阅读说明：本技术 人血白蛋白去除多聚体工艺 (Process for removing polymer from human serum albumin ) 是由 刘余江 张海梦 张宝献 滕世超 张建璀 李长明 肖炼峰 卿舟 于 2019-09-23 设计创作，主要内容包括：本发明提供了一种人血白蛋白去除多聚体工艺,包括如下步骤：S1、组分分离；S2、人血白蛋白纯化工序；S3、巴氏灭活工序；S4、层析工序；S5、二次巴氏灭活工序；S6、无菌灌装工序。本发明的提供的人血蛋白制造工艺在巴氏灭活工艺后增加离子交换层析工艺和二次巴氏灭活等工艺步骤,用双巴氏灭活法代替传统的巴氏灭活法制备人血白蛋白,提升了制品质量,有效降低了制品中多聚体的含量；通过有效去除制品中多聚体和杂蛋白含量,进而提高人血白蛋白质量,降低产品使用过程中的不良反应的发生,可以提高人血白蛋白的市场竞争力,具备了良好的市场前景。(The invention provides a process for removing polymers from human serum albumin, which comprises the following steps: s1, separating components; s2, purifying human serum albumin; s3, performing a pasteurization process; s4, a chromatography step; s5, performing secondary pasteurization; and S6, aseptic filling. According to the human blood protein manufacturing process, the ion exchange chromatography process, the secondary pasteurization and other process steps are added after the pasteurization process, the double pasteurization method is used for replacing the traditional pasteurization method to prepare the human blood albumin, the product quality is improved, and the content of polymers in the product is effectively reduced; by effectively removing the content of polymers and hetero-proteins in the product, the quality of the human serum albumin is improved, the occurrence of adverse reactions in the using process of the product is reduced, the market competitiveness of the human serum albumin can be improved, and the product has good market prospect.)

1. The process for removing the polymer from the human serum albumin is characterized by comprising the following steps of:

s1, separating components: disinfecting and cleaning the surface of the plasma discharged from the warehouse, breaking bags, fusing the plasma in a plasma fusing tank, separating and cryoprecipitating through a centrifuge, pumping the supernatant into a separating tank, and separating by using a human serum albumin separating tank and a filter press to obtain a component I, a component II + a component III, a component IV and a component V precipitate;

s2, human serum albumin purification step: dissolving, refining and filtering the component V precipitate in a human serum albumin separating tank, and then carrying out ultrafiltration on the human serum albumin by using an ultrafiltration machine;

s3, primary pasteurization: preparing the human serum albumin subjected to ultrafiltration in the last step in a pasteurization tank, and then performing pasteurization treatment in the pasteurization tank;

s4, chromatography step: performing ion exchange chromatography using a chromatography system;

s5, secondary pasteurization: carrying out pasteurization treatment on the analyzed human serum albumin in a pasteurization tank;

s6, sterile filling: and (5) sterilizing and filling on a filling line.

2. The process of claim 1, wherein the column is equilibrated with acetic acid-sodium acetate buffer solution before chromatography.

3. The process for removing multimers from human serum albumin according to claim 1 or 2, wherein the linear flow rate for chromatography is 1.0cm/min to 2 cm/min.

4. The process for removing multimers of human serum albumin according to claim 2, wherein the gel used in the chromatography column is an ion-exchange packing.

5. The process for removing multimers of human serum albumin of claim 4, wherein said ion-exchange-type filler is a strong anion or weak anion-exchange-type filler.

6. The process for removing multimers of human serum albumin according to claim 1, wherein the primary pasteurization step is performed at 60 ℃ for 10 hours.

7. The process of claim 6, wherein the product is ultrafiltered or diluted after a pasteurization step to achieve the desired concentration, pH and conductivity of human serum albumin for chromatographic loading.

8. The process for removing polymers from human serum albumin according to claim 7, wherein the ultrafiltration comprises 4-10 times dialysis of the product using a 10KD ultrafiltration membrane module.

9. The process for removing multimers of human serum albumin according to claim 7, wherein said dilution is by diluting the preparation with a balanced solution or water for injection.

Technical Field

The invention relates to the technical field of biological products and blood product production, in particular to a process for removing polymers from human serum albumin.

Background

Human serum albumin is a main component in plasma, and its main functions are to maintain the normal osmotic pressure of blood, to bind various substances such as calcium ions, fatty acids, bilirubin, tryptophan, and drugs present in blood, and to serve as carriers in the transport of these substances. The purified human serum albumin has better curative effect on shock caused by blood loss, trauma, burn and the like, cerebral pressure increase caused by cerebral edema and cerebral injury, and edema or ascites caused by hypoproteinemia, liver cirrhosis and nephropathy.

