Preparation method of horse anti-rabies immune protein

Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method of horse anti-rabies immune protein, which comprises the following steps: (1) digestion of immune plasma; (2) protein G affinity column chromatography; (3) cation exchange column chromatography; (4) anion exchange column chromatography; (5) performing molecular volume exclusion chromatography; and (6) preserving the horse anti-rabies immune protein. The purity of the prepared horse anti-rabies immune protein reaches 95% or above, the activity is improved to 273.42 IU / mg from 137.93 IU / mg, the total protein content is reduced to 40 mg / ml or below from 80 mg / ml on the basis of 200 international units (IU) per milliliter of preparation, and the medication safety is greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for preparing equine anti-rabies immune protein. Background Art

[0002] Rabies is a zoonotic disease caused by the rabies virus. The virus replicates in the bitten muscle tissue and then invades the central nervous system and enters the brain. Once rabies breaks out, there are no effective clinical treatment measures, and the mortality rate is close to 100%. Therefore, medical interventions such as injecting immune protein (passive immunization) and injecting vaccine (active immunization) are important preventive measures to avoid the onset of rabies.

[0003] In 1954, humans successfully used RIG to effectively prevent rabies. In the late 1960s, equine anti-rabies serum (ERIG) was prepared. However, due to the unpurified equine anti-rabies serum, serious side reaction events such as allergic reactions and serum sickness occurred during use. In 1971, the optimal dose of human anti-rabies immunoglobulin (HRIG) was determined. There is a lot of clinical accumulation in the prevention of rabies with anti-rabies serum in China. High ratios (13.0% and 12.5%) of positive cases were found during skin tests and injections respectively. Someone analyzed 1087 reported adverse reactions of rabies antisera from 1956 to 2016. 986 cases were caused by equine antisera and 101 cases were caused by human antisera. The adverse reactions are mainly serum reactions, which are caused by foreign proteins. Therefore, improving the purity of immune protein is very important for reducing drug side effects. Currently, there are three types of immune protein injection agents for rabies passive immunization clinically used in China: equine anti-rabies serum, human rabies immunoglobulin, and rabies monoclonal antibody (Omutivimab injection). The allergic reaction rate of human rabies immunoglobulin is relatively low, but the protein concentration is high, the viscosity of the injection agent is high, and the injection is difficult. Also, because it is a human blood product, there are concerns about blood pathogen infections and ethical restrictions. Rabies monoclonal antibody has no risk of blood pathogen infection, and is safe and efficient. However, monoclonal antibody can only specifically bind to a single virus strain, and the immune protection effect is not as complete as that of polyclonal antibody. Currently, it is only limited to being used in combination with human rabies immunoglobulin. Equine anti-rabies serum (ERIG) is easy to prepare in large quantities. Due to the overly rough existing preparation process of antisera, the purity of the prepared equine anti-rabies serum injection is low, the positive rate is high, and the drug side effects are serious, which greatly limits its application in medical practice.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention provides a new solution for preparing highly pure and highly active equine anti-rabies immune protein, overcomes the defects existing in the existing process technologies, and improves the effectiveness and safety of the equine anti-rabies immune protein product. The preparation process of the highly pure equine anti-rabies immune protein involved in the present invention includes the following steps:

[0006] (1) Digestion of immune plasma

[0007] The qualified immune equine plasma is diluted by adding an acidified buffer solution at 2-4 times the volume of the plasma under slow stirring, and the pH is adjusted to an appropriate range; after the reaction mixture reaches the appropriate temperature, pepsin is added; stirring is continued for an appropriate time until the digestion reaction ends; the pH of the reaction solution is adjusted to 3.8-4.5 to terminate the digestion reaction, and a digestion solution is obtained;

[0008] (2) Protein G affinity column chromatography

[0009] The Protein G affinity column is flushed with 5 column volumes of the loading buffer solution, and then the digestion solution obtained in step (1) is loaded onto the Protein G affinity column, and the Protein G affinity column is flushed with 5 column volumes of the loading buffer solution; then the Protein G affinity column is flushed with 5 column volumes of the elution buffer solution, the eluate is collected, and the pH of the eluate is adjusted to 4.5-5.5;

