CN105820233B - Preparation method of insulin derivative - Google Patents
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- CN105820233B CN105820233B CN201510000301.9A CN201510000301A CN105820233B CN 105820233 B CN105820233 B CN 105820233B CN 201510000301 A CN201510000301 A CN 201510000301A CN 105820233 B CN105820233 B CN 105820233B
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Abstract
The invention provides a method for preparing insulin derivatives, which aims at solving the problem of low yield in the existing preparation process of insulin derivatives by using an isoelectric precipitation method, and the method adds divalent or trivalent metal ions into a reaction solution after the coupling reaction of an insulin part and a modification group part is completed, so that crude products of the insulin derivatives or insulin derivative precursors can be completely precipitated from the reaction solution, the loss of the products remained in a supernatant can be reduced to be less than 7 percent of that of a control example (without adding metal ions), and can be reduced to be less than 1 percent or undetectable, and the product yield is greatly improved.
Description
Technical Field
The invention relates to a preparation method of an insulin derivative.
Background
Diabetes is a common endocrine metabolic disease. The prevalence rate of diabetes is increasing in recent years all over the world, and in China, along with the change of life styles and the acceleration of aging processes of people, the prevalence rate of diabetes is more rapidly increasing, and currently, the number of diabetes patients in China exceeds 1.14 hundred million, which brings heavy burden to individuals, families and society.
Diabetic patients generally have relative or absolute insulin secretion deficiency, so insulin therapy has been regarded as an important means for treating diabetes and achieving good blood sugar control. In the last 70 th century, recombinant human insulin was developed by genetic engineering techniques. Later, a series of insulin analogues and derivatives were developed in succession in order to obtain drugs with different time-of-action profiles, i.e. fast or long acting. Wherein an insulin analogue refers to an insulin product obtained by adding, deleting or changing one or more amino acids in the insulin sequence, and an insulin derivative refers to an insulin product obtained by introducing a modifying group such as a long chain fatty acid, polyethylene glycol, an alkyl chain or the like at one or more positions of insulin or an insulin analogue. Compared with the common human insulin, the insulin derivative has longer action time, and the pharmacokinetic curve is more stable and is closer to the secretion mode of the basal insulin under physiological conditions. Examples of the insulin derivatives known so far include insulin detemir and insulin degluin developed by Novonide, and acylated insulin and pegylated insulin developed by lygoden.
In terms of the preparation method, insulin analogs can be directly obtained by genetic engineering, generally by designing the nucleic acid sequence, since they involve only changes in the amino acid sequence of insulin. However, insulin derivatives require the attachment of other modifying groups outside the insulin sequence, which are not usually obtained by genetic engineering together with the insulin moiety, if not a protein or polypeptide. Therefore, the existing methods for preparing insulin derivatives are generally realized by the following steps: firstly, an insulin part and a modification group part are respectively obtained, and then the modification group part and the insulin part are coupled through corresponding chemical reaction to obtain a crude product of the insulin derivative or a crude product of the insulin derivative precursor. Since the volume of the solution for the coupling reaction is generally large and the concentration of the reaction product is relatively low, after the coupling reaction is completed, the reaction product is usually precipitated and separated from the reaction solution, and even dried, and then the subsequent preparation steps, such as further purification, or removal of the protecting group on insulin and/or a modifying group, followed by purification, etc., are performed.
The precipitation separation step described in the above-mentioned production method is usually carried out by using the principle of isoelectric precipitation of insulin in the prior art, i.e., the solution is adjusted to a pH near the isoelectric point of the insulin derivative to precipitate the insulin derivative, and then the precipitate is collected by centrifugation. However, this isoelectric precipitation method does not completely precipitate the reaction product, and still leaves a considerable amount of the insulin derivative or insulin derivative precursor in the supernatant without precipitation, so that a lot of reaction product is lost in this step, directly affecting the final product yield. For example, in example 4 of patent 200480021733.8, the final yield of the insulin derivative is only 12%.
