 12 CV was loaded. Elution was performed with 10 mmol/LPB and regeneration was performed with H ₂ O. As shown in FIG3 , when the protein content of the sample solution is about 2 mg/ml and the conductivity is about 36 mS/cm (equivalent to about 0.2 mol/L ammonium sulfate), the loading capacity of Phenyl HP is only about 3 CV, which is equivalent to about 6 mg protein/ml filler.

(2) Load confirmation

In order to further determine the loading capacity of Phenyl HP, the loading solution was diluted to about 1 mg/ml, and the loading amount of 5 mg/ml filler was used for confirmation using an XK16/40 chromatography column (column volume 40 ml). The results are shown in Figure 4-A-B. During the re-equilibrium process after loading, the penetration peak (FT2) was significantly raised. After continuing the equilibrium, obvious target protein shedding (FT3) was observed. As the equilibrium volume increased, the penetration peak was basically maintained at around 20 mAu (FT4), and the target protein began to fall off in large quantities. The purity of (Elu) in the eluate was relatively high. Considering the overload of loading according to 5 mg/ml filler, the loading amount was reduced to 3 mg/ml filler, and the results are shown in Figure 4-C-D. When the loading amount was reduced to 3 mg/ml filler, there was no obvious penetration of the target protein during the re-equilibrium process. The purity of the target protein in the main peak of the eluate (Elu1) was relatively high. According to calculation, the yield of insulin is 17.7% when the sample is loaded on 5 mg/ml filler, while the yield of insulin is 32.3% when the sample is loaded on 3 mg/ml filler.

(3) Low-load process validation

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

Example 3 OsrHSA-EMCS-Insulin high-load 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 scale-up of the later process. Therefore, the loading capacity of Phenyl HP is increased by increasing the conductivity of the loading solution.

According to Example 2, the reaction solution of OsrHSA and Insulin-EMCS was prepared, 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, pH6.5), and a 16ml Phenyl HP chromatography column (C10/40) was used for loading determination. The results are shown in Figure 6. The 23rd tube (15ml/tube) shows a relatively obvious OsrHSA-EMCS-Insulin coupling product. The sample collected in each tube is 15ml, that is, the loading capacity is (23×15ml)/16ml=21.56, that is, the maximum loading amount is 20CV (about 1CV remains in the pipeline), that is, 20mg (in terms of OsrHSA)/ml filler, which is significantly improved compared with Example 2.

Example 4 Verification of high-load preparation process of OsrHSA-EMCS-Insulin

(1) Insulin-EMCS coupling

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

(2) Excess EMCS removal

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

(3) Insulin-EMCS coupling with OsrHSA

The desalted Insulin-EMCS was diluted to about 1600 ml with coupling buffer, and then 50.9 ml of OsrHSA (153 mg/ml) was added according to the mass ratio of Insulin-EMCS: OsrHSA of 1:22.9 (molar ratio of 1:2) (the final concentration of OsrHSA in the system was 5 mg/ml after addition). After reacting at 25°C for 50 minutes, Cys was added with a molar amount of 5 times that of OsrHSA to terminate the reaction.

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

The reaction solution of the coupling product was diluted to about 1 mg/ml (calculated as OsrHSA) and purified using a Phenyl HP chromatography column. The chromatography conditions were as follows: chromatography column: GCC-50-400; column height 20 cm; column volume (CV) 390 ml; flow rate 35 ml/min. Sample volume: about 7.8 L, 20 CV; equilibrium solution: 10 mmol/LPB, 0.5 M ammonium sulfate, pH 6.5 (re-equilibrium volume: 3 CV, 1170 ml); eluent: 10 mmol/LPB, 0.025 M ammonium sulfate, pH 7.2 (collected until UV30 mAu); CIP: H ₂ O; the eluted collection solution was concentrated to a smaller volume using a 50 kDa ultrafiltration membrane, and then at least 2 volumes of 10 mmol/LPB (pH 7.2) were added, concentrated and dialyzed, and repeated 7 times to obtain the OsrHSA-EMCS-Insulin stock solution.