In the industrial production of human serum albumin, it is necessary to treat the human serum albumin under various conditions different from the human in vivo environment, and thus multimers of human serum albumin are produced. This is because although human serum albumin has very stable chemical properties, it is still subject to 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. The low-temperature ethanol method for producing human albumin has the inherent characteristics that the purity of the separated human albumin product is generally 96 percent, the existence of a small amount of hetero protein is difficult to avoid, and certain trace protein components contained in the product, such as glycoprotein, haptoglobin, hemopexin and the like, have the capacity of promoting the formation of protein polymers or polymers in the heating process of pasteurization.

In clinical use of human serum albumin, although reports on the adverse effects of such multimers on the human body have not been seen, it is suspected that such multimers can exhibit novel antigenicity. From the viewpoint of safety of medicine, the limit of the amount of polymer mixed is defined in the "human serum albumin" test standard for pharmaceutical use, and therefore it is a very important problem in production to reduce the polymer content in the preparation as much as possible.

The existing production process of human serum albumin removes viruses in the product by pasteurization, but the pasteurization process can affect the stability of the product, increase the content of polymers in the product and affect the quality of the product. Along with the increasing competition of the domestic blood product industry, the improvement of the quality of blood product products becomes a strategic measure for improving the benefit of enterprises. Meanwhile, as the social medical guarantee level is gradually improved, and the indications of human serum albumin products are increased, the market demand of human serum albumin is greatly increased, and the gap is enlarged, so that the quality of albumin is improved more importantly. Therefore, there is a need for a more scientific and reasonable process for reducing the polymer content in human serum albumin.

Disclosure of Invention

The invention aims to solve the problem that excessive polymers appear in the prior human serum albumin after the pasteurization heating process, and provides a process for removing the polymers from the human serum albumin aiming at the defects in the prior art.

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

the process for removing the polymer from the human serum albumin comprises the following steps:

s1, separating components: disinfecting and cleaning the surface of the plasma discharged from the warehouse, breaking bags, fusing the plasma in a plasma fusing tank, separating and cryoprecipitating through a centrifuge, pumping the supernatant into a separating tank, and separating by using a human serum albumin separating tank and a filter press to obtain a component I, a component II + a component III, a component IV and a component V precipitate;

s2, human serum albumin purification step: dissolving, refining and filtering the component V precipitate in a human serum albumin separating tank, and then carrying out ultrafiltration on the human serum albumin by using an ultrafiltration machine;

s3, primary pasteurization: preparing the human serum albumin subjected to ultrafiltration in the last step in a pasteurization tank, and then performing pasteurization treatment in the pasteurization tank;

s4, chromatography step: performing ion exchange chromatography using a chromatography system;

s5, secondary pasteurization: carrying out pasteurization treatment on the analyzed human serum albumin in a pasteurization tank;

s6, sterile filling: the sterilization filling is carried out on a filling line, and the traditional preparation method is adopted except for a chromatography process and a secondary pasteurization process, namely the prior art.

Furthermore, an acetic acid-sodium acetate buffer solution equilibrium chromatographic column is used before chromatography, and the chromatographic column can adopt specifications of a pre-packed column and a manual packed column.

Furthermore, the chromatography linear flow rate during chromatography is 1.0 cm/min-2 cm/min, so that the retention time of the product during chromatography can be ensured to be 10-20 min.

Furthermore, the gel used in the chromatographic column is an ion exchange type filler.

Further, the ion exchange type filler is a strong anion or weak anion exchange type filler; weak anion exchange resins and strong anion exchange fillers differ in that:

1) the working exchange capacity is different, and the working exchange capacity of the weak anion resin is more than twice that of the strong anion resin;

2) the weak anion resin can exchange strong acid radical anions (such as sulfate ions and chloride ions), but is difficult to exchange weak acid radical anions (such as silicate ions); the strong anion resin can be removed;

3) the organic pollution resistance of the weak anion resin is obviously higher than that of the strong anion resin, and the recovery capability is also good.

Further, in the primary pasteurization step, the adopted inactivation temperature is 60 ℃ and the time is 10 hours; the pasteurisation is to inactivate potential viruses that may be present in the protein product, to ensure the safety of the product. The pasteurization is also a step for generating polymers in a finished product of human serum albumin, and the primary pasteurization is to ensure that unstable proteins and impurities in the albumin preparation form polymers so as to be beneficial to chromatographic removal.