Disclosure of Invention
The invention provides a method for preparing insulin derivatives, which can completely precipitate crude products of insulin derivatives or insulin derivative precursors from reaction liquid after the coupling reaction (hereinafter referred to as coupling reaction) of an insulin part and a modification group part is finished, thereby greatly improving the product yield.
Specifically, the invention provides a preparation method of an insulin derivative, which comprises the step of adding divalent or trivalent metal ions into a reaction solution after the coupling reaction of an insulin part and a modification group part is completed.
Further, the present invention provides a method for preparing an insulin derivative, comprising:
1. respectively obtaining an insulin part and a modification group part of the insulin derivative;
2. carrying out coupling reaction on an insulin part and a modifying group part;
3. after the coupling reaction in the step 2 is finished, adjusting the pH value of the reaction liquid to be near the isoelectric point of the reaction product;
4. adding divalent or trivalent metal ions into the reaction liquid in the step 3;
5. collecting the precipitate obtained in step 4.
Different insulin derivatives have different synthetic strategies due to different insulin amino acid sequences or different types of modification groups. Some insulin derivatives can obtain crude products of the insulin derivatives after the coupling reaction is finished; some insulin derivatives need to protect some groups interfering with the coupling reaction in the insulin part and/or the modifying group part before the coupling reaction, crude products of insulin derivative precursors are obtained after the coupling reaction, and then the protecting groups are removed to obtain the crude products of the insulin derivatives. Aiming at the two different synthesis strategies, the preparation method of the insulin derivative provided by the invention is as follows (the specific flow chart is shown in figure 1).
In one aspect, the present invention provides a method for preparing an insulin derivative, comprising:
1. respectively obtaining an insulin part and a modification group part of the insulin derivative;
2. carrying out coupling reaction on an insulin part and a modifying group part;
3. after the coupling reaction in the step 2 is finished, adjusting the pH value of the reaction liquid to be near the isoelectric point of the reaction product;
4. adding divalent or trivalent metal ions into the reaction liquid in the step 3;
5. and (4) collecting the precipitate obtained in the step (4) to obtain a crude product of the insulin derivative.
In another aspect, the present invention provides a method for preparing an insulin derivative, comprising:
1. obtaining an insulin moiety and a modifying group moiety of an insulin derivative, respectively, wherein the insulin moiety and/or the modifying group moiety carries one or more protecting groups;
2. carrying out coupling reaction on an insulin part and a modifying group part;
3. after the coupling reaction in the step 2 is finished, adjusting the pH value of the reaction liquid to be near the isoelectric point of the reaction product;
4. adding divalent or trivalent metal ions into the reaction liquid in the step 3;
5. collecting the precipitate obtained in step 4;
6. and (4) carrying out deprotection reaction on the precipitate obtained in the step (5) to obtain a crude product of the insulin derivative.
Preferably, the preparation method of the present invention further comprises the step of, after obtaining the crude insulin derivative, post-treating the crude insulin derivative to obtain a pure insulin derivative.
The "insulin derivative" in the invention refers to an insulin product formed by introducing a modification group at one or more positions of the main chain of insulin or an insulin analogue.
The "insulin moiety" as referred to herein refers to an insulin or insulin analogue moiety in the structure of an insulin derivative, the sequence of which may be the sequence of all or part of human insulin, or all or part of an insulin analogue sequence in which amino acids have been added, deleted or changed. The insulin moiety of the present invention may be obtained commercially or may be prepared by any peptide/protein synthesis technique known in the art, such as solid phase synthesis, genetic engineering, recombinant DNA, and the like.
The "modifying group moiety" as used herein refers to a group chemically bonded to the insulin moiety in the structure of the insulin derivative, and examples thereof include, but are not limited to, fatty acid chains, polyethylene glycol chains, alkyl chains, and the like, preferably fatty acid chains. The modifier moiety of the invention is commercially available or can be prepared by any of the techniques known in the art, depending on the structure of the modifier moiety.
