CN112375137B - Preparation method of human serum albumin capable of effectively reducing PKA content - Google Patents

Abstract

According to the preparation method of the human serum albumin capable of effectively reducing the PKA content, phosphate is used for controlling the ionic strength in the FIV separation process, so that the solubility of the albumin is improved, and the solubility of the PKA is reduced at the same time, so that more PKA is removed by precipitation, and the human serum albumin with low PKA content is prepared; impurity removal is carried out on the downstream of product preparation through combination of A50 gel adsorption and activated carbon filter plate adsorption, A50 gel mainly adsorbs serine protease such as PKA, haptoglobin and alpha acid glycoprotein, and the activated carbon filter plate mainly adsorbs heme, endotoxin, serine protease and the like, so that the downstream of product preparation is combined through combination of A50 gel adsorption and activated carbon filter plate adsorption, PKA content can be effectively reduced, product appearance can be improved, endotoxin content can be reduced, polymer content can be reduced, albumin purity can be improved, and product safety can be improved; pretreatment of A50 gel can improve the adsorption effect of A50 gel.

Description

Preparation method of human serum albumin capable of effectively reducing PKA content

Technical Field

The invention relates to the technical field of preparation methods of human serum albumin, in particular to a preparation method of human serum albumin capable of effectively reducing PKA content.

Background

Since the establishment of COHN low-temperature ethanol process in 1940 s, human serum albumin has been clinically used safely for almost 80 years. The human serum albumin is widely applied to the treatment of hemorrhagic shock, hypoproteinemia and tumors, and is an irreplaceable therapeutic preparation. However, the PKA possibly remained in the human serum albumin preparation can cause the blood pressure of patients to be reduced during the infusion process, and causes serious adverse reaction, so the PKA content of the human serum albumin preparation is regulated by the pharmacopoeia to be not more than 35 IU/ml.

In the production and preparation process of human serum albumin, coagulation factor XII may be activated due to various reasons, activated XIIa fragment or modified form thereof is PKA, which can cause prekallikrein to be converted into kallikrein, which activates kininogen to be converted into bradykinin, and bradykinin causes blood vessel dilatation and capillary permeability increase to cause blood pressure reduction.

In the production process of human serum albumin, PKA is mainly distributed in FIV precipitate, so that FIV separation parameters are key parameters for controlling the PKA content of human serum albumin, and parameters such as ionic strength, reaction temperature, precipitation aging time and the like influence the solubility of PKA, so that the distribution of PKA in precipitate and supernatant is influenced, and the PKA content in a human serum albumin product is finally influenced; the quantity of filter aid and the selection of filter plate types also influence the adsorption of PKA in the deep filtration process.

In order to reduce the PKA content in a human serum albumin preparation, a protease inhibitor such as chymotrypsin or a C1 esterase inhibitor is used for inactivating PKA in some processes, but the use of the protease or the protease inhibitor can destroy the delicate equilibrium relationship between blood coagulation factors, so that a series of blood coagulation factors are activated, and the separation and purification of plasma proteins are difficult; there are processes that use fumed silica to adsorb serine proteases to remove PKA, but this method runs the risk of introducing heavy metals; in some processes, 58-65 ℃ is additionally added for heating to inactivate PKA for a plurality of hours in addition to 10-hour pasteurellosis virus inactivation at 60 ℃, but the method prolongs the exposure time of albumin molecules to harsh environment, and may cause protein molecule conformation change and generate new antigen.

In the plasma, a plurality of plasma proteins with physical and chemical properties similar to those of human serum albumin exist, such as haptoglobin, hemopexin, transferrin, beta 2-microglobulin and alpha-acid glycoprotein, these proteins are difficult to remove completely from albumin preparations by precipitation methods with varying solubility, become the major heteroproteins in albumin preparations, the albumin oligomer can reduce the osmotic pressure maintaining effect of the albumin oligomer, the polymer can be quickly eliminated by the body, the half life period of the polymer can be shortened, the blood volume expansion effect of the polymer can be influenced, the delivery function of the albumin also depends on the molecular integrity of the albumin oligomer, and the affinity of steroids, fatty acid, bilirubin and the like with the albumin oligomer is weaker than that with the monomer.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a preparation method of human serum albumin with simple process, high yield and effectively reduced PKA content.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a preparation method of human serum albumin capable of effectively reducing PKA content comprises the following steps:

(1) adjusting FIV separation ionic strength: adding 0.8-1.2 mol/L phosphate buffer solution into the FI + II + III/FII + III supernatant, and controlling the ionic strength to be 0.08-0.12 to obtain supernatant with adjusted ionic strength;

(2) adjusting the pH of the FIV separation liquid: adjusting the pH of the supernatant with the adjusted ionic strength to 5.8-6.2 by using an acetate buffer solution to obtain a supernatant with the adjusted pH;

(3) ethanol was added and the reaction temperature was adjusted: adding 95% volume fraction ethanol with the temperature below-15 ℃ to the supernatant with the adjusted pH until the volume of the ethanol reaches 38-42% of the volume of the mixed solution, adjusting the temperature of the mixed solution to-5-7 ℃ to obtain the temperature-adjusted mixed solution;

(4) precipitation and aging: standing the temperature-adjusted mixed solution for more than 6 hours to obtain a standing solution;

(5) centrifugal separation FIV: centrifuging the standing solution at-5 to-7 ℃, and separating to remove FIV precipitate to obtain supernatant after centrifugal separation;

(6) clarification and filtration: adding a filter aid into the supernatant obtained by centrifugal separation, so that the concentration of the filter aid reaches 5-15 g/L; filtering with a filter plate under a pressure not higher than 0.3Mpa, and collecting clear filtrate;

(7) isoelectric precipitation: adjusting the pH value of the clarified filtrate to 4.7-4.9, adjusting the volume of ethanol to 38-42% of the volume of the clarified filtrate, adjusting the temperature to-8-10 ℃, and standing for more than 6 hours to obtain a precipitate suspension;

(8) separating FV precipitate: centrifuging the precipitate suspension at-8-10 ℃ and collecting FV precipitate;

(9) FV precipitation refining: dissolving the FV precipitate by using 2-4 times of volume of 0-5 ℃ injection water, and keeping the temperature of the solution at-2 to-3 ℃ to obtain FV precipitate solution;

