阅读说明：本技术 一种人血白蛋白的纯化方法 (Method for purifying human serum albumin ) 是由 刘余江 张宝献 滕世超 李凯旋 彭建 于 2021-11-17 设计创作，主要内容包括：本发明涉及人血白蛋白制备技术领域,具体涉及一种人血白蛋白的纯化方法,包括以下依次进行的步骤：S1：原料血浆经过乙醇沉淀处理获得含有组分V的滤液；S2：所述滤液依次经过超滤和巴氏灭活后,获得蛋白制品B；S3：对蛋白制品B依次进行阴离子交换层析和阳离子交换层析,或者对蛋白制品B依次进行阳离子交换层析和阴离子交换层析,获得层析后制品；S4对层析后制品进行超滤处理,获得蛋白制品C。本方案可以解决经巴氏灭活的人血白蛋白制品中的多聚体难以充分去除的技术问题,以提升人血白蛋白制品的质量、有效性以及安全性,并同时保证人血白蛋白的提取纯化效率。(The invention relates to the technical field of preparation of human serum albumin, in particular to a purification method of human serum albumin, which comprises the following steps of: s1: carrying out ethanol precipitation treatment on raw plasma to obtain a filtrate containing a component V; s2: sequentially carrying out ultrafiltration and pasteurization on the filtrate to obtain a protein product B; s3: sequentially carrying out anion exchange chromatography and cation exchange chromatography on the protein product B, or sequentially carrying out cation exchange chromatography and anion exchange chromatography on the protein product B to obtain a chromatographed product; s4, carrying out ultrafiltration treatment on the product after chromatography to obtain a protein product C. The scheme can solve the technical problem that polymers in the human serum albumin product subjected to pasteurization are difficult to sufficiently remove, so that the quality, effectiveness and safety of the human serum albumin product are improved, and the extraction and purification efficiency of the human serum albumin is ensured.)

1. A method for purifying human serum albumin, which is characterized in that: comprises the following steps in sequence:

s1, obtaining a filtrate containing human serum albumin;

s2 first ultrafiltration and first pasteurization: sequentially carrying out ultrafiltration and pasteurization on the filtrate to obtain a protein product B;

s3 ion exchange chromatography: sequentially carrying out anion exchange chromatography and cation exchange chromatography on the protein product B, or sequentially carrying out cation exchange chromatography and anion exchange chromatography on the protein product B to obtain a chromatographed product;

s4 second ultrafiltration: and (4) carrying out ultrafiltration treatment on the product after chromatography to obtain a protein product C.

2. The method for purifying human serum albumin according to claim 1, wherein: also included is a second pasteurization S5: and (4) carrying out pasteurization on the protein product C to obtain a protein product D.

3. The method according to claim 2, wherein the purification step comprises: in S3, the anion exchange chromatography packing is Q, TMAE or DEAE.

4. The method according to claim 3, wherein the purification step comprises: in S3, the packing for cation exchange chromatography is SP.

5. The method according to claim 4, wherein the purification step comprises: in S3, when the protein product B is subjected to anion exchange chromatography and cation exchange chromatography in sequence, adjusting the pH value of the protein product B to 4.40-5.00, the conductivity to be less than or equal to 2.5mS/cm and the protein concentration to be less than or equal to 100 g/L; loading the adjusted protein product B onto an anion exchange chromatographic column at a flow rate of less than or equal to 3.0cm/min, and collecting a flow-through liquid A; adjusting the pH value of the flow-through liquid A to be 5.10-5.50 and the conductivity to be 2.0-6.0 mS/cm; and loading the adjusted flow-through liquid A onto a cation exchange chromatographic column at a flow rate of less than or equal to 3.0cm/min, and collecting the flow-through liquid B.

6. The method according to claim 5, wherein the purification step comprises: in S3, when the protein product B is subjected to anion exchange chromatography and cation exchange chromatography in sequence, adjusting the pH value of the protein product B to 4.60-4.80, the conductivity to 1.0-1.5 mS/cm and the protein concentration to be less than or equal to 100 g/L; loading the adjusted protein product B onto an anion exchange chromatographic column at a flow rate of less than or equal to 3.0cm/min, and collecting a flow-through liquid A; adjusting the pH value of the flow-through liquid A to be 5.10-5.50 and the conductivity to be 4.0-6.0 mS/cm; and loading the adjusted flow-through liquid A onto a cation exchange chromatographic column at a flow rate of less than or equal to 3.0cm/min, and collecting the flow-through liquid B.

7. The method according to claim 4, wherein the purification step comprises: in S3, when the protein product B is subjected to cation exchange chromatography and anion exchange chromatography in sequence, the pH value of the protein product B is adjusted to be 5.10-5.50, the conductivity is 2.0-6.0 mS/cm, and the protein concentration is less than or equal to 100 g/L; loading the adjusted protein product B onto a sample ion exchange chromatographic column at a flow rate of less than or equal to 3.0cm/min, and collecting a flow-through liquid B; adjusting the pH value of the flow-through liquid B to be 4.40-5.00 and the conductivity to be 1.0-1.5 mS/cm; and loading the adjusted flow-through liquid B onto a cation exchange chromatographic column at a flow speed of less than or equal to 3.0cm/min, and collecting the flow-through liquid A.

8. The method according to claim 2, wherein the purification step comprises: in S2 and S5, the conditions of pasteurization are constant temperature 60.0. + -. 0.5 ℃ for 10 h.