The term "coupling reaction" as used herein refers to a chemical reaction that connects the insulin moiety and the modifying group moiety via a chemical bond. Examples of the coupling reaction used in the present invention include, but are not limited to, esterification, acylation, alkylation, reduction, and the like, preferably acylation, depending on the reactive groups for chemical bond linkage in the insulin moiety and the modifying group moiety.
The term "divalent or trivalent metal ion" as used herein refers to any divalent or trivalent metal ion having water solubility, including but not limited to Cu2+、Co2+、Ni2+、Zn2+、Mn2+、Fe2+、Sr2+、Sn2+、Fe3+、Al3+. Preferably Cu2+、Co2+、Ni2+、Zn2+、Fe3+。
In the present invention, the divalent or trivalent metal ion is added based on the amount of the insulin moiety used. The amount of divalent or trivalent metal ion added in the present invention means the molar ratio of divalent or trivalent metal ion to insulin moiety, and may be in the range of 0.1 to 10:1, preferably 0.8 to 8:1, more preferably 0.8:1, 2:1, 4:1, 8: 1.
The "deprotection reaction" in the present invention means a reaction for removing a protecting group from an insulin derivative precursor. Deprotection reactions are conventional reactions in polypeptide/protein synthesis and the skilled person can select protecting groups, deprotection reagents and reaction conditions according to common general knowledge in the art.
The "post-treatment" in the present invention refers to obtaining a pure insulin derivative product by one or more processing steps commonly used in the art, such as extraction, washing, drying, purification, etc., after obtaining a crude insulin derivative product.
The prior art isoelectric precipitation method is well known to those skilled in the art for precipitation of isolated proteins. The principle is as follows: at isoelectric point, the net charge of protein molecules is zero (i.e. the positive and negative charges are equal), the protein molecules have no mutual repulsion in solution due to the same charges, and the molecules are more likely to collide with each other to generate precipitation. Thus, the protein has minimal solubility at the isoelectric point and precipitates readily.
However, isoelectric precipitation eliminates charge repulsion between protein molecules and does not actually impart forces to the protein molecules to aggregate with each other. Therefore, the isoelectric precipitation method alone does not effectively precipitate all protein molecules in the solution, the solubility is relatively high, and a large part of the protein is still dissolved in the supernatant after centrifugation, which directly results in low yield of the final product.
Surprisingly, the addition of divalent or trivalent metal ions during the manufacturing process significantly reduces the solubility of the insulin molecules, allowing the insulin molecules to precipitate out of solution. In the invention, after the coupling reaction is finished, divalent or trivalent metal ions are added, and then the precipitate is collected by centrifugation, the loss amount of the product remained in the supernatant can be reduced to below 7 percent of that of a control example (without adding metal ions), some products can be reduced to below 1 percent of that of the control example, or the product remained in the supernatant can not be detected at all, so that the product yield of the insulin derivative is greatly improved. Under the same other conditions, the product yield can be improved by about 5 times. Moreover, the metal ions can be removed after the crude product of the insulin derivative is purified, and the obtained pure product of the insulin derivative does not contain the metal ions and does not influence the use of subsequent products, such as preparation and the like.
In current insulin formulations, there are also some instances where divalent zinc ions are present in the formulation. The divalent zinc ion contained in the preparation can enable insulin molecules to form hexameric complexes, but the hexameric complexes cannot precipitate out of the solution, and the preparation solution is still in a clear state. Because the insulin molecule can only play a role in reducing blood sugar in a monomer or dimer form, the aim of containing divalent zinc ions in the insulin preparation is to delay the release speed of the insulin, prolong the action time, not reduce the solubility of the insulin molecule, and not relate to the preparation of the insulin molecule.
Drawings
FIG. 1 is a schematic flow diagram of a process for preparing an insulin derivative according to the present invention.
Detailed Description
Example 1 deglutaric insulin (control) was prepared without addition of metal ions.
The starting material Des (B30) -human insulin was prepared according to the method described in chinese patent 94193852.2, example 11.