(10) adsorption of A50: adding pretreated A50 gel into the FV precipitation solution, stirring and adsorbing for 4-5 hours to obtain suspension adsorbed by A50 gel; the addition amount of the pretreated A50 gel is 3-5 g per kg of protein;

(11) adsorption by an active carbon filter plate: filtering the suspension adsorbed by the A50 gel by using an activated carbon filter plate, collecting clear filtrate, and adjusting the pH to 7.0-7.5 to obtain clear filtrate with the pH adjusted;

(12) preparing ultrafiltration dialysis: concentrating the clarified filtrate with the adjusted pH value by using a 10KD ultrafiltration membrane to obtain a first concentrated solution; dialyzing with dialysate not less than 8 times of the volume of the dialysate at constant volume, and concentrating to obtain a second concentrated solution; adding sodium caprylate into the second concentrated solution to prepare a stock solution with the protein content of 100g/L or 200g/L, the sodium caprylate content of 0.16mmol/g protein and the pH value of 6.4-7.4;

(13) inactivation of pasteuria virus: the pasteurization temperature of the stock solution is 60 +/-0.5 ℃, and the pasteurization time is more than 10 hours;

(14) and (5) sterilizing, subpackaging and checking to obtain the human serum albumin.

Further, in the step (1), 1.0mol/L phosphate buffer solution is added to the FI + II + III/FII + III supernatant, and the ionic strength is controlled to be 0.10, so that the supernatant with the adjusted ionic strength is obtained.

Further, the pH of the supernatant of the adjusted ionic strength is adjusted to 6.0 with an acetate buffer in the step (2), to obtain a supernatant of adjusted pH.

Further, in the step (3), ethanol with a volume fraction of 95% at a temperature of below-15 ℃ is added to the supernatant liquid with the adjusted pH until the volume of the ethanol reaches 40% of the volume of the mixed liquid, and the temperature of the mixed liquid is adjusted to-6 ℃ to obtain the temperature-adjusted mixed liquid.

Further, the filter aid used in said step (6) is a CELITE C503 diatomaceous earth filter aid.

Further, in the step (7), the pH value of the clarified filtrate is adjusted to 4.8, the volume of ethanol reaches 40% of the volume of the clarified filtrate, the temperature reaches-9 ℃, and the clarified filtrate is kept stand for more than 6 hours to obtain a precipitate suspension.

Further, the centrifugation process parameters in the step (8) are as follows: the centrifuge speed was 15000 rpm.

Further, in the step (9), the FV precipitate is dissolved by 3 times of volume of water for injection at 2 ℃, and the temperature of the solution is kept at-2.5 ℃, thereby obtaining an FV precipitate solution.

Further, the method for gel pretreatment of A50 in the step (10) comprises the following steps: adding 250ml of water for injection into each gram of gel, expanding for 15 minutes, filtering with a 300um filter bag net, and treating the collected gel at 121 ℃ and 0.21Mpa for 30 minutes to obtain the pretreated A50 gel.

Further, in the step (12), the clarified filtrate with the adjusted pH value is concentrated by using a 10KD ultrafiltration membrane until the protein content is 14% -16%, so as to obtain a first concentrated solution; dialyzing with dialysate of not less than 8 times volume at constant volume to obtain second concentrated solution; adding sodium caprylate into the second concentrated solution to prepare a stock solution with the protein content of 100g/L or 200g/L, the sodium caprylate content of 0.16mmol/g protein and the pH value of 6.4-7.4.

The invention relates to a method for preparing human serum albumin capable of effectively reducing PKA content, wherein sodium chloride is usually used to adjust ionic strength and change the solubility of different proteins in a conventional low-temperature ethanol separation process, PKA is fragments alpha FXIIa and beta FXIIa after blood coagulation factor FXII (HAGEMAN factor) activation, the molecular weight is about 35KD, and the isoelectric point is 4.2-4.4, FIV separation step is a key step for removing PKA, ionic strength and salt species have great influence on PKA, albumin solubility and protein viscosity, and are key process parameters influencing PKA content, albumin solid-liquid separation efficiency and albumin solid-liquid separation efficiency, applicants research finds that phosphate has larger influence on protein solubility than sodium chloride under the condition of the same ionic strength, so that the invention uses phosphate to carry out ionic strength control in the FIV separation process, improves albumin solubility and simultaneously reduces PKA solubility, thereby precipitating and removing more PKA and preparing the human serum albumin with low PKA content.

The invention relates to a method for preparing human serum albumin capable of effectively reducing PKA content, wherein concomitant proteins such as haptoglobin, hemopexin and alpha-acid glycoprotein in an albumin preparation are main reasons for forming polymers and oligomers in the processes of pasteurization and long-term storage, in the traditional preparation process, the contents of haptoglobin, hemopexin and alpha-acid glycoprotein in a final product are more, so that the content of the polymers is higher, and the quality of the product is greatly influenced, in the method, impurities are removed by combining A50 gel adsorption and activated carbon filter plate adsorption at the downstream of product preparation, A50 gel mainly adsorbs serine protease such as PKA, haptoglobin and alpha-acid filter plate glycoprotein, activated carbon mainly adsorbs heme, endotoxin, serine protease and the like, so that the combination of the A50 gel adsorption and the activated carbon filter plate adsorption is carried out at the downstream of product preparation, can effectively reduce PKA content, improve product appearance, reduce endotoxin content, reduce polymer content and raise albumin purity and product safety.

The preparation method of human serum albumin capable of effectively reducing PKA content disclosed by the invention has the advantages that A50 gel is pretreated, so that more binding sites on the surface and in the gel are exposed, the adsorption effect of A50 gel is better, the PKA content is further reduced, the appearance of a product is improved, the endotoxin content is reduced, the polymer content is reduced, and the albumin purity and the product safety are improved.

Drawings

FIG. 1 is a process flow chart of a method for preparing human serum albumin capable of effectively reducing PKA content according to the present invention.

Detailed Description

The following examples may help one skilled in the art to more fully understand the invention, but are not intended to limit the invention in any way.