[0010] (3) Cation exchange column chromatography

[0011] The cation exchange column is flushed with 5 column volumes of buffer A, the eluate obtained in step (2) is loaded onto the cation exchange column, eluted with 5 column volumes of buffer A, the flow-through liquid is collected, and ultrafiltered and concentrated with buffer B to obtain a neutralized flow-through liquid;

[0012] (4) Anion exchange column chromatography

[0013] The anion exchange column is flushed with 5 column volumes of buffer B, the neutralized flow-through liquid obtained in step (3) is loaded onto the anion exchange column, eluted with 5 column volumes of buffer B, the flow-through liquid is collected, and the flow-through liquid is concentrated to 20-60 mg / ml to obtain a concentrated liquid;

[0014] (5) Molecular size exclusion chromatography

[0015] The size exclusion chromatography column is flushed with 5 column volumes of buffer C, the concentrated liquid obtained in step (4) is loaded onto the size exclusion chromatography column, eluted with 5 column volumes of buffer C, the target fraction is collected, the pH is adjusted to 5.5-6.5, and concentrated to 20-60 mg / ml to obtain an equine anti-rabies immune protein solution;

[0016] (6) Preservation of equine anti-rabies immune protein

[0017] The horse anti-rabies immunoprotein solution obtained in the pulping step (5) is sterilized by filtration with a 0.22 μm filter and stored refrigerated and protected from light at 2-8°C.

[0018] The acidified buffer solution in the step (1) is a citric acid buffer solution with a concentration of 50-250 mM and a pH value of 2.0-2.5.

[0019] After dilution and acidification in the step (1), the pH is adjusted to a range of 2.7-3.3; the amount of pepsin added is one-fifth to one-fortieth of the IgG antibody content in the immune plasma; the temperature of the digestive juice is controlled at 30±2°C, and the digestion time is 30-120 minutes.

[0020] The loading buffer solution in the step (2) is a phosphate buffer solution with a concentration of 10-100 mM and a pH of 3.8-4.5; the elution buffer solution is a citric acid buffer solution with a concentration of 10-100 mM and a pH value of 2.5-3.0.

[0021] The buffer solution A in the step (3) has a phosphate concentration of 10-100 mM and a sodium chloride concentration of 50-150 mM, and a pH value of 4.5-5.5.

[0022] The buffer solution B in the step (3) and the step (4) has a phosphate concentration of 10-100 mM and a sodium chloride concentration of 20-100 mM, and a pH value of 6.0-7.0.

[0023] The buffer solution C in the step (5) is a mixed solution with a phosphate concentration of 10-50 mM and a sodium chloride concentration of 50-150 mM, and the pH value of the mixed solution is 5.5-6.5.

[0024] The beneficial effects of the present invention are as follows:

[0025] The purity of the horse anti-rabies immunoprotein prepared in this application reaches more than 95%, and the activity is increased from 137.93 IU / mg of the original process product to 273.42 IU / mg. Based on 200 international units (IU) per milliliter of the preparation, the total protein content of the preparation is reduced from 80 mg / ml to less than 40 mg / ml, greatly improving the drug safety. Description of the Drawings

[0026] Figure 1 It is a comparison diagram of the molecular exclusion column chromatography analysis results of the horse anti-rabies immunoprotein prepared by the present invention and the horse anti-rabies immunoprotein prepared by the original process (Chinese Pharmacopoeia 2020 Edition: Anti-rabies Serum). Detailed Embodiments

[0027] The present invention will be further described below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0028] A method for preparing a horse anti-rabies immune protein, comprising the following steps:

[0029] (1) Digestion of the immune protein

[0030] Under slow stirring, 15 ml of acidified buffer (100 mM citric acid solution, pH 2.3) was added to the qualified horse immune plasma (5 ml, containing 150 mg IgG) for dilution and acidification (to pH = 3.0). The digestion solution was heated to 30 °C, and then 0.75 ml of 1% pepsin aqueous solution (7.5 mg of pepsin in total) was added, and stirring was continued at 30 °C for 30 minutes. The digestion solution was quickly placed in a cold water bath, and the pH of the digestion solution was adjusted to 4.0 (15 ml, 150 mM citric acid solution, pH 4.5) to terminate the digestion reaction, obtaining 35 ml of digestion solution;

[0031] (2) Protein G affinity column chromatography

[0032] The protein G affinity column (packing volume 15 ml) was rinsed with 75 ml of loading buffer (20 mM phosphate buffer, pH 4.0), then the digestion solution obtained in step (1) was loaded onto the protein G affinity column, and then the protein G affinity column was rinsed with 75 ml of loading buffer (20 mM phosphate buffer, pH 4.0), and then rinsed with 75 ml of elution buffer (20 mM citric acid buffer, pH 2.7). 28 ml of eluate was collected, and the pH of the eluate was adjusted to 4.8 to obtain 48 ml of protein solution;

[0033] (3) Cation exchange column chromatography

[0034] The cation exchange column (packing volume 20 ml) was rinsed with 100 ml of buffer A (20 mM phosphoric acid, 100 mM sodium chloride, pH 4.8), the eluate obtained in step (2) was loaded onto the cation exchange column, and then eluted with 100 ml of buffer A, and 65 ml of flow-through solution was collected; ultrafiltration replacement concentration was carried out with buffer B (10 mM sodium phosphate, 50 mM sodium chloride, pH 6.3) to obtain 30 ml of neutralized flow-through solution;

[0035] (4) Anion exchange column chromatography

[0036] Wash the anion exchange column (packing volume: 15 mL) with 75 mL of buffer B (10 mM sodium phosphate, 50 mM sodium chloride, pH 6.3); load the neutralized flow-through obtained in step (3) onto the anion exchange column, elute with 75 mL of buffer B, collect 35 mL of the flow-through, and ultrafiltrate and concentrate it to 30 mg / mL;

[0037] (5) Size exclusion chromatography

[0038] Wash the size exclusion chromatography column (packing volume: 60 mL) with 300 mL of buffer C (20 mM sodium phosphate, 140 mM sodium chloride, pH 6.1); load the concentrate obtained in step (4) onto the size exclusion chromatography column, elute with 300 mL of buffer C, collect 8 mL of the target fraction, adjust the pH to 6.0, and ultrafiltrate and concentrate it to 40 mg / mL to obtain equine anti-rabies immunoprotein;

[0039] (6) Preservation of equine anti-rabies immunoprotein

[0040] Perform sterilizing filtration using a 0.22 μm filter, and store it refrigerated and protected from light at 2 - 8°C.

[0041] The potency assay method uses the rapid fluorescent focus inhibition test (RFFIT) (Chinese Pharmacopoeia 2020 Edition: 3512) to analyze the protein purity and potency of the preparation method of the equine anti-rabies immunoprotein disclosed in this application and the equine anti-rabies immunoprotein in the prior art, and the following table is obtained:

[0042] Protein concentration (mg / ml) Potency (IU / ml) Potency (IU / mg) Original process 5.33 738.16 137.93 New process 4.74 1296.03 273.42

[0043] According to the above table, the activity of the equine anti-rabies immunoprotein prepared by the present invention is increased from 137.93 IU / mg to 273.42 IU / mg, which reduces the total protein content in the preparation with the same activity dose by half, reduces the side effects caused by foreign proteins, and improves the drug safety.

[0044] Figure 1 It shows that the equine anti-rabies immunoprotein prepared by the present invention has a high purity and slightly larger molecular particles. The purity of the equine anti-rabies immunoprotein prepared in this application reaches over 95% (detected by SE-HPLC), the product has a high purity, the molecular weight is close to 100 k, while the molecular weight of the market product is 96 k, with a difference of 4 k units. Therefore, the Fab’2 of the market product has at least lost some antibody fragments in the binding region of the antibody molecule.