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

Table 1 Chromatographic yields of three batches of high-load process validation

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

Example 5 Linker removal process

After the coupling of Insulin and Linker, the excess Linker needs to be removed to avoid direct reaction of the excess Linker with OsrHSA. Since the molecular weight of Linker is generally small, it can be removed by desalting, dialysis, ultrafiltration and other methods. Since dialysis is not conducive to industrial scale-up, desalting and ultrafiltration methods are selected for comparative study. The details are as follows: weigh 320 mg of Insulin dry powder, add about 20 ml of 4 mmol/L dilute hydrochloric acid (pH2.0) to fully dissolve, and under stirring conditions, use 0.5 mol/L NaOH solution to slowly adjust the pH to 7.2 (pH cannot exceed 8.0), and then add coupling buffer (20 mmol/LPB, 2 mmol/LEDTA, pH7.2) to 80 ml, stir and mix, the protein content is 4 mg/ml. Then the above insulin solution was divided into 2 equal parts, each of which was 40 ml. According to the molar ratio of insulin to EMCS of 1:5, 1.38 ml of EMCS solution (31 mg/ml) was slowly added dropwise under stirring conditions. The reaction was stirred at 25°C for 30 minutes. After the reaction was completed, 5 times the molar amount of EMCS was added to terminate the reaction. 10kDa and 5kDa ultrafiltration membrane packs were used to initially concentrate 40 ml of insulin-EMCS reaction product to the minimum volume (pipeline residue - 15 ml). After the initial concentration was completed, 50 ml of coupling buffer was added and the concentration was continued to the minimum volume. After the liquid was changed, the membrane was rinsed with an appropriate amount of dialysate and the volume was restored to 80 ml (twice the initial volume). The results are shown in Figure 8. Regardless of whether it is a 5kDa membrane package or a 10kDa membrane package, the aggregates increase after ultrafiltration dialysis; the yield of the 5kDa membrane package is about 80%, while the yield of the 10kDa membrane package is only 64%, both lower than the 90% yield of the G-25 desalting column; on the one hand, the low yield of the membrane package is related to the selected molecular weight cutoff and membrane package material. In order to improve the yield, a 1-3kDa ultrafiltration membrane package can be selected; but the ultrafiltration time required for the membrane package with a smaller molecular weight cutoff is longer; considering the yield and operation time, it is more appropriate to choose a desalting column such as G-25.

Example 6 OsrHSA-EMCS-Insulin polymer removal process

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

Based on the above results, the concentration of ammonium sulfate in the loading solution was optimized in order to obtain a process with higher loading capacity and lower content of polymers and dimers. OsrHSA and Insulin-EMCS reaction solutions were prepared according to the above examples, and the purification conditions were optimized according to the following conditions: (1) 0.45 mol/L ammonium sulfate loading, 0.4 mol/L ammonium sulfate washing; (2) 0.4 mol/L ammonium sulfate loading, 0.35 mol/L ammonium sulfate washing; (3) 0.35 mol/L ammonium sulfate loading, 0.3 mol/L ammonium sulfate washing. The results are shown in Figure 9. As the concentration of ammonium sulfate in the loading solution decreases, the maximum loading amount also decreases. The maximum loading amount of 0.35mol/L and 0.4mol/L ammonium sulfate loading is about 12mg/ml filler, while the maximum loading amount of 0.45mol/L ammonium sulfate loading is about 20mg/ml filler. The washing liquid and elution collection liquid under each condition were subjected to SEC-HPLC detection. When 0.45mol/L ammonium sulfate was loaded, the aggregate content in the washing liquid was 11.6%, and the polymer content in the eluate was 4.0%; when 0.4mol/L ammonium sulfate was loaded, the polymer content in the washing liquid was 7.6%, and the polymer content in the eluate was 3.6%; when 0.35mol/L ammonium sulfate was loaded, the polymer content in the washing liquid was 7.6%, and the polymer content in the eluate was 3.6%. The above results show that the lower the ammonium sulfate concentration in the loading solution, the lower the content of dimers and polymers in the eluent; although the electrophoretic bands of the washing solution show that it is mainly the target protein, the SEC-HPLC results show that the content of its aggregates is significantly higher than that of the eluent. Due to the addition of the washing step, the yield of Insulin is reduced to about 25%; through the above optimization, it can be seen that dimers and polymers are mainly removed by loading penetration and washing, and their content is negatively correlated with the chromatography yield and the amount of loading.