The preparation method of the human serum albumin for effectively reducing the PKA content has the following preparation requirements:

1. the water for preparing main reagents such as phosphate buffer solution, acetate buffer solution and the like is the water for injection;

2. the collection and quality requirements of raw material plasma used for preparing the FI + II + III/FII + III supernatant fluid must meet the related requirements of human plasma for blood product production in pharmacopoeia of the people's republic of China;

3. the production equipment facilities and environment meet the GMP requirements;

4. FI + II + III/FII + III supernatants refer to: removing supernatant of FII + III/FI + II + III precipitate, and enriching albumin;

5. the centrifugation equipment used was: a high capacity continuous flow centrifuge of the type: GQ142

6. The main reagents used are pharmaceutical grade, and the manufacturer information is as follows

Name of reagent	Manufacturer of the product
		Glacial acetic acid	TAISHAN XINNING PHARMACEUTICAL Co.,Ltd.
Sodium acetate	TAISHAN XINNING PHARMACEUTICAL Co.,Ltd.
		Sodium chloride	TIANJIN HAIGUANG PHARMACEUTICAL Co.,Ltd.
Sodium hydroxide	Hunan Er-Kang Pharmaceutical Co.,Ltd.
		95% ethanol	HUNAN XIANGYIKANG PHARMACEUTICAL Co.,Ltd.
Sodium caprylate	Hunan Jiu-Dian pharmaceutical Limited liability company
		Disodium hydrogen phosphate	Hunan Jiu-Dian pharmaceutical Limited liability company

Example 1

A preparation method of human serum albumin capable of effectively reducing PKA content comprises the following steps:

(1) adjusting FIV separation ionic strength: adding 0.8mol/L phosphate buffer solution into the supernatant FI + II + III/FII + III at the speed of 150ml/min, and controlling the ionic strength to be 0.08 to obtain the supernatant with the ionic strength adjusted;

(2) adjusting the pH of the FIV separation liquid: adjusting the pH of the supernatant with the adjusted ionic strength to 5.8 by using an acetate buffer solution with the pH of 4.0 to obtain a supernatant with the adjusted pH;

(3) ethanol was added and the reaction temperature was adjusted: adding 95% ethanol with volume fraction below-15 deg.C into the supernatant with adjusted pH until the volume of ethanol reaches 38% of the volume of the mixed solution, wherein the ethanol adding speed is 80L/h; gradually adjusting the temperature of the mixed solution to-5 ℃ while adding the ethanol to obtain a temperature-adjusted mixed solution;

(4) precipitation and aging: standing the temperature-adjusted mixed solution for more than 6 hours to obtain a standing solution;

(5) and (3) centrifugal separation FIV: centrifuging the standing solution at-5 ℃, and separating to remove FIV precipitate to obtain supernatant after centrifugal separation; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(6) clarification and filtration: adding a CELITE C503 diatomite filter aid to the supernatant of the centrifugal separation to ensure that the concentration of the filter aid reaches 5 g/L; performing deep filtration with a filter plate under the pressure not higher than 0.3Mpa, and collecting the clarified filtrate;

(7) isoelectric precipitation: adjusting the pH of the clarified filtrate to 4.7 by using 0.5mol/L acetic acid containing 40% ethanol, adjusting the volume of the ethanol to 38% of the volume of the clarified filtrate, adjusting the temperature to-8 ℃, stirring for reacting for 2 hours, and standing for more than 6 hours to obtain precipitate suspension;

(8) separating FV precipitate: centrifuging the precipitate suspension at-8 deg.C, and collecting FV precipitate; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(9) FV precipitation refining: dissolving the FV precipitate by using 2 times of volume of 0 ℃ water for injection, and keeping the temperature of the solution at-2 ℃ to obtain FV precipitate solution;

(10) adsorption of A50: adding pretreated A50 gel into the FV precipitation solution, stirring and adsorbing for 4 hours to obtain suspension adsorbed by A50 gel; the addition amount of the pretreated A50 gel is 3g per kilogram of protein; sampling after dissolving the precipitate, checking the protein content, and calculating the addition amount of A50 gel; the method for pretreating the A50 gel comprises the following steps: adding 250ml of water for injection with the temperature of 85-95 ℃ into each gram of gel, expanding for 15 minutes, filtering by using a 300-micron filter bag net, and treating the collected gel for 30 minutes at the temperature of 121 ℃ and under the pressure of 0.21Mpa to obtain pretreated A50 gel;

(11) adsorption by an activated carbon filter plate: filtering the suspension adsorbed by the A50 gel by using a filter plate containing activated carbon, collecting clear filtrate, and adjusting the pH to 7.0 by using 1mol/L sodium bicarbonate to obtain clear filtrate with the pH adjusted;

(12) preparing ultrafiltration dialysis: concentrating the clarified filtrate with the adjusted pH value by using a 10KD ultrafiltration membrane until the protein content is 14 percent to obtain a first concentrated solution; dialyzing with sodium chloride dialysate of not less than 8 times volume at constant volume, and concentrating until protein content is greater than 150g/L or greater than 250g/L to obtain second concentrated solution; adding sodium caprylate into the second concentrated solution to prepare an albumin stock solution with the protein content of 100g/L or 200g/L, the sodium caprylate content of 0.16mmol/g protein and the pH value of 6.4-7.4;

(13) inactivation of pasteuria virus: the pasteurization temperature of the albumin stock solution is 60 +/-0.5 ℃, and the pasteurization time is more than 10 hours;

(14) and (5) sterilizing, subpackaging and inspecting to obtain the human serum albumin.