9. The method according to claim 2, wherein the purification step comprises: in S2, sequentially concentrating the filtrate, performing isometric ultrafiltration treatment, and preparing a protein product A; the protein concentration in the protein product A is 10-25 wt%, and the pH value is 6.40-7.40; the protein product A contains sodium chloride and sodium caprylate, the final concentration of the sodium chloride is 60-100 mmol/L, and the addition amount of the sodium caprylate is 0.14-0.18 mmol per g of protein; the protein product A is subjected to pasteurization, filtration and ultrafiltration dialysis to obtain a protein product B.

10. The method for purifying human serum albumin according to claim 1, wherein: in S1, the method for obtaining the filtrate containing human serum albumin comprises: thawing and centrifuging the raw plasma to obtain supernatant A; adjusting the temperature of the supernatant A to be-3.0 to-1.0 ℃, the protein concentration to be 40-65 g/L, pH to be 6.80-7.30 and the conductivity to be 12-14 mS/cm, adding ethanol with the final concentration to be 7-10%, reacting for 1-3 h, and carrying out filter pressing to collect a supernatant B; adjusting the temperature of the supernatant B to be-6.0 to-4.0 ℃, the protein concentration to be 30-45 g/L, pH to be 5.70-6.30 and the conductivity to be 5.0-8.0 mS/cm, adding ethanol with the final concentration to be 18-22%, reacting for 1-3 h, and performing filter pressing to collect the supernatant C; adjusting the temperature of the supernatant C to be-6.0 to-2.0 ℃, the protein concentration to be 20-30 g/L, pH to be 5.70-6.30 and the conductivity to be 2.0-5.0 mS/cm, adding ethanol with the final concentration to be 38-42%, reacting for 1-3 h, and carrying out filter pressing to collect supernatant D; adjusting the temperature of the supernatant D to be-15.0 to-7.0 ℃, the protein concentration to be 10-20 g/L, pH to be 4.70-4.90 and the conductivity to be 2.0-5.0 mS/cm, adding ethanol with the final concentration to be 38-42%, reacting for 1-3 h, and performing filter pressing to collect precipitates; dissolving the precipitate to obtain a component V solution; adjusting the temperature of the component V solution to-4.0 to-1.0 ℃, the protein concentration to 20-60 g/L, pH to 4.50-4.70, adding ethanol with the final concentration of 9-13%, reacting for 1-3 h, filtering and collecting the filtrate.

Technical Field

The invention relates to the technical field of preparation of human serum albumin, and in particular relates to a purification method of human serum albumin.

Background

In human plasma protein, albumin accounts for over fifty percent, and human albumin accounts for 80 percent of plasma colloid osmotic pressure, and mainly regulates the dynamic balance of water between tissues and blood vessels, and is mainly used in the following aspects in clinical use: 1. treating malnutritional edema (hypoproteinemia, or malnutritional edema due to liver cirrhosis, viral hepatitis B, nephrotic syndrome, etc., or cerebral edema); 2. rescue shock (such as shock caused by hemorrhagic shock, toxic shock due to infection, trauma, burn, etc.); 3. for early and late treatment of burns; 4. can be used for preventing and treating hypovolemia.

The development of therapeutic and prophylactic blood products has reached a high level abroad in the last decade. This is reflected not only in the increase of new plasma protein species to meet various therapeutic needs, but also in the development of plasma protein separation technology, and increasingly stringent requirements on the quality of the product to ensure the safe and effective use of the product. For therapeutic protein products, the polymeric protein component can cause adverse reactions and other safety issues. When the protein polymer reaches a certain amount, the immune response reaction of the organism can be promoted. The function of human albumin is highly dependent on the integrity of its molecular structure, and when the albumin product containing polymer is injected into human body, the colloidal osmotic pressure of albumin in blood can be reduced, and its excretion in body is accelerated, and the albumin polymer does not exist in normal human blood. Therefore, the polymer content in the human serum albumin product directly influences the quality of the product and the safety and effectiveness of clinical medication.

Despite its extremely stable chemical properties, albumin is affected by temperature, shear force, protein concentration and pH during purification, preparation and storage, and the presence of some components in plasma itself can cause protein molecules to polymerize and form dimers, trimers and even multimers. Due to its inherent characteristics, the low temperature ethanol method generally produces an albumin product with a purity of 96%, and the presence of a small amount of hetero-protein is difficult to avoid, and some trace protein components contained therein, such as glycoprotein, haptoglobin, hemopexin, etc., have the ability to promote the formation of protein polymers or polymers during the heating process of pasteurization. In 2016, Zhangdan, Chenbei and the like, in the analysis of the formation tendency and preliminary analysis of polymers in a human serum albumin process, a human serum albumin product is prepared by a low-temperature ethanol method, and part of polymers formed after pasteurization are polymerized by heteroprotein in the product and albumin-bonded monomers.

Because plasma components are complex, protein types are various, and precipitation, denaturation and the like are easy to occur in the purification process, the temperature and conditions of the production process need to be strictly controlled in the production process of blood products, and violent reaction is avoided. Because ethanol is easy to obtain, low in cost and easy to amplify and produce in large scale, at present, most of domestic major blood product manufacturers adopt a low-temperature ethanol method to produce human serum albumin. The low-temperature ethanol precipitation method is to gradually separate proteins in plasma by adjusting five-variable parameters of the plasma, namely pH, conductivity, protein concentration, ethanol concentration and temperature to fractionate and precipitate plasma components. In the production process of human serum albumin, the fineness of the existing production technology is not enough, and various kinds of hetero-proteins are easy to generate coprecipitation. Due to the particularity of blood products, the protein needs to be subjected to virus inactivation in the production process, the virus inactivation adopts a classical pasteurization method, and lost protein, foreign protein and the like form polymers during the pasteurization.