Example 7 Insulin different linker coupling process

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

Weigh 340mg Insulin dry powder, add 34ml 4mmol/L dilute hydrochloric acid (pH2.0) to fully dissolve, and then slowly adjust the pH to 7.2 with 0.5mol/L NaOH solution under stirring conditions, then add PB-1 to 85ml (final concentration of Insulin 4mg/ml), adjust the pH to 7.10-7.20, and stir to mix; divide the above Insulin solution into 2 parts, and slowly add 1.242ml SMB solution (100mmol/L) or Br-EMCS solution (100mmol/L) under stirring conditions according to the molar ratio of Insulin to SMB or Br-EMCS of 1:5, and stir the reaction at room temperature for 30min. 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.

The desalted Insulin-SMB or Insulin-Br-EMCS was diluted to about 715 ml using coupling buffer, and then 15 ml of OsrHSA (240 mg/ml) was added at a mass ratio of 1:21 between Insulin and OsrHSA (the final concentration of OsrHSA in the system was 4-5 mg/ml after addition), and the reaction was stirred at room temperature for 2 hours. The conductivity of the reaction solution was adjusted to 77±2 mS/cm using sodium sulfate, and then diluted to about 1 mg/ml with equilibrium solution (calculated as OsrHSA, a total of about 3570 ml), and the pH was adjusted to 6.0-6.1 with dilute hydrochloric acid. After filtering with a 0.22 μm microporous filter membrane, the sample solution for Phenyl HP chromatography was obtained. The steps of Phenyl HP chromatography (GCC-40-200; column height 14 cm; column volume 175 ml; flow rate 30 ml/min) are as follows: Equilibration: flush the equilibrium column with 3-5 CV equilibrium solution (10 mmol/PB, 0.5 mol/L ammonium sulfate, conductivity 75-79 mS/cm, pH 6.0) until the baseline, elution pH and conductivity are stable; loading: load the sample into the column with a loading volume of about 3500 ml (20 CV ); Re-equilibration: flush the column with 5CV of equilibration solution until the baseline, elution pH and conductivity are stable; Wash: flush the column with 3CV of washing solution (10mM PB, 0.45mol/L ammonium sulfate, conductivity 62-66mS/cm, pH6.0); Elution: flush the column with eluent (10mmol/L, 50mmol/L ammonium sulfate, conductivity 10-12mS/cm, pH6.0) until the concentration drops to 20-30mAu and stops collection;

The collected liquid (-700ml) was concentrated to about 20ml using a 50kDa ultrafiltration membrane package (Sartorius, vivaflow200, PES material), and then at least 2 times the volume of dialysate was added, mixed evenly and concentrated again to 20ml, and repeated 7 times. After the dialysis, the concentrated liquid was concentrated to a smaller volume (about 15ml), and then the membrane package was emptied, the concentrated liquid was collected, and stored at -80℃ for later use. The coupling product of OsrHSA and Insulin with Br-EMCS or SMB as Linker and the purified electrophoresis spectrum are shown in Figure 10.