Example 2

A preparation method of human serum albumin capable of effectively reducing PKA content comprises the following steps:

(1) adjusting FIV separation ionic strength: adding 1.2mol/L phosphate buffer solution into the FI + II + III/FII + III supernatant at the speed of 150ml/min, and controlling the ionic strength to be 0.12 to obtain supernatant with the ionic strength adjusted;

(2) adjusting the pH of the FIV separation liquid: adjusting the pH of the supernatant with the adjusted ionic strength to 6.2 by using an acetate buffer solution with the pH of 4.0 to obtain a supernatant with the adjusted pH;

(3) ethanol was added and the reaction temperature was adjusted: adding 95% volume fraction ethanol at-15 deg.C or below into the supernatant with adjusted pH until the volume of ethanol reaches 42% of the volume of the mixture, wherein the ethanol adding speed is 80L/h; gradually adjusting the temperature of the mixed solution to-7 ℃ while adding the ethanol to obtain a temperature-adjusted mixed solution;

(4) precipitation and aging: standing the temperature-adjusted mixed solution for more than 6 hours to obtain a standing solution;

(5) centrifugal separation FIV: centrifuging the standing solution at-7 ℃, and separating to remove FIV precipitate to obtain supernatant after centrifugal separation; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(6) clarification and filtration: adding CELITE C503 diatomite filter aid into the supernatant liquid of the centrifugal separation so that the concentration of the filter aid reaches 15 g/L; performing deep filtration with a filter plate under a pressure not higher than 0.3Mpa, and collecting the clarified filtrate;

(7) isoelectric precipitation: adjusting the pH of the clear filtrate to 4.9 by using 0.5mol/L acetic acid containing 40% of ethanol, adjusting the volume of the ethanol to 42% of the volume of the clear filtrate, adjusting the temperature to-10 ℃, stirring for reacting for 2 hours, and standing for more than 6 hours to obtain precipitate suspension;

(8) separating FV precipitate: centrifuging the precipitate suspension at-10 deg.C, and collecting FV precipitate; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the clear liquid is 500-1000 ml/min;

(9) FV precipitation refining: dissolving the FV precipitate by 4 times of volume of 5 ℃ water for injection, and keeping the temperature of the solution at-3 ℃ to obtain FV precipitate solution;

(10) adsorption of A50: adding pretreated A50 gel into the FV precipitation solution, stirring and adsorbing for 5 hours to obtain suspension adsorbed by A50 gel; the addition amount of the pretreated A50 gel is 5g per kilogram of protein; sampling after dissolving the precipitate to check the protein content, and calculating the addition amount of A50 gel; the method for pretreating the A50 gel comprises the following steps: adding 250ml of water for injection with the temperature of 85-95 ℃ into each gram of gel, expanding for 15 minutes, filtering by using a 300-micron filter bag net, and treating the collected gel for 30 minutes at the temperature of 121 ℃ and under the pressure of 0.21Mpa to obtain pretreated A50 gel;

(11) adsorption by an activated carbon filter plate: filtering the suspension adsorbed by the A50 gel by using an activated carbon-containing filter plate, collecting clear filtrate, and adjusting the pH to 7.5 by using 1mol/L sodium bicarbonate to obtain clear filtrate with the pH adjusted;

(12) preparing ultrafiltration dialysis: concentrating the clarified filtrate with the adjusted pH value by using a 10KD ultrafiltration membrane until the protein content is 16 percent to obtain a first concentrated solution; dialyzing with sodium chloride dialysate of not less than 8 times volume at constant volume, and concentrating until protein content is greater than 150g/L or greater than 250g/L to obtain second concentrated solution; adding sodium caprylate into the second concentrated solution to prepare an albumin stock solution with the protein content of 100g/L or 200g/L, the sodium caprylate content of 0.16mmol/g protein and the pH value of 6.4-7.4;

(13) inactivation of pasteuria virus: the pasteurization temperature of the albumin stock solution is 60 +/-0.5 ℃, and the pasteurization time is more than 10 hours;

(14) and (5) sterilizing, subpackaging and checking to obtain the human serum albumin.

Example 3

A preparation method of human serum albumin capable of effectively reducing PKA content comprises the following steps:

(1) adjusting FIV separation ionic strength: adding 1mol/L phosphate buffer solution into the supernatant FI + II + III/FII + III at the speed of 150ml/min, and controlling the ionic strength at 0.1 to obtain the supernatant with the ionic strength adjusted;

(2) adjusting the pH of the FIV separation liquid: adjusting the pH of the supernatant with the adjusted ionic strength to 6 by using an acetate buffer solution with the pH of 4.0 to obtain a supernatant with the adjusted pH;

(3) ethanol was added and the reaction temperature was adjusted: adding 95% volume fraction ethanol at-15 deg.C or below into the supernatant with adjusted pH until the volume of ethanol reaches 40% of the volume of the mixed solution, wherein the ethanol adding speed is 80L/h; gradually adjusting the temperature of the mixed solution to-6 ℃ while adding the ethanol to obtain a temperature-adjusted mixed solution;

(4) precipitation and aging: standing the temperature-adjusted mixed solution for more than 6 hours to obtain a standing solution;

(5) centrifugal separation FIV: centrifuging the standing solution at-6 deg.C, separating to remove FIV precipitate to obtain supernatant; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(6) clarifying and filtering: adding CELITE C503 diatomite filter aid into the supernatant liquid of the centrifugal separation to ensure that the concentration of the filter aid reaches 10 g/L; performing deep filtration with a filter plate under a pressure not higher than 0.3Mpa, and collecting the clarified filtrate;

(7) isoelectric precipitation: adjusting the pH of the clear filtrate to 4.8 by using 0.5mol/L acetic acid containing 40% of ethanol, adjusting the volume of the ethanol to 40% of the volume of the clear filtrate, adjusting the temperature to-9 ℃, stirring for reacting for 2 hours, and standing for more than 6 hours to obtain precipitate suspension;

(8) separating FV precipitate: centrifuging the precipitate suspension at-9 deg.C, and collecting FV precipitate; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(9) FV precipitation refining: dissolving the FV precipitate by 3 times of volume of 2 ℃ water for injection, and keeping the temperature of the solution at-2.5 ℃ to obtain FV precipitate solution;

(10) adsorption of A50: adding pretreated A50 gel into the FV precipitation solution, stirring and adsorbing for 4.5 hours to obtain suspension adsorbed by A50 gel; the addition amount of the pretreated A50 gel is 4g per kg of protein; sampling after dissolving the precipitate to check the protein content, and calculating the addition amount of A50 gel; the method for pretreating the A50 gel comprises the following steps: adding 250ml of water for injection with the temperature of 85-95 ℃ into each gram of gel, expanding for 15 minutes, filtering by using a 300-micron filter bag net, and treating the collected gel for 30 minutes at the temperature of 121 ℃ and under the pressure of 0.21Mpa to obtain pretreated A50 gel;

(11) adsorption by an activated carbon filter plate: filtering the suspension adsorbed by the A50 gel by using a filter plate containing activated carbon, collecting clear filtrate, and adjusting the pH to 7.2 by using 1mol/L sodium bicarbonate to obtain clear filtrate with the pH adjusted;

(12) preparing ultrafiltration dialysis: concentrating the clarified filtrate with the adjusted pH value by using a 10KD ultrafiltration membrane until the protein content is 15% to obtain a first concentrated solution; dialyzing with sodium chloride dialysate with volume not less than 8 times, and concentrating until protein content is greater than 150g/L or greater than 250g/L to obtain second concentrated solution; adding sodium caprylate into the second concentrated solution to prepare an albumin stock solution with the protein content of 100g/L or 200g/L, the sodium caprylate content of 0.16mmol/g protein and the pH value of 6.4-7.4;

(13) inactivation of pasteur virus: the pasteurization temperature of the albumin stock solution is 60 +/-0.5 ℃, and the pasteurization time is more than 10 hours;

(14) and (5) sterilizing, subpackaging and inspecting to obtain the human serum albumin.

