阅读说明：本技术 一种从组分ii上清液中回收白蛋白的方法 (Method for recovering albumin from component II supernatant ) 是由 夏琦鸿 李凯旋 张宝献 滕世超 张建璀 李长明 肖岚 陈世洁 于 2019-10-31 设计创作，主要内容包括：本发明公开了一种从组分II上清液中回收白蛋白的方法,包括以下步骤,步骤一,取组分II上清液,用10KD的膜包浓缩上清液,浓缩蛋白浓度至80g/L,用2～8℃注射用水等体积10倍透析浓缩后得蛋白液。步骤二,DEAE Sephadex A-50(二乙基氨基乙基-葡聚糖A-50)准备,用0.01mol/L枸橼酸钠+0.08mol/L氯化钠缓冲液平衡A-50凝胶2～3次,缓冲液的pH为6.00±0.50,处理后,待用。本发明通过设置有一系列的步骤使本方法在使用过程中能够对血浆处理后的废液进行处理,使其中的白蛋白能够被重新回收利用,从而达到提高血浆利用率的目的。(The invention discloses a method for recovering albumin from component II supernatant, which comprises the following steps of taking component II supernatant, concentrating the supernatant by using a 10KD membrane package, concentrating the protein concentration to 80g/L, dialyzing and concentrating by using 2-8 ℃ injection water with the same volume of 10 times to obtain protein liquid. And step two, preparing DEAE Sephadex A-50 (diethylaminoethyl-dextran A-50), balancing A-50 gel for 2-3 times by using 0.01mol/L sodium citrate and 0.08mol/L sodium chloride buffer solution, wherein the pH value of the buffer solution is 6.00 +/-0.50, and treating for later use. The invention can treat the waste liquid after plasma treatment in the using process by arranging a series of steps, so that the albumin in the waste liquid can be recycled, thereby achieving the purpose of improving the utilization rate of the plasma.)

1. A process for recovering albumin from a component II supernatant comprising the steps of:

step one, taking component II supernatant, concentrating the supernatant by using a 10KD membrane package to obtain protein solution, concentrating the protein solution to 40-120 g/L, dialyzing and concentrating by using 2-8 ℃ injection water with the same volume of 10 times to obtain protein solution;

step two, preparing ion exchange gel, balancing A-50 gel for 2-3 times by using 0.01mol/L sodium citrate and 0.08mol/L sodium chloride buffer solution, wherein the pH value of the buffer solution is 5.0-7.0, and treating for later use;

adjusting the pH value of the concentrated protein solution to 5.0-7.0, the conductivity of the concentrated protein solution to 10ms/cm or less, the protein concentration of the concentrated protein solution to 50g/L or less, adsorbing the product by using A-50 gel after adjustment, enabling the flow-through solution to be discarded, balancing the A-50 gel for 2-3 times by using 0.01mol/L sodium citrate and 0.08mol/L sodium chloride buffer solution, washing the chromatographic column by using 0.01mol/L sodium citrate and 0.15-0.25 mol/L sodium chloride washing solution, and discarding the washing solution;

eluting the A-50 gel by using 0.01mol/L sodium citrate and 2mol/L sodium chloride buffer solution, collecting eluent, adjusting the pH of the product to 6.40-7.40 by using 0.1mol/L sodium hydroxide solution, dialyzing the eluent by a 10KD membrane package with 5-10 times of injection water in equal volume, and concentrating the protein concentration to 150g/L after dialysis;

calculating the preparation volume of the semi-finished product according to the total amount of the protein and the protein content of the final semi-finished product of 100g/L, adding sodium caprylate according to the protein content of 0.150-0.175 mmol/g and sodium chloride according to the semi-finished product volume of 90-110 mmol/L, and diluting the protein concentration of the product to 100g/L by using water;

step six, filtering and sterilizing the prepared product through a filter element with the terminal of 0.22 mu m, keeping the temperature at 60 ℃ for 10 hours, and performing pasteurization on the product;

step seven, split charging: the pasteurized inactivated product was dispensed into 50ml molded bottles.

2. A process according to claim 1 for the recovery of albumin from a component II supernatant, wherein: and (5) concentrating the protein concentration in the first step to 80 g/L.

3. A process according to claim 1 for the recovery of albumin from a component II supernatant, wherein: and in the second step, the ion exchange gel is an anion exchange gel.

4. A process according to claim 3 for the recovery of albumin from a component II supernatant, wherein: the anion exchange gel is DEAE Sephadex A-50 (diethylaminoethyl-dextran A-50).

5. A process according to claim 1 for the recovery of albumin from a component II supernatant, wherein: the pH values in the second step and the third step are both 6.0; the pH in step four was 6.9.

6. A process according to claim 1 for the recovery of albumin from a component II supernatant, wherein: in the fourth step, dialysis is carried out with 5 times of water for injection in equal volume.

7. A process according to claim 1 for the recovery of albumin from a component II supernatant, wherein: in the fifth step, sodium caprylate is added according to the volume of 0.175mmol/g protein, and sodium chloride is added according to the volume of 105mmol/L semi-finished product.

Technical Field

The invention relates to the field of protein separation and purification, in particular to a method for recovering albumin from component II supernatant.