The starting material tert-butylhexadecanediacyl-Glu (OSu) -OtBu was prepared according to the method described in example 4 of Chinese patent 200480021733.8.
Des (B30) human insulin (500 mg, 0.088 mmol) was added to 137.5ml of 50 mM/L NaHCO at 10-15 deg.C3To the acetonitrile/water mixed solution (acetonitrile: water =1:1), 3.0ml of 5% Na was added2CO3The (W/V) aqueous solution adjusted the solution to clear (pH = 10-11). tert-butylhexadecanediacyl-Glu (OSu) -OtBu (111mg, 0.176mmol) was further dissolved in 5ml of acetonitrile, followed by addition to the above des (B30) human insulin solution at 10-15 ℃ for coupling reaction. The reaction was carried out for 1 hour.
After the coupling reaction is finished, adjusting the pH of the reaction solution to 6-7 by using HCl, centrifuging, collecting precipitate, and freeze-drying to obtain 117.5 mg of the deglutaric insulin precursor crude product. The supernatant after centrifugation was subjected to chromatography, and 170.7 mg of deglutaric insulin precursor remained in the supernatant as measured by an external standard method.
Dissolving the freeze-dried product in 95% (V/V) trifluoroacetic acid (15 ml) at 10-15 ℃, stirring for deprotection reaction, pouring the reaction liquid into 150 ml methyl tert-butyl ether after reaction for 1 hour, collecting precipitate by centrifugation, and drying in the air to obtain 122 mg of the crude product of insulin deglutamide.
Molecular weight was 6104.3 by mass spectrometry, calculated 6103.97.
Example 2 addition of Zn2+Ion preparation of deglutition insulin (Zn)2+Molar ratio to insulin 0.8: 1).
The coupling reaction between des (B30) human insulin and tert-butylhexadecanediacyl-Glu (OSu) -OtBu was carried out according to the method of example 1.
After the coupling reaction is finished, adjusting the pH of the reaction solution to 6-7 by using HCl, and then adding 2 ml of ZnCl2Aqueous solution (ZnCl)2Concentration of 4.8 mg/mL, Zn2+The molar ratio of the precipitate to insulin is 0.8: 1), the precipitate is collected by centrifugation and freeze-dried to obtain 566 mg of crude deglutaric insulin precursor. The supernatant after centrifugation was subjected to chromatography, and 12.0 mg of deglutaric insulin precursor remained in the supernatant was measured by an external standard method. The amount of insulin deglutamide precursor remaining in the supernatant compared to the control example 1 was 7% (12.0 mg/170.7 mg) of the control example.
The deglutated insulin precursor is subjected to deprotection reaction according to the method of example 1 to obtain 599 mg of crude deglutated insulin product. The crude product yield was 4.91 times higher (599 mg/122 mg) compared to control example 1.
The product was confirmed to be deglutition insulin according to the mass spectrometry method of example 1.
Example 3 addition of Zn2+Ion preparation of deglutition insulin (Zn)2+The molar ratio to insulin was 2: 1).
The coupling reaction between des (B30) human insulin and tert-butylhexadecanediacyl-Glu (OSu) -OtBu was carried out according to the method of example 1.
After the coupling reaction is finished, adjusting the pH of the reaction solution to 6-7 by using HCl, and then adding 2 ml of ZnCl2Aqueous solution (ZnCl)2Concentration 12 mg/mL, Zn2+The molar ratio of the precipitate to insulin is 2: 1), the precipitate is collected by centrifugation and freeze-dried to obtain 569 mg of a deglutaric insulin precursor crude product. The supernatant after centrifugation was subjected to chromatography, and 6.0 mg of deglutaric insulin precursor remained in the supernatant was measured by an external standard method. The amount of insulin deglutamide precursor remaining in the supernatant compared to the control example 1 was 3.5% (6.0 mg/170.7 mg) of the control example.
