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CN-121270679-B - Crystallization process and product of recombinant insulin glargine

CN121270679BCN 121270679 BCN121270679 BCN 121270679BCN-121270679-B

Abstract

The invention provides a crystallization process and a product of recombinant insulin glargine, which relate to the technical field of biological medicines, wherein the crystallization process comprises the steps of mixing and dissolving the recombinant insulin glargine, organic acid, an organic solvent, salts and water, adding zinc substances to obtain a crystallization solution, regulating pH, stirring at a low temperature, standing, separating recombinant insulin glargine crystals, wherein in the crystallization solution, when the concentration of insulin is 5g/L, the concentration of the organic solvent is 10% -30%, the pH=8.0-9.3, and when the concentration of insulin is 5< 10g/L, the concentration of the organic solvent is 15% -30%, and the pH=8.8-9.5. The invention realizes successful crystallization of high-concentration recombinant insulin glargine by adopting specific recombinant insulin glargine concentration, organic solvent concentration, pH adjustment and other conditions under the condition of no phenol substances, and the crystal forms are similar to those of the phenol-containing recombinant insulin glargine, so that the obtained crystalline recombinant insulin glargine is applied to preparation of the medicine for treating diabetes.

Inventors

  • WEN JIE
  • MA TIANRAN
  • WANG XIANGYANG
  • YU PING

Assignees

  • 合肥亿帆生物制药有限公司
  • 亿帆医药(上海)有限公司
  • 合肥欣竹生物科技有限公司

Dates

Publication Date
20260508
Application Date
20251208

Claims (7)

  1. 1. A crystallization process of recombinant insulin glargine is characterized by comprising the steps of mixing and dissolving recombinant insulin glargine, organic acid, organic solvent, salts and water, adding zinc substance components, uniformly mixing to obtain a crystallization solution, regulating pH of the crystallization solution, stirring at low temperature, standing, and separating solids to obtain recombinant insulin glargine crystals; the organic acid is at least one of acetic acid and citric acid; The organic solvent is at least one selected from isopropanol, acetonitrile and ethanol; the salt is at least one selected from sodium citrate, sodium chloride and sodium acetate; the zinc substance component is at least one selected from zinc acetate, zinc chloride and zinc oxide; The concentration of the recombinant insulin glargine in the crystallization solution is 5-10 g/L, when the concentration of the recombinant insulin glargine in the crystallization solution is 5-g/L, the volume fraction of the organic solvent is 10-20%, and the pH is adjusted to 8.0-9.3; when the concentration of the recombinant insulin glargine in the crystallization solution is 5-10 g/L and 5 g/L is not included, the volume fraction of the organic solvent is 15-30%, and the pH is adjusted to 8.8-9.5; In the crystallization solution, the concentration of the organic acid is 0.3-1mol/L, the molar concentration of the salt is 10-200 mmol/L, and the molar ratio of the zinc ion to the recombinant insulin glargine is 1-10:1.
  2. 2. The crystallization process according to claim 1, wherein the mixing is uniform, the mixing stirring speed is 100-200 rpm, and the temperature of the crystallization solution is 15-30 ℃.
  3. 3. The crystallization process according to claim 1, wherein the temperature of the low temperature agitation is 2-10 ℃.
  4. 4. A crystallization process according to claim 3, wherein the temperature of the low temperature agitation is 4-8 ℃.
  5. 5. The crystallization process according to claim 1, wherein the low temperature agitation is performed for a period of time ranging from 20 to 40 min.
  6. 6. The crystallization process according to claim 5, wherein the low temperature agitation time is 30 min.
  7. 7. The crystallization process according to claim 1, wherein the standing time is 2-8 h.