Example 8 Glucose Tolerance Test (IPGTT)

OsrHSA-EMCS-Insulin (prepared according to Example 4) was subjected to a rat glucose tolerance test (IPGTT). The experiment was divided into 3 groups, a control group and an experimental group, with 6 SD rats in each group. The experimental group was administered subcutaneously at a dose of 125nmol/kg, and the control group was given a placebo saline of the same volume. 16h before the glucose tolerance test, fasting but not water. 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, 6h; wherein -0.5h was the fasting blood glucose concentration before administration, 0h was the blood glucose concentration before intraperitoneal injection of 20g/kg glucose 0.5h after administration, and the blood glucose concentration was measured at 0.5h, 1h, 2h, 4h, 6h after the glucose injection. The results showed (Figure 11) that compared with the Insulin group and the negative control group, the efficacy of OsrHSA-EMCS-Insulin could last up to 45-49 hours, indicating that OsrHSA-EMCS-Insulin had a longer efficacy; after 5 rounds of IPGTT, the blood glucose concentration of OsrHSA-EMCS-Insulin was basically the same as that of the control group, indicating that the action time of OsrHSA-EMCS-Insulin in rats was about 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 research subjects for testing. The specific operation is as follows: 7-week-old SD male rats weighing 200g-250g were randomly divided into groups, and different doses of OsrHSA-EMCS-Insulin (prepared according to Example 4) and an equal volume of normal saline (negative control) were subcutaneously injected under non-fasting conditions, and then blood was collected from the rat orbital venous plexus at 4h, 8h, 24h, 32h and 48h after administration, respectively, treated at 37℃ for 30min, and then centrifuged at 4000rpm for 15min, and the supernatant was taken and stored at -80℃. Blood glucose test strips (Roche) and Insulin ELISA kits (R&D, DY8056-05) were used to measure blood glucose and insulin content. As shown in Figure 12, OsrHSA-EMCS-Insulin showed a significant dose effect. The higher the dose, the better the hypoglycemic effect and the longer the duration. At the highest dose of 1000nmol/kg, the hypoglycemic effect in SD rats can be maintained for 32-48h. The results of blood drug (Insulin) concentration determination showed that the blood insulin concentration was positively correlated with the dose within 0-32h. After 48h, the insulin content in the blood of the three groups tended to the background level; the changes in the insulin content in the blood were basically consistent with the changes in blood glucose concentration.

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

Recombinant human insulin (Insulin) coupled with SPDP

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

Insulin-SPDP coupled to OsrHSA

After the reaction, the excess SPDP and DMSO were removed using a Bestdex G-25 M desalting column. The Insulin-SPDP obtained after desalting was added to 3.88 ml of OsrHSA solution (3 mmol/L) at a molecular molar ratio of 1:1 with OsrHSA, mixed thoroughly, and allowed to react at 4 °C for 18 h.

Purification of OsrHSA-SPDP-Insulin coupling product

The above reaction solution was adjusted to be consistent with the conductivity of the equilibrium solution (10mM PB, 0.2M ammonium sulfate, pH6.5) using 3M ammonium sulfate, and then diluted to about 1mg/ml (in terms of OsrHSA) with the equilibrium solution, and loaded onto a chromatography column filled with 176ml Phenyl Bestrose HP chromatography medium and 25cm in height at a flow rate of 15ml/min. After the loading, the chromatography column was re-equilibrated with the equilibrium solution until the UV was basically consistent with the baseline. The dimer was removed using a washing solution of 10mM PB, 0.18M ammonium sulfate, pH6.5, and finally the target protein was eluted using an elution solution of 10mM PB, pH7.2. The elution collection solution was concentrated and replaced with a 50kDa ultrafiltration membrane to obtain the OsrHSA-SPDP-Insulin conjugate stock solution. The purification chromatogram (A) and electrophoresis detection results (B) of the OsrHSA-SPDP-Insulin conjugate 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, a male BSK-DB diabetic mouse model was selected for study. OsrHSA-SPDP-Insulin was prepared according to implementation 7. Mice were fasted overnight but not water-deprived, with 5 mice in each test group. A single dose was administered by subcutaneous injection, wherein the OsrHSA-SPDP-Insulin dosage was set at 25IU/kg, 50IU/kg (assuming that the activity of Insulin remained unchanged after coupling with OsrHSA, and the activity of Insulin was 28IU/mg), the dosage of the positive control Insulin was 10IU/kg, and the negative control group was given an equal volume of normal saline. Blood was taken from the tail vein of mice before and 8h after administration, and blood glucose was measured using a Roche blood glucose meter and test strips. The blood glucose change curve was drawn 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 Figure 14, the blood sugar level of the insulin control group dropped to the lowest level 2 hours after administration, and recovered to the same level as the saline group at about 4 hours; while OsrHSA-SPDP-Insulin (OsrHSA-Insulin in the legend) showed a certain dose effect, and the hypoglycemic effect was more obvious, and the duration of action was significantly prolonged (greater than 8 hours). This example proves that HSA conjugates can significantly prolong the duration of action of the conjugated drugs.