The 'Chinese pharmacopoeia' 2020 edition stipulates that the polymer content of a human serum albumin product cannot exceed 5%, so that the polymer content is also a key quality index of human serum albumin. Wujing discusses the main reason why pasteurization is responsible for the generation of multimers in the "Effect of different conditions pasteurization and thermal stability test on human serum albumin multimers", Satoshi Adachi removes human serum albumin multimers using anion exchange chromatography in patent US7001885B2, and we performed tests under the same conditions, which failed to completely remove multimers and extended a tailing peak in multimers to dimers. Therefore, there is a need to develop a purification method capable of sufficiently removing multimers in human serum albumin to improve the quality, effectiveness and safety of human serum albumin preparations and ensure the extraction and purification efficiency of human serum albumin.

Disclosure of Invention

The invention aims to provide a method for purifying human serum albumin, which solves the technical problem that multimers in a pasteurized inactivated human serum albumin product are difficult to remove sufficiently.

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

a method for purifying human serum albumin comprises the following steps in sequence:

s1, obtaining a filtrate containing human serum albumin;

s2 first ultrafiltration and first pasteurization: sequentially carrying out ultrafiltration and pasteurization on the filtrate to obtain a protein product B;

s3 ion exchange chromatography: sequentially carrying out anion exchange chromatography and cation exchange chromatography on the protein product B to obtain flow-through liquid B; or sequentially carrying out cation exchange chromatography and anion exchange chromatography on the protein product B to obtain flow-through liquid A;

s4 second ultrafiltration: and carrying out ultrafiltration treatment on the flow-through liquid A or the flow-through liquid B to obtain a protein product C.

The principle and the advantages of the scheme are as follows: the scheme adopts the conventional low-temperature ethanol method in the prior art to extract human serum albumin (S1), and because of the particularity of blood products, the protein needs to be subjected to virus inactivation in the production process (S2), the virus inactivation adopts the classical pasteurization method, and lost protein, foreign protein and the like form polymers during pasteurization. In order to reduce polymers caused by operation means such as Pasteur inactivation and the like, the technical scheme adopts the modes of anion exchange chromatography and cation exchange chromatography, successfully realizes the removal of the polymers and simultaneously ensures the more ideal protein yield.

According to the scheme, the inventor analyzes impurity components in human blood albumin, and removes polymer peaks and tailing peaks by respectively adopting anion exchange chromatography and cation exchange chromatography according to the difference of the hetero-protein and albumin polymer components and the difference of isoelectric points. The ion exchange chromatography principle is to realize protein separation and purification by means of ion interaction. When the solution environment of the target protein and the impurity protein is between the isoelectric points, the net charges on the surfaces of different protein molecules are opposite in electric property, and the net charges on the surfaces of different protein molecules show the behavior difference that one type of protein molecules are combined with ion exchange groups on the surface of a specific ion exchange chromatography medium, and the other type of protein molecules are free in an aqueous phase. According to the difference, the target protein and the impurity protein can be effectively separated by an ion exchange chromatography method.

In the technical scheme, the extraction method of the human serum albumin is a conventional method in the prior art. Roughly: performing ethanol precipitation treatment on raw plasma, removing a component I, a component II + III and a component IV, and refining to obtain a filtrate containing a component V, wherein the filtrate containing the component V is a filtrate containing human serum albumin.

Further, a second pasteurization S5 is also included: and (4) carrying out pasteurization on the protein product C to obtain a protein product D. By further pasteurisation, the sterility of the product is ensured.

Further, in S3, the filler for anion exchange chromatography is Q, TMAE or DEAE.

Experimental studies have found that the bond influence of the selection of the filler for anion exchange chromatography on the purification of human serum albumin and the removal of dimers is involved. Q, DEAE and TMAE anion exchange chromatography medium can reach the effect, the particle size of the chromatography medium is 50-80 μm, TMAE > Q > DEAE in the sequence of the human serum albumin polymer removing effect, and TMAE < Q ═ DEAE in the protein yield.

Further, in S3, the packing for cation exchange chromatography is SP. The polymer impurities in the human serum albumin can be sufficiently removed by cation exchange chromatography with SP as a filler.

Further, in S3, when the protein product B is subjected to anion exchange chromatography and cation exchange chromatography in sequence, the pH value of the protein product B is adjusted to 4.40-5.00, the conductivity is less than or equal to 2.5mS/cm, and the protein concentration is less than or equal to 100 g/L; loading the adjusted protein product B onto an anion exchange chromatographic column at a flow rate of less than or equal to 3.0cm/min, and collecting a flow-through liquid A; adjusting the pH value of the flow-through liquid A to be 5.10-5.50 and the conductivity to be 2.0-6.0 mS/cm; and loading the adjusted flow-through liquid A onto a cation exchange chromatographic column at a flow rate of less than or equal to 3.0cm/min, and collecting the flow-through liquid B. The flow-through liquid B is the chromatographic product prepared by the scheme.

It has been found that in anion exchange chromatography and cation exchange chromatography, the choice of pH and the choice of conductivity are critical to the purification performance. The pH value and the conductivity are adjusted to the levels, so that polymers in the human blood albumin can be fully removed, and the more ideal protein yield is ensured.