Deprotection of the deglutaric insulin precursor was carried out as described in example 1 to obtain 602 mg of deglutaric insulin crude product. The crude product yield was 4.93 times higher compared to control example 1.
The product was confirmed to be deglutition insulin according to the mass spectrometry method of example 1.
Example 4 addition of Zn2+Ion preparation of deglutition insulin (Zn)2+At a 4:1 molar ratio to insulin).
The coupling reaction between des (B30) human insulin and tert-butylhexadecanediacyl-Glu (OSu) -OtBu was carried out according to the method of example 1.
After the coupling reaction is finished, adjusting the pH of the reaction solution to 6-7 by using HCl, and then adding 2 ml of ZnCl2Aqueous solution (ZnCl)2Concentration of 24 mg/mL, Zn2+The molar ratio of the precipitate to insulin is 4: 1), and the precipitate is collected by centrifugation and freeze-dried to obtain 573 mg of a deglutaric insulin precursor crude product. The supernatant after centrifugation was subjected to chromatography, and 0.07 mg of deglutaric insulin precursor remained in the supernatant was measured by an external standard method. The amount of insulin deglutamide precursor remaining in the supernatant compared to the control example 1 was 0.04% (0.07 mg/170.7 mg) of the control example.
Deprotection of the deglutaric insulin precursor was carried out as described in example 1 to obtain 606 mg of crude deglutaric insulin. The crude product yield was 4.97 times higher compared to control example 1.
The product was confirmed to be deglutition insulin according to the mass spectrometry method of example 1.
The crude deglutated insulin product was purified by RP-HPLC using C8-column and lyophilized to obtain pure deglutated insulin 267mg with a pure yield of 50%. Purity by HPLC was 99.8%. The zinc was removed by chromatographic purification after the reaction was complete. The pure deglutated insulin prepared according to this method has a yield 4.17 times higher (267 mg/64mg = 4.17) than that of example 4 (64 mg, 12%) of patent 200480021733.8.
Example 5 addition of Zn2+Ion preparation of deglutition insulin (Zn)2+The molar ratio to insulin was 8: 1).
The coupling reaction between des (B30) human insulin and tert-butylhexadecanediacyl-Glu (OSu) -OtBu was carried out according to the method of example 1.
After the coupling reaction is finished, adjusting the pH of the reaction solution to 6-7 by using HCl, and then adding 2 ml of ZnCl2Aqueous solution (ZnCl)2Concentration of 48 mg/mL, Zn2+The molar ratio of the precipitate to insulin is 8: 1), the precipitate is collected by centrifugation and freeze-dried to obtain 608 mg of crude deglutaric insulin precursor. The supernatant after centrifugation was subjected to chromatography, and deglutamic insulin precursor was not detected in the supernatant. The loss amount of deglutition insulin precursor remaining in the supernatant was reduced to substantially 0 compared to comparative example 1.
Deprotection of the deglutaric insulin precursor was carried out as described in example 1 to obtain 607 mg of crude deglutaric insulin. The crude product yield was 4.98 times higher compared to control example 1.
The product was confirmed to be deglutition insulin according to the mass spectrometry method of example 1.
Example 6 Co addition2+Ion preparation of degu insulin (Co)2+At a 4:1 molar ratio to insulin).
The coupling reaction between des (B30) human insulin and tert-butylhexadecanediacyl-Glu (OSu) -OtBu was carried out according to the method of example 1.
After the coupling reaction, the pH of the reaction solution was adjusted to 6-7 with HCl, and 2 mL of an aqueous solution of cobalt citrate dihydrate (104.1 mg/mL of cobalt citrate dihydrate, Co)2+With insulinThe molar ratio is 4: 1), and the precipitate is collected by centrifugation and freeze-dried to obtain 571 mg of crude degummed insulin precursor. The supernatant after centrifugation was subjected to chromatography, and 6.53 mg of deglutaric insulin precursor remained in the supernatant was measured by an external standard method. The amount of insulin deglutamide precursor remaining in the supernatant compared to the control example 1 was 3.8% (6.53 mg/170.7 mg) of the control example.