Description

Crystallization process and product of recombinant insulin glargine Technical Field The invention belongs to the technical field of biological medicine, and particularly relates to a crystallization process and a product of recombinant insulin glargine. Background Human insulin analogues (Insulin Analogues) are a type of insulin produced by local modification of the amino acid sequence of human insulin by genetic engineering techniques. The modification is mainly aimed at optimizing the pharmacokinetic properties of human insulin. Depending on the half-life, such insulin analogues are further classified into (1) fast acting insulin analogues such as insulin lispro and insulin aspart, which act 10-15 minutes after injection, and (2) long acting insulin analogues such as insulin glargine, insulin detention and insulin deglutide, which provide a smooth glycemic control for 24 hours. As a widely used drug for diabetics, the storage and preparation process of insulin analogues is also being further improved to better accommodate the needs of the process production. In terms of the prior art, the solid insulin analogue crystal form is more convenient than the liquid insulin analogue bulk drug in terms of the storage performance of the bulk drug, the formulation design of the subsequent preparation and the like. The crystal prepared by crystallization has a uniform and stable solid form, can reduce the area requirement of a freeze dryer, shortens the subsequent freeze drying time, has high sample stability and long storage time, and is more suitable for industrial production. Crystallization is a process of forming an ordered lattice structure by utilizing the reduced solubility of a target protein in a specific solvent. For human insulin and human insulin analogues, the highly purified insulin solution is typically concentrated to a suitable concentration (e.g. 2-5 g/L), the pH is adjusted to 6.0-8.0, and a buffer system (e.g. histidine, citric acid) and crystallization aid are added. The commonly used crystallization aids include (1) zinc salts, which form a complex with human insulin or human insulin analogues through Zn 2+ to promote the formation of hexamers and crystal growth, (2) organic solvents such as ethanol and acetone to reduce the solubility of insulin and induce crystallization, and (3) salts such as NaCl to regulate the ionic strength and influence the solubility. In addition, phenols such as phenol, m-cresol or phenol derivatives, which serve as stabilizers and crystal form modifiers, play an important key role in the crystallization of human insulin or human insulin analogues, and are therefore widely used in the crystallization process of insulin products. This is because the phenolic derivative binds to a specific site on the insulin hexamer and acts as an allosteric effector, inducing the transition of the T6 hexamer to the R6 hexamer through the T3R3 intermediate (B1-B8 of the B chain of the T structure of insulin is in the extended state, B1-B3 of the B chain of the R structure is in the extended state, and B4-B19 is in the helical state). Typically, six phenol molecules are bound per insulin hexamer, and in the presence of a phenolic derivative, the hexamer form of Zn-insulin will be more stable and thus more prone to crystal formation. Recombinant insulin glargine is recombinant GlyA21-ArgB31-ArgB 32-human insulin, which is a human insulin analogue obtained by mutating asparagine (Asn) at position A21 of the human insulin A chain to glycine (Gly) and adding two arginines (Arg) at the carboxy terminus of the B chain. Unlike human insulin or other human insulin analogues, insulin glargine has its isoelectric point raised from 5.4 to nearly neutral 6.7 due to the introduction of two positively charged arginines at the end, further making the crystallization step difficult during production. Chinese patent CN102219851a states that recombinant insulin glargine can only exist in an amorphous precipitate form and cannot form a stable and uniform crystal form by using conventional human insulin crystallization methods or conventional crystallization methods of other human insulin analog products (e.g., insulin lispro). Although there have been some reports on the process of recombinant insulin glargine crystallization, phenols are regarded as important crystal stabilizers and crystal form modifiers, and are still widely used in the process of insulin glargine crystallization. Chinese patent No. 102219851A discloses a preparation method of recombinant insulin glargine crystal, which comprises the steps of adjusting pH=7.0-9.0 from crystal liquid containing recombinant insulin glargine, organic solvent, zinc, phenol derivative, salt and organic acid to crystallize the recombinant insulin glargine, wherein the volume concentration of the organic solvent is 10-30%, the initial pH of the crystal liquid is 8.0-9.0, and the pH of the crystal liquid in the crystallization process is 7.0-8.0. In addition,