Further, in S3, when the protein product B is subjected to anion exchange chromatography and cation exchange chromatography in sequence, the pH value of the protein product B is adjusted to 4.60-4.80, the conductivity is 1.0-1.5 mS/cm, and the protein concentration is less than or equal to 100 g/L; loading the adjusted protein product B onto an anion exchange chromatographic column at a flow rate of less than or equal to 3.0cm/min, and collecting a flow-through liquid A; adjusting the pH value of the flow-through liquid A to be 5.10-5.50 and the conductivity to be 4.0-6.0 mS/cm; and loading the adjusted flow-through liquid A onto a cation exchange chromatographic column at a flow rate of less than or equal to 3.0cm/min, and collecting the flow-through liquid B. The flow-through liquid B is the chromatographic product prepared by the scheme.

Experiments prove that the pH value of a sample subjected to the anion exchange chromatography column is adjusted to 4.60-4.80, the conductivity is adjusted to 1.0-1.5 mS/cm, the protein yield of the obtained flow-through liquid A is higher than 90%, and the content of the obtained polymer is lower than 0.33%. When cation exchange chromatography is used, the conductivity is 4.0-6.0 mS/cm, the protein yield can be ensured to be more than 95%, and a polymer peak and a tailing peak can not occur.

Further, in S3, when the protein product B is subjected to cation exchange chromatography and anion exchange chromatography in sequence, the pH value of the protein product B is adjusted to be 5.10-5.50, the conductivity is 2.0-6.0 mS/cm, and the protein concentration is less than or equal to 100 g/L; loading the adjusted protein product B onto a sample ion exchange chromatographic column at a flow rate of less than or equal to 3.0cm/min, and collecting a flow-through liquid B; adjusting the pH value of the flow-through liquid B to be 4.40-5.00 and the conductivity to be 1.0-1.5 mS/cm; and loading the adjusted flow-through liquid B onto a cation exchange chromatographic column at a flow speed of less than or equal to 3.0cm/min, and collecting the flow-through liquid A. The flow-through liquid A is the chromatographic product prepared by the scheme. The anion-cation exchange chromatography can be carried out in an exchange sequence, and the effect of extracting and purifying the human serum albumin is not influenced.

Further, in S2 and S5, the conditions of pasteurization are constant temperature of 60.0 + -0.5 deg.C for 10 h. Due to the particularity of blood products, the proteins need to be subjected to virus inactivation in the production process, and the pasteurization condition can ensure that possible viruses in human serum albumin can be removed.

Further, in S2, the filtrate is sequentially subjected to concentration and equal-volume ultrafiltration treatment, and then a protein product A is prepared; the protein concentration in the protein product A is 10-25 wt%, and the pH value is 6.40-7.40; the protein product A contains sodium chloride and sodium caprylate, the final concentration of the sodium chloride is 60-100 mmol/L, and the addition amount of the sodium caprylate is 0.14-0.18 mmol per g of protein; the protein product A is subjected to pasteurization, filtration and ultrafiltration dialysis to obtain a protein product B. And removing partial impurities and inactivating viruses through the operation to obtain a protein product B for a subsequent ion chromatography purification process.

Further, in S1, the method for obtaining the filtrate containing human serum albumin comprises: thawing and centrifuging the raw plasma to obtain supernatant A; adjusting the temperature of the supernatant A to be-3.0 to-1.0 ℃, the protein concentration to be 40-65 g/L, pH to be 6.80-7.30 and the conductivity to be 12-14 mS/cm, adding ethanol with the final concentration to be 7-10%, reacting for 1-3 h, and carrying out filter pressing to collect a supernatant B; adjusting the temperature of the supernatant B to be-6.0 to-4.0 ℃, the protein concentration to be 30-45 g/L, pH to be 5.70-6.30 and the conductivity to be 5.0-8.0 mS/cm, adding ethanol with the final concentration to be 18-22%, reacting for 1-3 h, and performing filter pressing to collect the supernatant C; adjusting the temperature of the supernatant C to be-6.0 to-2.0 ℃, the protein concentration to be 20-30 g/L, pH to be 5.70-6.30 and the conductivity to be 2.0-5.0 mS/cm, adding ethanol with the final concentration to be 38-42%, reacting for 1-3 h, and carrying out filter pressing to collect supernatant D; adjusting the temperature of the supernatant D to be-15.0 to-7.0 ℃, the protein concentration to be 10-20 g/L, pH to be 4.70-4.90 and the conductivity to be 2.0-5.0 mS/cm, adding ethanol with the final concentration to be 38-42%, reacting for 1-3 h, and performing filter pressing to collect precipitates; dissolving the precipitate to obtain a component V solution; adjusting the temperature of the component V solution to-4.0 to-1.0 ℃, the protein concentration to 20-60 g/L, pH to 4.50-4.70, adding ethanol with the final concentration of 9-13%, reacting for 1-3 h, filtering and collecting the filtrate.

The low-temperature ethanol method is a common method for separating and purifying human serum albumin, and because ethanol is easy to obtain, low in cost and easy to amplify and produce in a large scale, most of domestic blood product manufacturers produce human serum albumin by using the low-temperature ethanol method. The low-temperature ethanol precipitation method is to gradually separate proteins in plasma by adjusting five-variable parameters of the plasma, namely pH, conductivity, protein concentration, ethanol concentration and temperature to fractionate and precipitate plasma components.

Drawings

FIG. 1 is a general process flow of a purification method of human blood albumin.