Furthermore, after a primary pasteurization process, the product can be ultrafiltered or diluted so as to enable the concentration, pH and conductivity of human serum albumin to meet the requirements of chromatographic sample loading.

Further, the ultrafiltration comprises the step of carrying out 4-10 times dialysis on the product by adopting a 10KD ultrafiltration membrane package, wherein the ultrafiltration dialysis is used for removing ions in the solution, reducing the conductivity and concentrating the protein concentration.

Further, the dilution can be to dilute the product by using a balance solution or water for injection, and the dilution is to adjust the product parameters, including conductivity and protein concentration; and (3) a secondary pasteurization step, wherein the adopted inactivation temperature is 60 ℃, and the time is 10 hours. The method aims to form a double-pasteurization process together with the primary pasteurization, and is used for inspecting the change condition of the polymer after secondary pasteurization and verifying the removal condition of the polymer by primary pasteurization and chromatography.

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

the invention provides a process for removing polymers from human serum albumin, wherein a secondary pasteurization process step is added after a primary pasteurization process for a product, and a double pasteurization method is used for replacing the traditional pasteurization method for preparing the human serum albumin, so that the polymer content is reduced, and the quality of the product is effectively improved; polymers are not generated any more after secondary inactivation, so that the safety of the product is improved;

the invention also adds an ion exchange chromatography process in the steps of the primary pasteurization process and the secondary pasteurization process, and the product flows through a chromatographic column filled with weak anion exchange filler, and polymer and foreign protein content in the product are effectively removed by using a simple process, so that the quality of the human serum albumin is improved, the occurrence of adverse reaction in the using process of the product is reduced, the market competitiveness of the human serum albumin can be improved, and the product has better market prospect.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the functions of the invention clearer and easier to understand, the invention is further described by combining the following specific embodiments:

the process for removing polymer from human serum albumin in the prior art comprises the following steps:

s1, separating components: disinfecting and cleaning the surface of the plasma discharged from the warehouse, breaking bags, fusing the plasma in a plasma fusing tank, separating cryoprecipitate through a centrifuge, pumping the supernatant into a separating tank, specifically, separating the cryoprecipitate through the centrifuge, pumping the supernatant into the separating tank, separating the supernatant by using a human serum albumin separating tank and a filter press, and separating the supernatant by using the human serum albumin separating tank and the filter press to obtain a component I, a component II + a component III, a component IV and a component V precipitate; s2, human serum albumin purification step: dissolving, refining and filtering the component V precipitate in a human serum albumin separating tank, and then carrying out ultrafiltration on the human serum albumin by using an ultrafiltration machine; s3, primary pasteurization: preparing the human serum albumin subjected to ultrafiltration in the last step in a pasteurization tank, and then performing pasteurization treatment in the pasteurization tank; s4, chromatography step: performing ion exchange chromatography using a chromatography system; s5, secondary pasteurization: carrying out pasteurization treatment on the human serum albumin stock solution in the previous process in a pasteurization tank; s6, sterile filling: and (5) sterilizing and filling on a filling line.

And refining the component V precipitate, ultrafiltering, dealcoholizing to obtain human serum albumin stock solution, adding sodium caprylate and sodium chloride into the human serum albumin stock solution, and performing pasteurization at 60 ℃ for 10 h. The human serum albumin polymer content after pasteurization is obviously increased, and the polymer generated after pasteurization can be removed by strong anion exchange chromatography.

Further, the step of screening for PH:

after the human serum albumin is inactivated by pasteurization, polymers in a product can be obviously removed through chromatography, and the effects on the improvement of protein purity and the reduction of PKA are realized, so that in order to screen out an optimal pH value, the protein concentration of 80g/L, the conductivity of 1.50ms/cm, the retention time of 20min and the loading capacity of 1000g/L are used for sampling, and the pH values are respectively adjusted to be 4.40, 4.60 and 4.80.

The finished human serum albumin is taken, and the specification is 5 g/bottle (10%, 50 ml).

And (3) disinfection solution: 0.5mol/L sodium hydroxide solution.

Balancing mother liquor: acetic acid-sodium acetate buffer, pH4.60 + -0.02, prepared at 15 ms/cm.

Balance liquid: acetic acid-sodium acetate buffer solution, pH4.60 + -0.02, diluted according to the conductivity of 1.50 ms/cm.

Eluent: 1.0mol/L NaCl solution +1/10 balance the mother liquor. pH 4.60. + -. 0.02

CIP solution: 1.0mol/L sodium chloride, 0.5mol/L sodium hydroxide and 20% ethanol.