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

Claims

A human serum albumin-insulin conjugate having the following structure: HSA-Linker-Insulin

in:

HSA is plant-derived recombinant human serum albumin, Linker is a small molecule linker with bifunctional groups, and Insulin is insulin;

The small molecule linker with a bifunctional group is one of the following: 6-(maleimido)hexanoic acid succinimidyl ester (EMCS), 6-(3-bromomaleimido)hexanoic acid succinimidyl ester (Br-EMCS), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), 4-maleimidobenzoic acid succinimidyl ester (SMB), 3-(2-pyridyldithiol)propionic acid N-hydroxysuccinimidyl ester (SPDP) or their derivatives.
The human serum albumin-insulin conjugate according to claim 1, characterized in that the linker is 6-(maleimido)hexanoic acid succinimidyl ester EMCS or N-succinimidyl 3-(2-pyridyldithio) propionate SPDP.
The method for preparing the human serum albumin-insulin conjugate according to claim 1 comprises the following steps:

1) subjecting insulin to a coupling reaction with a bifunctional linker to obtain an insulin-bifunctional linker intermediate coupling product;

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

3) Purifying the final coupling product obtained in step 2) to obtain the human serum albumin-insulin conjugate.
The method according to claim 3, characterized in that the bifunctional linker is EMCS or SPDP, and in the coupling reaction of step 1) and step 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.
The method according to claim 3, wherein after step 1), further comprising the step of removing excess bifunctional linker and organic solvent in the reaction by desalting or ultrafiltration concentration of the insulin-bifunctional linker reaction solution of step 1), to obtain an insulin-bifunctional linker intermediate coupling product.
The method according to claim 3, wherein the step 3) comprises:

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

3b) concentrating the purified solution obtained in step 3a) using an ultrafiltration membrane to obtain a human serum albumin-insulin conjugate.
The method according to claim 3, comprising the steps of:

(1) Insulin coupling with EMCS;

According to the molar ratio of insulin to EMCS of 1:5, the EMCS solution was added to the insulin solution for coupling reaction. After the reaction was completed, glycine was added to terminate the reaction;

(2) Excess EMCS removal;

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

(3) Insulin-EMCS coupled to recombinant human serum albumin;

The insulin-EMCS desalted in step (2) is added with recombinant human serum albumin at a molar ratio of 1:2 of insulin-EMCS to human serum albumin for coupling reaction, and Cys is added after the reaction is completed to terminate the reaction;

(4) Purification of the coupling product;

The coupling product obtained in step (3) was purified using a Phenyl HP chromatography column under the following chromatography conditions:

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

Eluent: 10 mmol/LPB, 0.025 M ammonium sulfate, pH 7.2;

CIP: H 2 O;

The eluted liquid was concentrated using a 50 kDa ultrafiltration membrane, and then concentrated and dialyzed by adding 10 mmol/LPB at pH 7.2. The concentration was repeated to obtain a human serum albumin-insulin conjugate.
[Corrected 01.12.2023 in accordance with Rule 26]
A pharmaceutical composition comprising the human serum albumin-insulin conjugate according to any one of claims 1 to 2.