FIG. 2 is a contour plot of pH and conductivity of a flow-through liquid polymer of example 2 of the present invention.

FIG. 3 is a contour plot of protein yield versus pH and conductivity for example 2 of the present invention.

FIG. 4 is a HPLC detection result chart of protein preparation B of example 4 of the present invention.

FIG. 5 is a HPLC detection result chart of flow-through liquid A of example 4 of the present invention.

FIG. 6 is an enlarged view of the multimer peak of FIG. 5.

FIG. 7 shows the HPLC detection result of flow-through liquid B of example 4 of the present invention.

FIG. 8 is an enlarged overlay of the multimer peaks of FIGS. 5 and 7.

FIG. 9 is a HPLC standard spectrum of the positions of peaks (i.e., monomer, dimer, and multimer) in the "Chinese pharmacopoeia" for measuring human albumin multimers.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used therein are commercially available.

The overall process flow of the scheme is shown in figure 1, and the specific process is as follows:

1. raw plasma treatment: after the raw material plasma is taken out of a warehouse, 70% -75% ethanol solution is used for disinfecting the surface of the plasma bag, then the plasma bag is broken, the temperature is controlled to be 0-4 ℃ for melting, the plasma is merged after melting, a centrifugal machine is used for centrifuging the plasma (the centrifugal force is 10000RCF), and supernatant A is collected. The raw plasma is a supernatant obtained by centrifuging blood to remove cells, contains proteins, inorganic salts, water and the like, and does not contain blood cells. More specifically, the raw plasma is human plasma indicated in "chinese pharmacopoeia": blood product production human plasma is healthy human plasma collected by apheresis for the production of plasma protein products.

2. And (3) precipitating and separating a component I: adjusting the temperature of the supernatant A to be-3.0 to-1.0 ℃, the protein concentration to be 40-65 g/L, pH to be 6.80-7.30, the conductivity to be 12-14 mS/cm, the volume percentage concentration of ethanol (pure ethanol) to be 7-10 vol.% (final concentration), reacting for 1-3 h, and performing filter pressing to collect the supernatant B.

3. And (3) precipitating and separating components II and III: adjusting the temperature of the supernatant B to be-6.0 to-4.0 ℃, the protein concentration to be 30-45 g/L, pH to be 5.70-6.30, the conductivity to be 5.0-8.0 mS/cm, the ethanol volume percentage concentration to be 18-22 vol%, reacting for 1-3 h, and performing filter pressing to collect the supernatant C.

4. And (3) precipitating and separating a component IV: adjusting the temperature of the supernatant C to be-6.0 to-2.0 ℃, the protein concentration to be 20-30 g/L, pH to be 5.70-6.30, the conductivity to be 2.0-5.0 mS/cm, the ethanol volume percentage concentration to be 38-42 vol%, reacting for 1-3 h, and performing filter pressing to collect the supernatant D.

5. And (3) precipitating and separating a component V: adjusting the temperature of the supernatant D to be-15.0 to-7.0 ℃, the protein concentration to be 10-20 g/L, pH to be 4.70-4.90, the conductivity to be 2.0-5.0 mS/cm, the ethanol volume percentage concentration to be 38-42 vol%, reacting for 1-3 h, and performing filter pressing to collect precipitates (namely a component V).

6. Refining a component V: and (3) dissolving the component V in water for injection with the precipitation amount being 2-4 times of that of the component V, and precipitating for 2-4 h (namely, precipitating, namely, the water for injection is 1g: 2-4 ml), so as to obtain a component V solution. Adjusting the temperature of the component V solution to-4.0 to-1.0 ℃, the protein concentration to 20-60 g/L, pH to 4.50-4.70 and the volume percentage concentration of ethanol to 9-13 vol.%, reacting for 1-3 h, filtering by using a depth filter, and collecting the filtrate.

7. And (3) ultrafiltration preparation: the protein product A is obtained after ultrafiltration and preparation of the filtrate, and specifically comprises the following steps: adjusting the pH value of the filtrate to 6.4-7.4 by using a sodium hydroxide solution, and concentrating the filtrate after the pH value is adjusted by using a 10KD ultrafiltration membrane package to ensure that the protein content of the filtrate reaches 180-240 g/L to obtain the concentrated filtrate. Then, the volume of the concentrated filtrate is maintained, and 10-15 times of water is used for equal volume ultrafiltration (i.e. water is added while water is filtered to keep the volume unchanged), so as to obtain equal volume of the filtrate after ultrafiltration. And then concentrating the filtrate after the equal-volume ultrafiltration, and adding auxiliary materials into the concentrated filtrate after the equal-volume ultrafiltration to prepare the protein product A with the pH value of 6.40-7.40. The auxiliary materials comprise sodium caprylate, sodium chloride and the like, the addition amount of the sodium caprylate in the protein product A is 0.14-0.18 mmol of sodium caprylate per g of protein, and the final concentration of the sodium chloride in the protein product A is 60-100 mmol/L. During the preparation process, the pH of protein product a was adjusted with sodium hydroxide solution and adjusted to the preparation volume with water for injection. The present solution is formulated at a protein concentration of 10 wt.% to 25 wt.%, e.g., 10 wt.%, 20 wt.%, or 25 wt.% in protein preparation a.