[0045] The above content is a further detailed description of the provided technical solution in combination with the preferred implementation mode of the present invention. It cannot be determined that the specific implementation of the present invention is only limited to the above descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can be made, and all should be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for preparing an anti-rabies immune protein for horses, characterized in that: The following steps are involved: (1) Digestion of immune plasma The qualified immune horse plasma is diluted with an acidified buffer solution at 24 times the plasma volume under slow stirring, and the pH is adjusted to a suitable range; pepsin is added when the reaction mixture reaches a suitable temperature; stirring is continued for a suitable time until the digestion reaction is completed; the pH of the reaction solution is adjusted to 3.8-4.5 to terminate the digestion reaction and obtain a digestion solution; (2) Protein G affinity column chromatography The protein G affinity column is rinsed with 5 column volumes of loading buffer, and then the digestion solution obtained in step (1) is loaded onto the protein G affinity column, and the protein G affinity column is rinsed with 5 column volumes of loading buffer; then the protein G affinity column is rinsed with 5 column volumes of elution buffer, the eluate is collected, and the pH of the eluate is adjusted to 4.5-5.5; (3) Cation exchange column chromatography The cation exchange column is rinsed with 5 column volumes of buffer A, the eluate obtained in step (2) is loaded onto the cation exchange column, eluted with 5 column volumes of buffer A, the flow-through is collected, and the flow-through is concentrated by ultrafiltration with buffer B to obtain a neutralized flow-through; (4) Anion exchange column chromatography The anion exchange column is rinsed with 5 column volumes of buffer B, the neutralized flow-through obtained in step (3) is loaded onto the anion exchange column, and the flow-through is eluted with 5 column volumes of buffer B, the flow-through is collected, and the flow-through is concentrated to 20-60 mg / ml to obtain a concentrated solution; (5) Size Exclusion Chromatography The size exclusion chromatography column is rinsed with 5 column volumes of buffer C, the concentrated solution obtained in step (4) is loaded onto the size exclusion chromatography column, and eluted with 5 column volumes of buffer C, the target fraction is collected, the pH is adjusted to 5.5-6.5, and ultrafiltration is concentrated to 20-60 mg / ml to obtain horse anti-rabies immune protein; (6) Preservation of equine anti-rabies immune proteins The solution was sterilized by filtration using a 0.22 μm filter and stored at 2-8°C in a dark place.

2. The method for preparing an anti-rabies immune protein for horses according to claim 1, characterized in that: The acidification buffer in step (1) uses a citric acid buffer with a concentration of 50-250 mM and a pH value of 2.0-2.

5.

3. The method for preparing an anti-rabies immune protein for horses according to claim 1, characterized in that: After dilution and acidification in step (1), the pH is adjusted to a range of 2.7-3.

3.

4. The method for preparing an anti-rabies immune protein for horses according to claim 1, characterized in that: The step (2) uses a loading buffer with a concentration of 10-100 mM phosphate buffer and a pH of 3.8-4.

5.

5. The method for preparing an anti-rabies immune protein for horses according to claim 1, characterized in that: The elution buffer in step (2) uses a citric acid buffer with a concentration of 10-100 mM and a pH value of 2.5-3.

0.

6. The method for preparing an anti-rabies immune protein for horses according to claim 1, characterized in that: The buffer A in step (3) has a phosphate concentration of 10-100 mM and a sodium chloride concentration of 50-150 mM, and a pH value of 4.5-5.

5.

7. The method for preparing an anti-rabies immune protein for horses according to claim 1, characterized in that: The buffer B in step (3) and step (4) has a phosphate concentration of 10-100 mM and a sodium chloride concentration of 20-100 mM, and a pH value of 6.0-7.

0.

8. The method for preparing an anti-rabies immune protein for horses according to claim 1, characterized in that: The buffer C in step (5) is a mixed solution of 10-50 mM phosphoric acid and 50-150 mM sodium chloride, and the pH value of the mixed solution is 5.5-6.5.

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