Background

The factors influencing the protein precipitation reaction in the low-temperature ethanol method are mainly 5, namely pH value, temperature, protein concentration, ionic strength and ethanol concentration. By controlling these parameters, the different protein components of plasma can be precipitated step by step, typically by precipitating components I, II + III, IV and V from plasma, and finally obtaining the albumin product from component V.

In the Chinese invention patent with the application number of 201110240072.X, a method for recovering albumin from component IV precipitation by using a low-temperature ethanol method, components I, II + III, IV and V are disclosed.

And precipitating and separating the component II + III by a low-temperature ethanol method again to obtain a component III, and finally separating a component II precipitate, wherein the component II precipitate is used for producing globulin products, and the supernatant of the component II is generally directly discarded. In fact, the component II supernatant also contains a certain amount of albumin and some foreign proteins, and when the albumin is extracted from the component II supernatant, the foreign proteins not only affect the purity of the product, but also promote the albumin to aggregate or react with the albumin to generate polymers.

The present invention provides a process for recovering high purity albumin from the supernatant of fraction II.

In the "identification requirement of human serum albumin finished product" in the third part of "Chinese pharmacopoeia" 2015 edition, the requirements for albumin are that the purity of albumin is not less than 96.0% of protein content, polymer is not more than 5.0%, and the content of kinase-releasing proenzyme activator is not more than 35 IU/ml. The detection result of the albumin finished product recovered by the method can reach the indexes, and a reference idea is provided for recovering albumin in production and improving economic benefits.

Disclosure of Invention

The present invention has been made to solve the above problems, and an object of the present invention is to provide a method for recovering albumin from a fraction II supernatant, which is capable of treating a waste liquid generated when plasma is treated by a low-temperature ethanol method, so as to recover and utilize human serum albumin in the waste liquid, thereby improving the utilization rate of plasma.

The technical scheme of the invention is as follows: a method for recovering albumin from component II supernatant comprises the following steps of taking component II supernatant, concentrating the supernatant by using a 10KD membrane package, concentrating the protein to 80g/L, dialyzing and concentrating by using 2-8 ℃ injection water with the same volume of 10 times to obtain protein liquid. And step two, preparing DEAE Sephadex A-50 (diethylaminoethyl-dextran A-50), balancing A-50 gel for 2-3 times by using 0.01mol/L sodium citrate and 0.08mol/L sodium chloride buffer solution, wherein the pH value of the buffer solution is 6.00 +/-0.50, and treating for later use. And step three, adjusting the pH value of the concentrated protein solution to 6.00 +/-0.50, the conductivity to 5.00 +/-2.00 ms/cm and the protein concentration to 10g/L +/-2 g/L, adsorbing the product by A-50 gel at the flow rate of 10L/min, and discarding the flow-through solution. And then balancing the A-50 gel for 2-3 times by using 0.01mol/L sodium citrate and 0.08mol/L sodium chloride buffer solution, washing the chromatographic column by using 0.01mol/L sodium citrate and 0.15-0.25 mol/L sodium chloride washing solution, and discarding the washing solution. And step four, eluting the A-50 gel by using 0.01mol/L sodium citrate and 2mol/L sodium chloride buffer solution, collecting eluent, and adjusting the pH value of the product to 6.90-7.00 by using 0.1mol/L sodium hydroxide solution. The eluate was dialyzed through a 10KD membrane filter with 5 times the volume of water for injection and the concentration of the protein was 150g/L after dialysis. And step five, calculating the preparation volume of the semi-finished product according to the total amount of the protein and the content of the final semi-finished product protein of 100g/L, adding sodium caprylate according to 0.175mmol/g protein and sodium chloride according to 105mmol/L semi-finished product volume, and diluting the protein concentration of the product to 100g/L by using water. And step six, filtering and sterilizing the prepared product through a filter element with the terminal of 0.22 mu m, keeping the temperature for 10 hours at 60 ℃, and performing pasteurization on the product. Step seven, split charging: the pasteurized inactivated product was dispensed into 50ml molded bottles.

Preferably, the concentration of the protein in the first concentration step is up to 80 g/L.

Preferably, the ion exchange gel in step two is an anion exchange gel.

Preferably, the anion exchange gel is DEAE Sephadex A-50 (diethylaminoethyl-dextran A-50).

Preferably, the pH values in the second step and the third step are both 6.0; the pH in step four was 6.9.

Preferably, in step four, dialysis is performed with 5 times of water for injection in equal volume.

Preferably, in step five, sodium caprylate is added according to the proportion of 0.175mmol/g protein and sodium chloride is added according to the proportion of 105mmol/L semi-finished product volume.

The invention has the beneficial effects that:

when the method is used, the waste liquid generated in the process of treating the plasma by the low-temperature ethanol method can be treated, and impurity ions and polymers in the waste liquid are removed, so that the albumin in the waste liquid is recycled, the utilization rate of the plasma is improved, and the waste of the albumin is reduced. And after the supernatant of the component II is treated according to the method, all indexes of the albumin can meet the requirements in the third part of 'Chinese pharmacopoeia' 2015 edition. The content of human serum albumin in the component II supernatant waste liquid generated by each ton of plasma is different, and the content of albumin is about 100-600 g per ton approximately, and by the method, a user can obtain about 300g of albumin in each ton of component II supernatant.

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.

The invention is further illustrated by the following examples:

a process for recovering albumin from a fraction II supernatant comprising the steps of,