8. And (3) pasteurizing inactivation: putting the protein product A into an inactivation tank, controlling the temperature of the product to be 60.0 +/-0.5 ℃, and keeping the temperature for 10 hours. Filtering the pasteurized product with 0.2 μm filter element, ultrafiltering and dialyzing with 10KD ultrafiltration membrane to remove ion (such as chloride ion, sodium ion, caprylic acid ion, etc.) to obtain protein product B. The protein concentration of the protein product B is more than 100g/L, and the conductivity is less than 0.30 ms/cm.

9. Anion exchange chromatography: adjusting the pH value of the protein product B to be 4.40-5.00, adjusting the protein concentration to be not higher than 100g/L and the conductivity to be not higher than 2.5mS/cm, loading the protein product B onto an anion exchange chromatographic column at the flow rate of not higher than 3.0cm/min, and collecting the flow-through liquid A. Q and DEAE, TMAE anion exchange chromatography medium can both reach the effect, and the size of chromatography medium particle is 50 ~ 80 μm, wherein the sequencing of human serum albumin polymer removal effect: TMAE (michiganmerck) > Q (available from each manufacturer) > DEAE (available from each manufacturer), ranking of protein yields: TMAE (Merck) < Q (owned by various manufacturers) ═ DEAE (owned by various manufacturers). Wherein the TMAE may be Fractogel EMD TMAE (M). Anion exchange chromatography media (packing) DEAE is an anion exchange chromatography column packing material formed by covalently attaching diethylaminoethyl ion exchange groups to a matrix, such as capto DEAE, unigel 80DEAE and unigel 50 DEAE. The anion exchange chromatography medium (filler) Q refers to an anion exchange chromatography column packing material formed by connecting quaternary ammonium groups with a matrix (such as 6% agarose) through chemically stable ether bonds, and the anion exchange chromatography column packing material can be selected from capto Q, nanogel 50Q and unigel 80Q.

10. Cation exchange chromatography: adjusting the pH value of the protein product B to be 5.10-5.50, the conductivity to be 2.0-6.0 mS/cm and the protein concentration to be not higher than 100g/L, loading the protein product B onto a cation exchange chromatographic column at a flow rate of not higher than 3.0cm/min, and collecting the flow-through liquid B. The cation exchange chromatography medium used was SP. Cation exchange chromatography medium (packing) SP is a chromatographic separation medium with strong acid cation groups formed by binding carboxymethyl groups to a matrix (e.g., 6% agarose), and specifically, Unigel 80SP, Unigel 50SP, capto SP, Eshmuno S can be used.

In addition, step 9 and step 10 are not in sequence, namely step 9 can be carried out first and then step 10 can be carried out, or step 10 can be carried out first and then step 9 can be carried out, and the removing effect of the polymer is not different. When step 9 is performed first, chromatography is performed using the flow-through liquid a in step 10; when step 10 is performed first, a chromatography operation is performed in step 9 using the flow-through solution B.

11. And (3) ultrafiltration preparation: and (3) after anion exchange chromatography and cation exchange chromatography, repeating the step (7) on the flow-through liquid B or the flow-through liquid A to obtain a protein product C.

12. Performing pasteurization and filling: putting the protein product C into an inactivation tank, controlling the temperature of the product to be 60.0 +/-0.5 ℃, and keeping the temperature for 10 hours. After the constant temperature is finished, performing centrifugal filtration by a filter element with the terminal of 0.2 mu m, and filling the product to obtain a protein product D, namely a finished product.

Step 11 may be directly prepared and then filled, and the operation of step 12 may not be adopted.

13. The protein concentration is detected by an ultraviolet spectrophotometer, and the detection method is general rule 0731 of Chinese pharmacopoeia. The polymer content is detected by a high performance liquid chromatography system, and the detection method is 'Chinese pharmacopoeia' Tong rule 3123.

Example 1:

in this example, the specific parameter selection and index test conditions of the process steps 1 to 8 adopted without subsequent ion exchange chromatography are detailed in table 1:

example 2

This example was carried out on the basis of example 1, and the protein preparation B obtained was prepared according to the method of test 2 via steps 1 to 8, and then subjected to anion exchange chromatography column chromatography using the protein preparation B, i.e., the process in step 9 was carried out. Adjusting the pH value of the protein product B to 4.70 (the optional range is 4.60-4.80), the conductivity to 1.2 (the optional range is 1.0-1.5 mS/cm), the protein concentration to 30g/L, respectively loading the protein product B to Q, TMAE and DEAE anion exchange chromatography columns at the flow rate of 1.5cm/min, wherein the loading amount is not higher than 1500g/L (namely the loading amount of each liter of ion exchange chromatography medium is not higher than 1500g of protein, in the embodiment, 1000g/L is specifically selected), and then collecting the flow-through liquid A. The fillers for the Q, TMAE and DEAE anion exchange chromatography columns are commercially available, and in this example, the specific types of the three fillers are unigel 80 qfractgel EMD TMAE (M) and unigel 50 DEAE. The packing material of Q, TMAE and DEAE is manually packed according to the conventional technical means in the field, and the specification of the chromatographic column is 16X 200 mm. Three columns were first sterilized with 5 column volumes of 0.5M sodium hydroxide solution at a flow rate of 1.5cm/min, and then column equilibration treatment was performed using 5 column volumes of acetic acid-sodium acetate buffer (pH 4.60. + -. 0.02, conductivity 1.50ms/cm, flow rate 1.5cm/min) as equilibration fluid. The filling, the disinfection and the column balance operation of the chromatographic column are conventional means in the prior art, different disinfection solutions, balance solutions, flow rates, concentrations and the like can be selected according to actual conditions, the pH value is maintained at 4.60 +/-0.02, and the aim effect can be achieved. For example, citric acid-sodium citrate buffer and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer can be used as the equilibrium solution. After the above operation is completed, anion exchange chromatography column chromatography is performed on the protein preparation B. The adjusted protein preparation B was loaded onto the column and the flow through (first fraction) started to collect at the same time as the loading. After the protein product B is loaded, the chromatographic column is treated again by using the equilibrium solution and is used for top washing the product, so that the protein recovery rate is improved. The flow-through (second fraction) was collected using 5 column volumes of acetic acid-sodium acetate buffer (flow rate 1.5cm/min) as the post equilibration solution, but at a pH and conductivity consistent with protein preparation B. Combining the first part and the second part to obtain a flow-through liquid A, and then carrying out sampling detection. Then, the subsequent operation is carried out according to the technical means commonly used in the field, so as to realize the regeneration of the chromatographic column, and the approximate operation process is as follows: the adsorbed multimers and hetero-proteins were eluted using 2M sodium chloride as eluent. Then, the remaining strongly adsorbed proteins in the column were washed with 8M urea as a washing solution. CIP solution (1M sodium chloride +0.5M sodium hydroxide + 20% ethanol) was then used to disinfect and thoroughly clean the residual protein. Next, the equilibrium solution was used to balance the problem of pH rise during CIP solution treatment. Finally, the column was sealed with 20% ethanol for the next use. The flow-through liquid A was sampled to detect the yields of polymer and protein, and the experimental results are shown in Table 2.

