阅读说明：本技术 海藻糖在制备重组人促红素液体制剂中的应用及重组人促红素液体制剂、制备方法和应用 (Application of trehalose in preparation of recombinant human erythropoietin liquid preparation, preparation method and application ) 是由 庞甲佩 解福生 郝向慧 马晓英 姜栋 徐兴贵 秦绪才 吴化斌 于 2020-05-27 设计创作，主要内容包括：本发明涉及海藻糖在制备重组人促红素液体制剂中的应用及重组人促红素液体制剂、制备方法和应用。一种重组人促红素液体制剂,包括重组人促红素,海藻糖,渗透压调节剂,pH缓冲体系和水。本发明所用海藻糖不存在潜在的病毒性污染,能保护重组人促红素药物的活性和稳定性,也能阻止玻璃容器将药物蛋白吸附至瓶壁上,而影响药物蛋白含量的稳定性,使得该药物制剂更易于保存和运输。本发明的药物制剂经过稳定性实验,在常温下也能很好保持蛋白质活性和稳定性；由于不使用人血白蛋白,消除了病毒血源性污染的可能；海藻糖容易获得,降低了重组人促红素成品的生产成本。(The invention relates to an application of trehalose in preparation of a recombinant human erythropoietin liquid preparation, the recombinant human erythropoietin liquid preparation, a preparation method and an application. A liquid preparation of recombinant human erythropoietin comprises recombinant human erythropoietin, trehalose, an osmotic pressure regulator, a pH buffer system and water. The trehalose used by the invention has no potential viral pollution, can protect the activity and stability of the recombinant human erythropoietin medicament, and can prevent the medicament protein from being adsorbed on the bottle wall by a glass container to influence the stability of the content of the medicament protein, so that the medicament preparation is easier to store and transport. The pharmaceutical preparation of the invention can well maintain the activity and stability of protein at normal temperature through stability experiments; because human serum albumin is not used, the possibility of virus blood-borne pollution is eliminated; the trehalose is easy to obtain, and the production cost of the recombinant human erythropoietin finished product is reduced.)

1. The application of trehalose as a protective agent of recombinant human erythropoietin in the preparation of a liquid preparation of recombinant human erythropoietin.

2. The use according to claim 1, wherein the trehalose is present in the recombinant human erythropoietin liquid preparation in a mass concentration of 1-5% g/mL.

3. A recombinant human erythropoietin liquid preparation is characterized by comprising the following components:

the recombinant human erythropoietin is 2000-12000 IU/mL,

1-5% g/mL of trehalose,

50-200 mmol/L of osmotic pressure regulator,

a pH buffer system of 5-50 mmol/L and a pH of 5-8,

and (3) water.

4. The liquid preparation of recombinant human erythropoietin according to claim 3, wherein the content of the recombinant human erythropoietin is 2000-5000 IU/mL.

5. The liquid formulation of recombinant human erythropoietin according to claim 3, wherein the osmotic pressure regulator is sodium chloride.

6. The liquid preparation of recombinant human erythropoietin according to claim 3, wherein the concentration of the osmotic pressure regulator is 80 mmol/L.

7. A liquid formulation of recombinant human erythropoietin according to claim 3, wherein the pH buffer system is a sodium dihydrogen phosphate/disodium hydrogen phosphate buffer system.

8. The liquid preparation of recombinant human erythropoietin according to claim 3, wherein the liquid preparation of recombinant human erythropoietin is a liquid injection.

9. A method for preparing a liquid preparation of recombinant human erythropoietin according to any one of claims 3 to 8, which comprises the following steps: mixing the recombinant human erythropoietin, trehalose, an osmotic pressure regulator, a pH regulating system and water to obtain the composition.

10. Use of a liquid formulation of recombinant human erythropoietin according to any one of claims 3 to 8 for the preparation of a medicament for promoting haemoglobin or erythropoiesis.

Technical Field

The invention belongs to the field of biological pharmaceutical engineering, and particularly relates to application of trehalose in preparation of a recombinant human erythropoietin liquid preparation, the recombinant human erythropoietin liquid preparation, a preparation method and application.