Example 4

A preparation method of human serum albumin capable of effectively reducing PKA content comprises the following steps:

(1) adjusting FIV separation ionic strength: adding 1mol/L phosphate buffer solution into the FI + II + III/FII + III supernatant at the speed of 150ml/min, and controlling the ionic strength to be 0.1 to obtain supernatant with the ionic strength adjusted;

(2) adjusting the pH of the FIV separation liquid: adjusting the pH of the supernatant with the adjusted ionic strength to 6 by using an acetate buffer solution with the pH of 4.0 to obtain a supernatant with the adjusted pH;

(3) ethanol was added and the reaction temperature was adjusted: adding 95% volume fraction ethanol at-15 deg.C or below into the supernatant with adjusted pH until the volume of ethanol reaches 40% of the volume of the mixed solution, wherein the ethanol adding speed is 80L/h; gradually adjusting the temperature of the mixed solution to-6 ℃ while adding ethanol to obtain a temperature-adjusted mixed solution;

(4) precipitation and aging: standing the temperature-adjusted mixed solution for more than 6 hours to obtain a standing solution;

(5) centrifugal separation FIV: centrifuging the standing solution at-6 deg.C, separating to remove FIV precipitate to obtain supernatant; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(6) clarifying and filtering: adding a CELITE C503 diatomite filter aid to the supernatant of the centrifugal separation to ensure that the concentration of the filter aid reaches 10 g/L; performing deep filtration with a filter plate under a pressure not higher than 0.3Mpa, and collecting the clarified filtrate;

(7) isoelectric precipitation: adjusting the pH of the clarified filtrate to 4.8 by using 0.5mol/L acetic acid containing 40% ethanol, adjusting the volume of the ethanol to 40% of the volume of the clarified filtrate, adjusting the temperature to-9 ℃, stirring for reacting for 2 hours, and standing for more than 6 hours to obtain precipitate suspension;

(8) separating FV precipitate: centrifuging the precipitate suspension at-9 deg.C, and collecting FV precipitate; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(9) FV precipitation refining: dissolving the FV precipitate by 3 times of volume of 2 ℃ water for injection, and keeping the temperature of the solution at-2.5 ℃ to obtain FV precipitate solution;

(10) adsorption of A50: adding pretreated A50 gel into the FV precipitation solution, stirring and adsorbing for 4.5 hours to obtain suspension adsorbed by A50 gel; the addition amount of the pretreated A50 gel is 4g per kg of protein; sampling after dissolving the precipitate to check the protein content, and calculating the addition amount of A50 gel; the method for pretreating the A50 gel comprises the following steps: adding 250ml of water for injection with the temperature of 85-95 ℃ into each gram of gel, expanding for 15 minutes, and filtering by using a 300-micron filter bag net to obtain pretreated A50 gel; the method for pretreating the A50 gel is a traditional pretreatment method of A50 gel;

(11) adsorption by an activated carbon filter plate: filtering the suspension adsorbed by the A50 gel by using a filter plate containing activated carbon, collecting clear filtrate, and adjusting the pH to 7.2 by using 1mol/L sodium bicarbonate to obtain clear filtrate with the pH adjusted;

(12) preparing ultrafiltration dialysis: concentrating the clarified filtrate with the adjusted pH value by using a 10KD ultrafiltration membrane until the protein content is 15 percent to obtain a first concentrated solution; dialyzing with sodium chloride dialysate with volume not less than 8 times, and concentrating until protein content is greater than 150g/L or greater than 250g/L to obtain second concentrated solution; adding sodium caprylate into the second concentrated solution to prepare an albumin stock solution with the protein content of 100g/L or 200g/L, the sodium caprylate content of 0.16mmol/g protein and the pH value of 6.4-7.4;

(13) inactivation of pasteuria virus: the pasteurization temperature of the albumin stock solution is 60 +/-0.5 ℃, and the pasteurization time is more than 10 hours;

(14) and (5) sterilizing, subpackaging and checking to obtain the human serum albumin.

Comparative example 1

A preparation method of human serum albumin capable of effectively reducing PKA content comprises the following steps:

(1) adjusting FIV separation ionic strength: adding 1mol/L sodium chloride solution into the FI + II + III/FII + III supernatant at the speed of 150ml/min, and controlling the ionic strength to be 0.1 to obtain supernatant with the ionic strength adjusted;

(2) adjusting the pH of the FIV separation liquid: adjusting the pH of the supernatant with the adjusted ionic strength to 6 by using an acetate buffer solution with the pH of 4.0 to obtain a supernatant with the adjusted pH;

(3) ethanol was added and the reaction temperature was adjusted: adding 95% ethanol with volume fraction below-15 deg.C into the supernatant with adjusted pH until the volume of ethanol reaches 40% of the volume of the mixed solution, wherein the ethanol adding speed is 80L/h; gradually adjusting the temperature of the mixed solution to-6 ℃ while adding ethanol to obtain a temperature-adjusted mixed solution;

(4) precipitation and aging: standing the temperature-adjusted mixed solution for more than 6 hours to obtain a standing solution;

(5) centrifugal separation FIV: centrifuging the standing solution at-6 deg.C, separating to remove FIV precipitate to obtain supernatant; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(6) clarification and filtration: adding CELITE C503 diatomite filter aid into the supernatant liquid of the centrifugal separation to ensure that the concentration of the filter aid reaches 10 g/L; performing deep filtration with a filter plate under a pressure not higher than 0.3Mpa, and collecting the clarified filtrate;