Table 2: polymer content of three different fillers and protein yield test results.

As can be seen from Table 2, different fillers have great differences in the removal effect of polymers in human serum albumin and the influence of protein yield, the removal effect of polymers TMAE is more than Q and more than DEAE, and the protein yield effect of TMAE is less than Q.

Example 3

With reference to the operation flow of experimental example 2, a DOE experiment was designed to examine the pH and conductivity of anion exchange chromatography. The protein product B obtained in example 2 after the Barcol inactivation ultrafiltration was taken, the pH and conductivity of the product were adjusted according to the DOE design shown in Table 3 below, and the product was loaded onto a Q anion exchange chromatography column (16X 200mm), and the flow-through liquid A was collected and sampled to detect the polymer and protein yields. The experimental results are detailed in table 3, fig. 2 and fig. 3.

Table 3: DOE experimental design and results of protein yield and polymer content test

As can be seen from Table 3, FIG. 2 and FIG. 3, Q anion exchange chromatography can remove the multimeric peaks in human serum albumin, where the results are the results of the system auto-integration. During detection of multimers, the polymer peak tailing peak is severe and extends to the side of the dimer peak, so that it is recognized as noise during automatic integration, and further cation exchange chromatography is required for removal.

Example 4

This example adds a cation exchange chromatography step, step 10 in the process, to example 3. In this example, the protein preparation B specifically selected for use in step 9 had a pH of 4.6 and a conductivity of 1.5mS/cm, and the procedure was otherwise as in example 3. The flow-through liquid A is obtained through the step 9, and the operation of the step 10 is carried out on the flow-through liquid A. Adjusting the pH value of the flow-through liquid A to 5.30 (an optional range of 5.10-5.50), the conductivity to 4.0mS/cm (an optional range of 2.0-6.0 mS/cm) and the protein concentration to be not adjusted, loading the SP cation exchange chromatography column at the flow rate of 1.5cm/min, wherein the loading amount of the flow-through liquid A is not higher than 1000g/L (namely the loading amount of each liter of ion exchange chromatography medium is not higher than 1000g of protein, in the embodiment, 800g/L is specifically selected), and collecting the flow-through liquid B for sampling and detecting the polymer.

A specific type of packing for SP cation exchange chromatography columns is Unigel 80 SP. The packing material SP is used for manual filling according to the conventional technical means in the field, and the specification of a chromatographic column is 16X 200 mm. Three columns were first sterilized with 5 column volumes of 0.5M sodium hydroxide solution at a flow rate of 1.5cm/min, and then column equilibration treatment was performed using 5 column volumes of acetic acid-sodium acetate buffer (pH 4.60. + -. 0.02, conductivity 1.50ms/cm, flow rate 1.5cm/min) as equilibration fluid. The filling, the disinfection and the column balance operation of the chromatographic column are conventional means in the prior art, different disinfection solutions, balance solutions, flow rates, concentrations and the like can be selected according to actual conditions, the pH value is maintained at 4.60 +/-0.02, and the aim effect can be achieved. For example, a disodium hydrogenphosphate-sodium dihydrogenphosphate buffer can also be used as the equilibrium solution. After the above operation, cation exchange chromatography on the flow-through solution A was performed. The adjusted flow-through solution a was loaded onto the column and the flow-through solution (first fraction) was started to collect at the same time as the loading. After the flow-through liquid A is loaded, the chromatographic column is treated again by using the equilibrium liquid, and the chromatographic column is used for top washing products, so that the protein recovery rate is improved. The flow-through (second fraction) was collected using 5 column volumes of acetic acid-sodium acetate buffer (flow rate 1.5cm/min) as the post equilibration solution, but at a pH and conductivity consistent with protein preparation B. Combining the first part and the second part to obtain a flow-through liquid B, and then carrying out sampling detection. Then, the subsequent operation is carried out according to the technical means commonly used in the field, so as to realize the regeneration of the chromatographic column, and the approximate operation process is as follows: the adsorbed multimers and hetero-proteins were eluted using 2M sodium chloride as eluent. CIP solution (1M sodium chloride +0.5M sodium hydroxide + 20% ethanol) was then used to disinfect and thoroughly clean the residual protein. Next, the equilibrium solution was used to balance the problem of pH rise during CIP solution treatment. Finally, the column was sealed with 20% ethanol for the next use.