Deprotection of the deglutaric insulin precursor was carried out as described in example 1 to obtain 604 mg of deglutaric insulin crude product. The crude product yield was 4.95 times higher compared to control example 1.
The product was confirmed to be deglutition insulin according to the mass spectrometry method of example 1.
Example 7 addition of Cu2+Ion preparation of deglutaric insulin (Cu)2+At a 4:1 molar ratio to insulin).
The coupling reaction between des (B30) human insulin and tert-butylhexadecanediacyl-Glu (OSu) -OtBu was carried out according to the method of example 1.
After the coupling reaction, the reaction solution was adjusted to pH 6-7 with HCl, and 2 ml of CuCl was added2Aqueous solution (CuCl)2Has a concentration of 23.7 mg/mL, Cu2+The molar ratio of the precipitate to insulin is 4: 1), and the precipitate is collected by centrifugation and freeze-dried to obtain 582mg of a crude deglutaric insulin precursor. The supernatant after centrifugation was subjected to chromatography, and 2.05 mg of deglutaric insulin precursor remained in the supernatant was measured by an external standard method. The amount of insulin deglutamide precursor remaining in the supernatant compared to the control example 1 was 1.2% (2.05 mg/170.7 mg) of the control example.
Deprotection of the deglutaric insulin precursor was carried out as described in example 1 to obtain 611 mg of crude deglutaric insulin. The crude product yield was 5.01 times higher compared to control example 1.
The product was confirmed to be deglutition insulin according to the mass spectrometry method of example 1.
Example 8 addition of Ni2+Ion preparation of degu insulin (Ni)2+At a 4:1 molar ratio to insulin).
The coupling reaction between des (B30) human insulin and tert-butylhexadecanediacyl-Glu (OSu) -OtBu was carried out according to the method of example 1.
After the coupling reaction, the pH of the reaction solution was adjusted to 6-7 with HCl, and 2 ml of NiCl was added2Aqueous solution (NiCl)2·6H2The concentration of O is 41.8 mg/mL, Ni2+The molar ratio of the precipitate to insulin is 4: 1), the precipitate is collected by centrifugation and freeze-dried to obtain 572mg of crude deglutaric insulin precursor. The supernatant after centrifugation was subjected to chromatography, and 0.23 mg of deglutaric insulin precursor remained in the supernatant was measured by an external standard method. The amount of insulin deglutamide precursor remaining in the supernatant compared to the control example 1 was 0.13% (0.23 mg/170.7 mg) of the control example.
Deprotection of the deglutaric insulin precursor was carried out as described in example 1 to obtain 605 mg of deglutaric insulin crude product. The crude product yield was 4.96 times higher compared to control example 1.
The product was confirmed to be deglutition insulin according to the mass spectrometry method of example 1.
Example 9 addition of Fe3+Ion preparation of deglutition insulin (Fe)3+At a 4:1 molar ratio to insulin).
The coupling reaction between des (B30) human insulin and tert-butylhexadecanediacyl-Glu (OSu) -OtBu was carried out according to the method of example 1.
After the coupling reaction is finished, the pH of the reaction solution is adjusted to 6-7 by HCl, and 2 ml of FeCl is added3Aqueous solution (FeCl)3Has a concentration of 28.6 mg/mL, Fe3+The molar ratio of the precipitate to insulin is 4: 1), and the precipitate is collected by centrifugation and freeze-dried to obtain 574 mg of crude deglutaric insulin precursor. The supernatant after centrifugation was subjected to chromatography, and 4.64 mg of deglutaric insulin precursor remained in the supernatant was measured by an external standard method. The amount of insulin deglutamide precursor remaining in the supernatant compared to the control example 1 was 2.7% (4.64 mg/170.7 mg) of the control example.
Deprotection of the deglutaric insulin precursor was carried out as described in example 1 to obtain 606 mg of crude deglutaric insulin. The crude product yield was 4.97 times higher compared to control example 1.