Background

Human Erythropoietin (EPO) is a glycoprotein, a human hormone secreted by the kidney, and its physiological role is mainly to stimulate the production of hemoglobin and erythrocytes in human bone marrow. The recombinant human erythropoietin (rhEPO) is a medicinal protein produced by applying a genetic engineering method, has the same physiological functions in vivo and in vitro as the natural human erythropoietin, and is mainly used for treating anemia caused by renal insufficiency patients clinically, including dialysis and non-dialysis patients; treatment of perisurgical red blood cell mobilization; the treatment of anemia in tumor patients after radiotherapy and chemotherapy, etc. Recombinant human erythropoietin is one of the most successful genetic engineering drugs developed by human beings so far.

Human albumin is a common and effective protective agent in recombinant human erythropoietin drugs (see patent US 4703008). However, in recent years, the supply of human serum albumin as a blood product directly extracted from human blood is increasingly tense, the price is rapidly increased, and the possibility of being polluted by exogenous viruses such as hepatitis virus and HIV exists; in addition, some people are clinically found to be allergic to human blood albumin, which causes urticaria and skin rash, and the use of the medicine for patients is also limited.

In order to solve these problems, efforts have been made to develop recombinant human erythropoietin preparations which are free of human serum albumin. For example, patent CN1247257C discloses a novel recombinant human erythropoietin preparation, which comprises mannitol, metal chelating agent, L-histidine salt, pH buffer, sodium chloride and other exudates. Patent CN100502943C discloses a pharmaceutical preparation comprising tween 80, tween 20, sodium chloride, pH buffer solution and the like. Although these patents avoid the use of human serum albumin, metal chelators may cause hypocalcemia, and tween surfactants may stimulate the body's immune system, leading to the production of antibodies. In addition, the patent preparations have more auxiliary materials and complex production process, and adverse drug reactions caused by the auxiliary materials increasingly attract attention of people.

Therefore, the development of a novel recombinant human erythropoietin preparation which has simple formula and production process, does not contain human serum albumin, has stable long-term storage property, is safe and reliable has important research significance and application value.

Disclosure of Invention

The invention aims to overcome the defects or shortcomings of poor safety and reliability, more auxiliary materials and complex production process of a recombinant human erythropoietin preparation without human albumin in the prior art, and provides the application of trehalose as a protective agent of recombinant human erythropoietin in preparing a recombinant human erythropoietin liquid preparation. The research of the invention finds that the trehalose can be used as a protective agent of the recombinant human erythropoietin and is suitable for preparing a liquid preparation of the recombinant human erythropoietin.

Another object of the present invention is to provide a recombinant human erythropoietin liquid preparation. The recombinant human erythropoietin liquid preparation provided by the invention is simple in formula, free of human serum albumin, stable in long-term storage property, safe and reliable.

The invention also aims to provide a preparation method of the recombinant human erythropoietin liquid preparation.

The invention also aims to provide the application of the recombinant human erythropoietin liquid preparation in preparing medicaments for promoting the production of hemoglobin or red blood cells.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of trehalose as a protective agent of recombinant human erythropoietin in the preparation of a liquid preparation of recombinant human erythropoietin.

Trehalose is a safe and reliable natural saccharide. Trehalose is a non-reducing sugar composed of two glucose molecules with 1, 1-glycosidic bonds, has 3 isomers, namely trehalose (alpha ), isophyiosaccharide (beta ) and neotrehalose (alpha, beta), and has non-specific protective effects on various bioactive substances.

The research of the invention finds that the trehalose can protect the activity and stability of the recombinant human erythropoietin medicament, can be used as a protective agent of the recombinant human erythropoietin to be added into a liquid preparation of the recombinant human erythropoietin, is safe and nontoxic, and has no potential viral pollution; in addition, the trehalose can prevent the glass container from adsorbing the drug protein to the bottle wall, so that the stability of the content of the drug protein is influenced, and the drug preparation is easier to store and transport.