Through detection, the polymer content after two times of chromatography is 0, and no polymer peak and tailing peak exist. The HPLC detection result of protein preparation B is shown in FIG. 4, that of flow-through liquid A is shown in FIG. 5, that of multimer of FIG. 5 is shown in FIG. 6, and that of flow-through liquid B is shown in FIG. 7. Figure 8 shows the polymer peak amplification of figure 7 and the polymer peak amplification of figure 5. It can be seen that the content of multimers was reduced and there were no multimer peaks and tailing peaks after cation exchange chromatography. See FIG. 9 for a standard map of the positions of peaks (i.e., monomer, dimer, multimer) in the measurement of human albumin multimers in the Chinese pharmacopoeia. The scheme aims to remove polymers and other protein impurities in human serum albumin as much as possible, retain monomers and partial dimers, obtain high-purity human serum albumin products and manufacture standard products different from the human serum albumin (the polymers, the dimers and other protein impurities are required to be removed completely).

Example 5

In this example, the order of step 9 and step 10 was reversed, i.e., cation exchange chromatography as described in example 4 was performed on protein preparation B (step 10) to obtain flow-through solution B, and then anion exchange chromatography as described in example 4 was performed on flow-through solution B (step 9) without protein concentration adjustment. After two times of chromatography, the polymer content is 0, and no polymer peak and tailing peak exist.

Example 6

The Q anion exchange chromatography column was performed according to the method in example 4 to obtain flow-through solution A. Flow-through A was adjusted according to the parameter conditions in example 4, the conductivity was adjusted according to Table 3, an SP cation exchange chromatography column was loaded at a flow rate of 1.5cm/min, and a sample of flow-through B was collected for HPLC detection and the protein yield was calculated. The experimental results in Table 4 show that the sample loading conductivity is 1.5-6.0 mS/cm, and a relatively ideal polymer removing effect can be obtained. However, the lower the conductivity of the sample is, the lower the protein yield is, the higher the conductivity is, the poorer the polymer removal effect is, and the range of the selected conductivity is 4.0-6.0 ms/cm, so that the protein yield can be ensured, and the polymer can be removed.

Table 4: conductivity settings and results of protein yield and multimer content testing

Example 7

This example was carried out on the basis of example 1, and the obtained protein product B was subjected to process step 9/10/11/12/13 in the following order:

9. anion exchange chromatography: taking a protein product B subjected to pasteurization and ultrafiltration filtration, adjusting the pH value of the protein product B to be 4.6 (the optional range is 4.60-4.80), the conductivity to be 1.5mS/cm (the optional range is 1.0-1.5 mS/cm), the protein concentration to be 30g/L, loading a Q anion exchange chromatographic column at the flow rate of 1.5cm/min, and collecting a flow-through liquid A. The specification of the chromatographic column adopted in the step is 100 multiplied by 500mm, and the sample loading amount of the protein product B is not higher than 1500g/L (namely, the sample loading amount of each liter of ion exchange chromatographic medium is not higher than 1500g of protein, and the sample loading amount is 1000g/L specifically in the embodiment).

10. Cation exchange chromatography: adjusting the pH value of the flow-through liquid A to be 5.30 (the optional range is 5.10-5.50), the conductivity to be 4.0mS/cm (the optional range is 2.0-6.0 mS/cm), and the protein concentration to be not adjusted, loading the SP cation exchange chromatographic column at the flow rate of 2.5cm/min, and collecting the flow-through liquid B. The specification of the chromatographic column adopted in the step is 100 multiplied by 500mm, and the sample loading amount of the flow-through liquid A is not higher than 1000g/L (namely, the sample loading amount of each liter of ion exchange chromatographic medium is not higher than 1000g protein, the embodiment specifically selects 800g/L)

11. And (3) ultrafiltration preparation: ultrafiltration was performed with reference to the specific parameters of step 7 of test 2 in example 1 to obtain protein preparation C.

12. Performing pasteurization and filling: putting the protein product C into an inactivation tank, controlling the temperature of the product to be 60.0 +/-0.5 ℃, and keeping the temperature for 10 hours. And after the constant temperature is finished, performing centrifugal filtration by a filter element with the terminal diameter of 0.2 mu m, and filling the product. Obtaining a protein product D, namely a finished product.

The experimental result shows that human serum albumin is subjected to pasteurization, anion exchange chromatography and cation exchange chromatography, the product is subjected to ultrafiltration preparation and pasteurization again, the product is not visibly changed with naked eyes, and the product after secondary pasteurization does not have polymers and polymer tailing peaks. Indicating that the product has removed impurity protein and damaged unstable protein after chromatography, and no polymer and tailing peak are formed when the product is pasteurized and inactivated again. The key items of the human serum albumin finished product are shown in Table 5, wherein the polymer content can be reduced to 0, polymer peaks and tailing peaks do not exist, and the curve is smooth and flat. Other test items are all in the specified range of 'Chinese pharmacopoeia' 2020 edition.

Table 5: test result of finished product

Note: the inspection method of each item is from 'Chinese pharmacopoeia' 2020 edition.

The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.