(7) isoelectric precipitation: adjusting the pH value of the clear filtrate to 4.8 by using 0.5mol/L acetic acid containing 40% of ethanol, adjusting the volume of the ethanol to 40% of the volume of the clear filtrate, adjusting the temperature to-9 ℃, stirring for reacting for 2 hours, and standing for more than 6 hours to obtain precipitate suspension;

(8) separating FV precipitate: centrifuging the precipitate suspension at-9 deg.C, and collecting FV precipitate; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(9) FV precipitation refining: dissolving the FV precipitate by 3 times of volume of 2 ℃ water for injection, and keeping the temperature of the solution at-2.5 ℃ to obtain FV precipitate solution;

(10) adsorption of A50: adding pretreated A50 gel into the FV precipitate solution, stirring and adsorbing for 4.5 hours to obtain suspension adsorbed by A50 gel; the addition amount of the pretreated A50 gel is 4g per kg of protein; sampling after dissolving the precipitate, checking the protein content, and calculating the addition amount of A50 gel; the method for pretreating the A50 gel comprises the following steps: adding 250ml of water for injection with the temperature of 85-95 ℃ into each gram of gel, expanding for 15 minutes, filtering by using a 300-micron filter bag net, and treating the collected gel for 30 minutes at the temperature of 121 ℃ and under the pressure of 0.21Mpa to obtain pretreated A50 gel;

(11) adsorption by an active carbon filter plate: filtering the suspension adsorbed by the A50 gel by using a filter plate containing activated carbon, collecting clear filtrate, and adjusting the pH to 7.2 by using 1mol/L sodium bicarbonate to obtain clear filtrate with the pH adjusted;

(12) preparing ultrafiltration dialysis: concentrating the clarified filtrate with the adjusted pH value by using a 10KD ultrafiltration membrane until the protein content is 15% to obtain a first concentrated solution; dialyzing with sodium chloride dialysate with volume not less than 8 times, and concentrating until protein content is greater than 150g/L or greater than 250g/L to obtain second concentrated solution; adding sodium caprylate into the second concentrated solution to prepare an albumin stock solution with the protein content of 100g/L or 200g/L, the sodium caprylate content of 0.16mmol/g protein and the pH value of 6.4-7.4;

(13) inactivation of pasteuria virus: the pasteurization temperature of the albumin stock solution is 60 +/-0.5 ℃, and the pasteurization time is more than 10 hours;

(14) and (5) sterilizing, subpackaging and inspecting to obtain the human serum albumin.

Comparative example 2

A preparation method of human serum albumin capable of effectively reducing PKA content comprises the following steps:

(1) adjusting FIV separation ionic strength: adding 1mol/L acetate buffer solution into the FI + II + III/FII + III supernatant at the speed of 150ml/min, and controlling the ionic strength at 0.1 to obtain supernatant with adjusted ionic strength;

(2) adjusting the pH of the FIV separation liquid: adjusting the pH of the supernatant with the adjusted ionic strength to 6 by using an acetate buffer solution with the pH of 4.0 to obtain a supernatant with the adjusted pH;

(3) ethanol was added and the reaction temperature was adjusted: adding 95% volume fraction ethanol at-15 deg.C or below into the supernatant with adjusted pH until the volume of ethanol reaches 40% of the volume of the mixed solution, wherein the ethanol adding speed is 80L/h; gradually adjusting the temperature of the mixed solution to-6 ℃ while adding the ethanol to obtain a temperature-adjusted mixed solution;

(4) precipitation and aging: standing the temperature-adjusted mixed solution for more than 6 hours to obtain a standing solution;

(5) centrifugal separation FIV: centrifuging the standing solution at-6 deg.C, separating to remove FIV precipitate to obtain supernatant; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(6) clarification and filtration: adding a CELITE C503 diatomite filter aid to the supernatant of the centrifugal separation to ensure that the concentration of the filter aid reaches 10 g/L; performing deep filtration with a filter plate under a pressure not higher than 0.3Mpa, and collecting the clarified filtrate;

(7) isoelectric precipitation: adjusting the pH of the clear filtrate to 4.8 by using 0.5mol/L acetic acid containing 40% of ethanol, adjusting the volume of the ethanol to 40% of the volume of the clear filtrate, adjusting the temperature to-9 ℃, stirring for reacting for 2 hours, and standing for more than 6 hours to obtain precipitate suspension;

(8) separating FV precipitate: centrifuging the precipitate suspension at-9 deg.C, and collecting FV precipitate; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(9) FV precipitation refining: dissolving the FV precipitate by 3 times of volume of 2 ℃ water for injection, and keeping the temperature of the solution at-2.5 ℃ to obtain FV precipitate solution;

(10) adsorption of A50: adding pretreated A50 gel into the FV precipitation solution, stirring and adsorbing for 4.5 hours to obtain suspension adsorbed by A50 gel; the addition amount of the pretreated A50 gel is 4g per kg of protein; sampling after dissolving the precipitate, checking the protein content, and calculating the addition amount of A50 gel; the method for pretreating the A50 gel comprises the following steps: adding 250ml of water for injection with the temperature of 85-95 ℃ into each gram of gel, expanding for 15 minutes, filtering by using a 300-micron filter bag net, and treating the collected gel for 30 minutes at the temperature of 121 ℃ and under the pressure of 0.21Mpa to obtain pretreated A50 gel;

(11) adsorption by an activated carbon filter plate: filtering the suspension adsorbed by the A50 gel by using an activated carbon-containing filter plate, collecting clear filtrate, and adjusting the pH to 7.2 by using 1mol/L sodium bicarbonate to obtain clear filtrate with the pH adjusted;

(12) preparing ultrafiltration dialysis: concentrating the clarified filtrate with the adjusted pH value by using a 10KD ultrafiltration membrane until the protein content is 15% to obtain a first concentrated solution; dialyzing with sodium chloride dialysate of not less than 8 times volume at constant volume, and concentrating until protein content is greater than 150g/L or greater than 250g/L to obtain second concentrated solution; adding sodium caprylate into the second concentrated solution to prepare an albumin stock solution with the protein content of 100g/L or 200g/L, the sodium caprylate content of 0.16mmol/g protein and the pH value of 6.4-7.4;

(13) inactivation of pasteur virus: the pasteurization temperature of the albumin stock solution is 60 +/-0.5 ℃, and the pasteurization time is more than 10 hours;

(14) and (5) sterilizing, subpackaging and inspecting to obtain the human serum albumin.