The protective agent refers to a substance for stabilizing and protecting the biological activity of protein.

Preferably, the trehalose is medicinal trehalose.

More preferably, the trehalose is an injection grade pharmaceutically acceptable trehalose.

Preferably, the mass concentration of trehalose in the recombinant human erythropoietin liquid preparation is 1-5% g/mL.

A recombinant human erythropoietin liquid preparation comprises the following components:

the recombinant human erythropoietin is 2000-12000 IU/mL,

1-5% g/mL of trehalose,

50-200 mmol/L of osmotic pressure regulator,

a pH buffer system of 5-50 mmol/L and a pH of 5-8,

and (3) water.

The invention uses trehalose as a protective agent of the recombinant human erythropoietin liquid preparation, and can well maintain the activity and stability of protein at normal temperature under the condition of not adding human serum albumin; because human serum albumin is not used, the possibility of virus blood-borne pollution is eliminated; the commercial medicinal trehalose is easy to obtain, and the production cost of the recombinant human erythropoietin finished product is reduced.

Preferably, the content of the recombinant human erythropoietin is 2000-5000 IU/mL.

Osmo-regulators, pH buffer systems conventional in the art may be used in the present invention.

Preferably, the osmolality adjusting agent is sodium chloride.

More preferably, the osmotic pressure regulator is sodium chloride for injection.

Preferably, the concentration of the osmotic pressure regulator is 80 mmol/L.

Preferably, the pH buffer system is a sodium dihydrogen phosphate/disodium hydrogen phosphate buffer system.

The medicinal preparation can be a water injection or a freeze-dried preparation, but is preferably a water injection.

Preferably, the recombinant human erythropoietin liquid preparation is a water injection or freeze-dried preparation.

More preferably, the recombinant human erythropoietin liquid preparation is an injection.

The invention also claims a preparation method of the recombinant human erythropoietin liquid preparation, which comprises the following steps: mixing the recombinant human erythropoietin, trehalose, an osmotic pressure regulator, a pH regulating system and water to obtain the composition.

The preparation method of the invention has simple process and is suitable for batch production.

Specifically, trehalose, an osmotic pressure regulator (such as sodium chloride) and a pH buffer system (such as sodium dihydrogen phosphate and disodium hydrogen phosphate) are weighed according to the formula, dissolved by a certain amount of water for injection, and the pH value is detected and finely adjusted; weighing and slowly adding the recombinant human erythropoietin stock solution, supplementing the injection water to a constant volume, and uniformly mixing to obtain a semi-finished product solution; sterilizing and filtering the prepared semi-finished product solution by using a sterilized 0.22 mu m microporous filter membrane, and sampling to detect endotoxin and sterility; and (3) filling after the endotoxin is detected to be qualified, performing online sterilization and filtration by using a sterilized 0.22-micron bag filter during filling, and performing corking and capping during filling to obtain a finished product of the recombinant human erythropoietin preparation.

The application of the recombinant human erythropoietin liquid preparation in preparing the medicines for promoting the production of the hemoglobin or the red blood cells is also within the protection scope of the invention.

Compared with the prior art, the invention has the following beneficial effects:

the trehalose used by the invention has no potential viral pollution, can protect the activity and stability of the recombinant human erythropoietin medicament, and can prevent the medicament protein from being adsorbed on the bottle wall by a glass container to influence the stability of the content of the medicament protein, so that the medicament preparation is easier to store and transport.

The pharmaceutical preparation of the invention can well maintain the activity and stability of protein at normal temperature through stability experiments; because human serum albumin is not used, the possibility of virus blood-borne pollution is eliminated; the trehalose is easy to obtain, and the production cost of the recombinant human erythropoietin finished product is reduced.

Detailed Description

The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples below, generally according to conditions conventional in the art or as suggested by the manufacturer; the raw materials, reagents and the like used are, unless otherwise specified, those commercially available from the conventional markets and the like. Any insubstantial changes and substitutions made by those skilled in the art based on the present invention are intended to be covered by the claims.