The product was confirmed to be deglutition insulin according to the mass spectrometry method of example 1.
Example 10 addition of Zn2+Ionic preparation of insulin detemir (Zn)2+At a 4:1 molar ratio to insulin).
The starting material Des (B30) -human insulin was prepared according to the method described in chinese patent 94193852.2, example 11.
The starting material, myristic acid N-hydroxysuccinimide ester, is commercially available or can be prepared from myristic acid and N-hydroxysuccinimide as starting materials according to methods known to those skilled in the art.
Des (B30) human insulin (500 mg, 0.088 mmol) was added to 137.5ml of 50 mM/L NaHCO at 10-15 deg.C3To the acetonitrile/water mixed solution (acetonitrile: water =1:1), 3.0mL of 5% Na was added2CO3The (W/V) aqueous solution adjusted the solution to clear (pH = 10-11). N-hydroxysuccinimide myristate (57 mg, 0.176mmol) was dissolved in 5ml of acetonitrile, and then added to the des (B30) human insulin solution at 10 to 15 ℃ to conduct coupling reaction for 1 hour.
After the coupling reaction was completed, the reaction solution was divided into two equal volumes.
And adjusting the pH of one part of the crude insulin to 6-7 by using HCl, centrifuging, collecting precipitate, and freeze-drying to obtain 66 mg of crude insulin detemir. The supernatant after centrifugation was subjected to chromatography, and 135.1 mg of insulin detemir remaining in the supernatant was measured by an external standard method.
The other part was adjusted to pH 6-7 with HCl and 1 ml ZnCl was added2Aqueous solution (ZnCl)2Concentration of 24 mg/mL, Zn2+The molar ratio of the precipitate to insulin is 4: 1), and the precipitate is collected by centrifugation and freeze-dried to obtain 263 mg crude insulin detemir. The supernatant after centrifugation was subjected to chromatography, and insulin residue in the supernatant was not detected.
Compared with the method without adding metal ions, the loss amount of the insulin detemir remained in the supernatant is reduced to be basically 0, and the yield of the insulin detemir crude product is 3.98 times higher (263 mg/66 mg).
Molecular weight was 5915.6 by mass spectrometry, calculated 5916.9.
And (3) carrying out RP-HPLC purification on the second part of the crude insulin detemir product by using a C8-column, and freeze-drying to obtain 155 mg of pure insulin product with the pure insulin yield of 60%. Purity by HPLC was 99.8%. The zinc was removed by chromatographic purification after the reaction was complete.
Example 11 addition of Zn2+The insulin derivative with the modification group connected to the A1 position is prepared (A1 modified insulin).
Des (B30) -human insulin was prepared according to the method described in chinese patent 94193852.2, example 11, and Boc protection of amino acid B29 of Des (B30) -human insulin was performed according to methods well known to those skilled in the art.
The starting material, myristic acid N-hydroxysuccinimide ester, is commercially available or can be prepared from myristic acid and N-hydroxysuccinimide as starting materials according to methods known to those skilled in the art.
Boc-protected des (B30) human insulin (500 mg, 0.086 mmol) at B29 was added to 137.5ml of 50 mM/L NaHCO at 10-15 deg.C3To the acetonitrile/water mixed solution (acetonitrile: water =1:1), 3.0ml of 5% Na was added2CO3The (W/V) aqueous solution adjusted the solution to clear (pH = 10-11). N-hydroxysuccinimide myristate (56 mg, 0.172 mmol) was further dissolved in 5ml of acetonitrile, and then added to the above insulin solution at 10 to 15 ℃ to conduct coupling reaction for 1 hour.
After the coupling reaction was completed, the reaction solution was divided into two equal volumes.
One part of the solution is adjusted to pH 6-7 by HCl, and precipitates are collected by centrifugation and freeze-dried to obtain 71 mg of crude A1 modified insulin precursor. The centrifuged supernatant was subjected to chromatography, and 119 mg of A1-modified insulin precursor remained in the supernatant as measured by an external standard method.