Comparative example 3

A preparation method of human serum albumin capable of effectively reducing PKA content comprises the following steps:

(1) adjusting FIV separation ionic strength: adding 1mol/L phosphate buffer solution into the FI + II + III/FII + III supernatant at the speed of 150ml/min, and controlling the ionic strength to be 0.1 to obtain supernatant with the ionic strength adjusted;

(2) adjusting the pH of the FIV separation liquid: adjusting the pH of the supernatant with the adjusted ionic strength to 6 by using an acetate buffer solution with the pH of 4.0 to obtain a supernatant with the adjusted pH;

(3) ethanol was added and the reaction temperature was adjusted: adding 95% volume fraction ethanol at-15 deg.C or below into the supernatant with adjusted pH until the volume of ethanol reaches 40% of the volume of the mixed solution, wherein the ethanol adding speed is 80L/h; gradually adjusting the temperature of the mixed solution to-6 ℃ while adding ethanol to obtain a temperature-adjusted mixed solution;

(4) precipitation and aging: standing the temperature-adjusted mixed solution for more than 6 hours to obtain a standing solution;

(5) centrifugal separation FIV: centrifuging the standing solution at-6 deg.C, separating to remove FIV precipitate to obtain supernatant; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(6) clarification and filtration: adding a CELITE C503 diatomite filter aid to the supernatant of the centrifugal separation to ensure that the concentration of the filter aid reaches 10 g/L; performing deep filtration with a filter plate under a pressure not higher than 0.3Mpa, and collecting the clarified filtrate;

(7) isoelectric precipitation: adjusting the pH of the clear filtrate to 4.8 by using 0.5mol/L acetic acid containing 40% of ethanol, adjusting the volume of the ethanol to 40% of the volume of the clear filtrate, adjusting the temperature to-9 ℃, stirring for reacting for 2 hours, and standing for more than 6 hours to obtain precipitate suspension;

(8) separating FV precipitate: centrifuging the precipitate suspension at-9 deg.C, and collecting FV precipitate; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the clear liquid is 500-1000 ml/min;

(9) FV precipitation refining: dissolving the FV precipitate by 3 times of volume of 2 ℃ injection water, and keeping the temperature of the solution at-2.5 ℃ to obtain FV precipitate solution;

(10) adsorption of A50: adding pretreated A50 gel into the FV precipitate solution, stirring and adsorbing for 4.5 hours to obtain suspension adsorbed by A50 gel; the addition amount of the pretreated A50 gel is 4g per kg of protein; sampling after dissolving the precipitate, checking the protein content, and calculating the addition amount of A50 gel; the method for pretreating the A50 gel comprises the following steps: adding 250ml of water for injection with the temperature of 85-95 ℃ into each gram of gel, expanding for 15 minutes, filtering by using a 300-micron filter bag net, and treating the collected gel for 30 minutes at the temperature of 121 ℃ and under the pressure of 0.21Mpa to obtain pretreated A50 gel;

(11) filtering by a deep filter plate: filtering the suspension adsorbed by the A50 gel by using a deep filter plate, collecting clear filtrate, and adjusting the pH to 7.2 by using 1mol/L sodium bicarbonate to obtain clear filtrate with the pH adjusted;

(12) preparing ultrafiltration dialysis: concentrating the clarified filtrate with the adjusted pH value by using a 10KD ultrafiltration membrane until the protein content is 15% to obtain a first concentrated solution; dialyzing with sodium chloride dialysate with volume not less than 8 times, and concentrating until protein content is greater than 150g/L or greater than 250g/L to obtain second concentrated solution; adding sodium caprylate into the second concentrated solution to prepare an albumin stock solution with the protein content of 100g/L or 200g/L, the sodium caprylate content of 0.16mmol/g protein and the pH value of 6.4-7.4;

(13) inactivation of pasteur virus: the pasteurization temperature of the albumin stock solution is 60 +/-0.5 ℃, and the pasteurization time is more than 10 hours;

(14) and (5) sterilizing, subpackaging and inspecting to obtain the human serum albumin.

Comparative example 4

A preparation method of human serum albumin capable of effectively reducing PKA content comprises the following steps:

(1) adjusting FIV separation ionic strength: adding 1mol/L phosphate buffer solution into the supernatant FI + II + III/FII + III at the speed of 150ml/min, and controlling the ionic strength at 0.1 to obtain the supernatant with the ionic strength adjusted;

(2) adjusting the pH of the FIV separation liquid: adjusting the pH of the supernatant with the adjusted ionic strength to 6 by using an acetate buffer solution with the pH of 4.0 to obtain a supernatant with the adjusted pH;

(3) ethanol was added and the reaction temperature was adjusted: adding 95% volume fraction ethanol at-15 deg.C or below into the supernatant with adjusted pH until the volume of ethanol reaches 40% of the volume of the mixed solution, wherein the ethanol adding speed is 80L/h; gradually adjusting the temperature of the mixed solution to-6 ℃ while adding ethanol to obtain a temperature-adjusted mixed solution;

(4) precipitation and aging: standing the temperature-adjusted mixed solution for more than 6 hours to obtain a standing solution;

(5) centrifugal separation FIV: centrifuging the standing solution at-6 deg.C, separating to remove FIV precipitate to obtain supernatant; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(6) clarifying and filtering: adding CELITE C503 diatomite filter aid into the supernatant liquid of the centrifugal separation to ensure that the concentration of the filter aid reaches 10 g/L; performing deep filtration with a filter plate under the pressure not higher than 0.3Mpa, and collecting the clarified filtrate;

(7) isoelectric precipitation: adjusting the pH of the clarified filtrate to 4.8 by using 0.5mol/L acetic acid containing 40% ethanol, adjusting the volume of the ethanol to 40% of the volume of the clarified filtrate, adjusting the temperature to-9 ℃, stirring for reacting for 2 hours, and standing for more than 6 hours to obtain precipitate suspension;