Sealing and storing liquid: 0.15mol/L sodium chloride + 20% ethanol.

Buffer solution: 1.0mol/L acetic acid solution and 0.5mol/L sodium hydroxide solution for adjusting the pH of the product and the solution.

The columns used are specifically referred to in table 1:

TABLE 1 chromatographic column information

Gel name	NanoGel-50DEAE 4.654ml
		Specification of chromatographic column	7.7×100mm
Withstand voltage	≤0.5MPa
		Gel volume	4.654ml
Capacity of carrying capacity	1000g/L
		Protein amount per chromatography	4.654g

TABLE 2 chromatographic Loading procedure

Name of solution	Column Volume (CV)	Flow rate (ml/min)	Estimated time (min)
				0.5mol/L sodium hydroxide solution	3～5	0.5	30～50
Equilibrium mother liquor	3～5	0.5	30～50
				Balancing liquid	3～5	0.5	30～50
Article of manufacture	Calculated by load	0.23	/
				Balancing liquid	3～5	0.23	60～100
Eluent	3～5	0.5	30～50
				CIP liquid	3～5	0.5	30～50
Sealing liquid	3～5	0.5	30～50

The preparation process comprises the following steps:

s1, preparation of products: taking a plurality of bottles of finished human serum albumin, prying the bottles, pouring out, filtering by using a 0.22 mu m filter element, and adjusting the pH value of the product to 4.80 +/-0.02 by using 1.0mol/L acetic acid solution; s2, ultrafiltration of the product: concentrating the protein concentration of the product to 70 +/-10 g/L, carrying out equal-volume ultrafiltration dialysis on 6 times of purified water, sampling and detecting the concentration, the conductivity, the pH value, the purity and the polymer content of the protein after ultrafiltration, dividing the detected human serum albumin into three parts, and respectively adjusting the protein concentration, the conductivity and the pH value of the product to the following conditions:

firstly, the protein concentration is 80 plus or minus 2g/L, the conductivity is 1.50 plus or minus 0.02ms/cm, and the pH value is 4.80 plus or minus 0.02;

② the protein concentration is 80 plus or minus 2g/L, the conductivity is 1.50 plus or minus 0.02ms/cm, the pH value is 4.60 plus or minus 0.02;

③, the protein concentration is 80 plus or minus 2g/L, the conductivity is 1.50 plus or minus 0.02ms/cm, and the pH value is 4.40 plus or minus 0.02;

s3, chromatography: and (3) calculating the chromatographic sample loading volume according to the concentration and the loading capacity of the human serum albumin, loading each sample twice, collecting flow-through liquid when the sample loading is started, sampling to detect the concentration, the polymer and the purity of the protein, and collecting the eluent to detect the concentration of the polymer and the protein.

Further, the chromatography procedure comprises a screening step for conductivity:

s1, preparation of products: taking a finished product of human serum albumin 6 bottle, prying the bottle, pouring out, filtering by using a 0.22 mu m filter element, and adjusting the pH value of the product to 4.60 +/-0.02 by using 1.0mol/L acetic acid solution; s2, ultrafiltration of the product: concentrating the concentration of the human serum albumin of the product to 70 +/-10 g/L, wherein the volume is 350-400 ml, carrying out equal-volume ultrafiltration dialysis on 5 times of purified water, sampling and detecting the concentration, the conductivity, the pH value, the purity and the polymer of the protein after ultrafiltration, dividing the detected human serum albumin into three parts, and respectively adjusting the concentration, the conductivity and the pH value of the product to the following conditions:

firstly, the protein concentration is 70 plus or minus 2g/L, the conductivity is 4.00 plus or minus 0.02ms/cm, and the pH value is 4.60 plus or minus 0.02;

② the protein concentration is 70 plus or minus 2g/L, the conductivity is 2.50 plus or minus 0.02ms/cm, the pH value is 4.60 plus or minus 0.02;

③, the protein concentration is 70 plus or minus 2g/L, the conductivity is 1.00 plus or minus 0.02ms/cm, and the pH value is 4.60 plus or minus 0.02;

s3, chromatography: calculating the chromatographic sample loading volume according to the protein concentration and the loading capacity, loading each sample twice, starting to collect flow-through liquid when the sample loading is started, sampling to detect the protein concentration, polymer and purity, collecting the detected protein concentration by eluent, and reserving the residual eluent by a sampling bag and storing the eluent in an environment at the temperature of-30 ℃; s4, analysis result: according to the protein recovery rate, purity and polymer analysis experiment results.