The other part was adjusted to pH 6-7 with HCl and 1 ml ZnCl was added2Aqueous solution (ZnCl)2Concentration of 24 mg/mL, Zn2+The molar ratio of the A1 modified insulin precursor to insulin is 4: 1), and the A1 modified insulin precursor crude product is obtained by precipitating, collecting the precipitate by centrifugation and freeze-drying. Subjecting the centrifuged supernatant to chromatographic analysisThe supernatant was assayed for 3.4 mg of residual A1 modified insulin precursor by external standard.
Compared with the method without adding metal ions, the loss amount of the A1 modified insulin precursor remained in the supernatant is reduced to 2.8 percent (3.4 mg/119 mg) of the control example, and the yield of the crude product of the A1 modified insulin precursor is 3.69 times higher (262 mg/71 mg).
Molecular weight was 6015.8 by mass spectrometry, calculated 6016.9.
This example omits the deprotection step and directly post-processes the a1 modified insulin precursor to give a1 modified insulin precursor in pure form: and (3) carrying out RP-HPLC purification on the second part of the crude product of the A1 modified insulin precursor by using a C8-column, and freeze-drying to obtain 115 mg of a pure A1 modified insulin precursor, wherein the pure product yield is 44.4%. Purity by HPLC was 99.8%. The zinc was removed by chromatographic purification after the reaction was complete.
Comparative data for the products obtained by the preparation methods of examples 1-11 are shown in Table 1 below.
Table 1 comparative statistics of product data for examples 1-11:
Claims (3)
1. a process for the preparation of an insulin derivative, said process comprising:
(1) obtaining an insulin moiety and a modifying group moiety, respectively, of an insulin derivative, wherein the insulin moiety and/or the modifying group moiety bears one or more protecting groups, the insulin moiety is des (B30) human insulin, and the modifying group moiety is tert-butylhexadecanediacyl-glu (osu) -OtBu;
(2) performing coupling reaction on an insulin part and a modification group part, wherein in a reaction liquid, the content of the insulin part is 0.60mM/L, the content of the modification group part is 1.21mM/L, the content of acetonitrile is 50.7 percent (volume/volume), the content of water is 49.3 percent (volume/volume), and NaHCO is3Has a concentration of 47.25mM/L, and Na2CO3The concentration of (A) is 1.03 g/L;
(3) after the coupling reaction in the step 2 is finished, adjusting the pH value of the reaction solution to be near the isoelectric point of the reaction product, wherein the pH value near the isoelectric point is 6-7;
(4) adding divalent or trivalent metal ions into the reaction liquid in the step 3, wherein the molar ratio of the divalent or trivalent metal ions to the insulin part is 0.8-2:1, and the divalent or trivalent metal ions are selected from Cu2+、Co2+、Ni2+、Zn2+Or Fe3+；
(5) Collecting the precipitate obtained in step 4;
(6) and (3) carrying out deprotection reaction on the precipitate obtained in the step (5) to obtain a crude product of the insulin derivative, wherein the insulin derivative is selected from deglutaric insulin.
2. The method according to claim 1, further comprising a step of, after obtaining the crude insulin derivative, subjecting the crude insulin derivative to a post-treatment to obtain a pure insulin derivative.
3. The preparation method according to claim 1 or 2, wherein the molar ratio of the divalent or trivalent metal ion to the insulin moiety is 0.8:1 or 2: 1.
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| US20240025957A1 (en) | 2019-12-30 | 2024-01-25 | Gan & Lee Pharmaceuticals Co., Ltd. | Insulin Derivative |
| CN113773398B (en) * | 2020-06-10 | 2023-05-23 | 宁波鲲鹏生物科技有限公司 | De-glu insulin derivative and application thereof |
| CN113773397B (en) * | 2020-06-10 | 2023-10-20 | 宁波鲲鹏生物科技有限公司 | Preparation method of insulin diglucoside |
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