(8) separating FV precipitate: centrifuging the precipitate suspension at-9 deg.C, and collecting FV precipitate; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;

(9) FV precipitation refining: dissolving the FV precipitate by 3 times of volume of 2 ℃ water for injection, and keeping the temperature of the solution at-2.5 ℃ to obtain FV precipitate solution;

(10) adsorption by an active carbon filter plate: filtering the FV precipitate dissolved solution by using a filter plate containing activated carbon, collecting clear filtrate, and adjusting the pH to 7.2 by using 1mol/L sodium bicarbonate to obtain clear filtrate with the pH adjusted;

(11) preparing ultrafiltration dialysis: concentrating the clarified filtrate with the adjusted pH value by using a 10KD ultrafiltration membrane until the protein content is 15% to obtain a first concentrated solution; dialyzing with sodium chloride dialysate of not less than 8 times volume at constant volume, and concentrating until protein content is greater than 150g/L or greater than 250g/L to obtain second concentrated solution; adding sodium caprylate into the second concentrated solution to prepare an albumin stock solution with the protein content of 100g/L or 200g/L, the sodium caprylate content of 0.16mmol/g protein and the pH value of 6.4-7.4;

(12) inactivation of pasteuria virus: the pasteurization temperature of the albumin stock solution is 60 +/-0.5 ℃, and the pasteurization time is more than 10 hours;

(13) and (5) sterilizing, subpackaging and inspecting to obtain the human serum albumin.

Tables 1 and 2 below show the measurement results of various indexes of the human serum albumin prepared in examples 1 to 4 and comparative examples 1 to 4;

the detection method comprises the following steps: the detection method of the haptoglobin, the alpha-acid glycoprotein and the hemopexin is an immunoturbidimetry (IMMAGE method), the detection method of the content of the polymer is SEC-HPLC, the detection method of the absorbance is an ultraviolet-visible spectrophotometry, the detection method of the purity is a cellulose acetate membrane electrophoresis method, the detection method of the turbidity is a nephelometer method, and the detection method of the content of PKA is a display substrate method.

TABLE 1

Serial number	Albumin yield (g/L plasma)	Haptoglobin content (g/L)	Alpha acid glycoprotein (g/L)	Heme binding protein (g/L)
					Example 1	28.9	0.51	0.25	0.44
Example 2	28.8	0.52	0.23	0.45
					Example 3	29.3	0.50	0.22	0.42
Example 4	28.7	0.61	0.28	0.38
					Comparative example 1	28.3	0.54	0.33	0.36
Comparative example 2	28.2	0.57	0.31	0.47
					Comparative example 3	28.6	0.53	0.32	1.11
Comparative example 4	28.9	1.25	0.75	0.48

TABLE 2

Serial number	Polymer content (%)	Albumin purity (%)	Absorbance of the solution	PKA content (IU/ml)	Turbidity (NTU)
						Example 1	1.8	98.5	0.02	<10	3.31
Example 2	1.7	98.4	0.02	<10	3.26
						Example 3	1.7	98.5	0.02	<10	3.24
Example 4	1.7	98.6	0.03	<10	3.25
						Comparative example 1	2.2	98.3	0.02	18	3.38
Comparative example 2	2.1	98.5	0.02	25	3.22
						Comparative example 3	2.0	98.1	0.06	<10	3.50
Comparative example 4	2.9	97.5	0.03	11	3.58

According to the determination results of various indexes of the human serum albumin, the preparation method of the human serum albumin capable of effectively reducing the PKA content has the advantages that phosphate is used for controlling the ionic strength in the FIV separation process, the solubility of albumin is improved, and meanwhile, the solubility of PKA is reduced, so that more PKA is removed through precipitation, and the human serum albumin with low PKA content is prepared; impurity removal is carried out on the downstream of product preparation through combination of A50 gel adsorption and activated carbon filter plate adsorption, A50 gel mainly adsorbs serine protease such as PKA, haptoglobin and alpha acid glycoprotein, and the activated carbon filter plate mainly adsorbs heme, endotoxin, serine protease and the like, so that the downstream of product preparation is combined through combination of A50 gel adsorption and activated carbon filter plate adsorption, PKA content can be effectively reduced, product appearance can be improved, endotoxin content can be reduced, polymer content can be reduced, albumin purity can be improved, and product safety can be improved; pretreatment of A50 gel can improve the adsorption effect of A50 gel.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (9)

1. A method for preparing human albumin capable of effectively reducing PKA content is characterized by comprising the following steps:

(1) adjusting ionic strength of FIV separation liquid: adding 0.8-1.2 mol/L phosphate buffer solution into the FI + II + III/FII + III supernatant, and controlling the ionic strength to be 0.08-0.12 to obtain supernatant with adjusted ionic strength;

(2) adjusting the pH of the FIV separation liquid: adjusting the pH of the supernatant with the adjusted ionic strength to 5.8-6.2 by using an acetate buffer solution to obtain a supernatant with the adjusted pH;

(3) ethanol was added and the reaction temperature was adjusted: adding 95% volume fraction ethanol with the temperature below-15 ℃ to the supernatant with the adjusted pH until the volume of the ethanol reaches 38-42% of the volume of the mixed solution, adjusting the temperature of the mixed solution to-5-7 ℃ to obtain the temperature-adjusted mixed solution;

(4) precipitation and aging: standing the temperature-adjusted mixed solution for more than 6 hours to obtain a standing solution;

(5) and (3) centrifugal separation FIV: centrifuging the standing solution at the temperature of-5 to-7 ℃, and separating to remove FIV precipitate to obtain supernatant after centrifugal separation;

(6) clarification and filtration: adding a filter aid into the supernatant obtained by centrifugal separation, so that the concentration of the filter aid reaches 5-15 g/L; filtering with a filter plate under a pressure not higher than 0.3Mpa, and collecting clear filtrate;

(7) isoelectric precipitation: adjusting the pH value of the clarified filtrate to 4.7-4.9, adjusting the volume of ethanol to 38-42% of the volume of the clarified filtrate, adjusting the temperature to-8-10 ℃, and standing for more than 6 hours to obtain a precipitate suspension;

(8) separating FV precipitate: centrifuging the precipitate suspension at-8-10 ℃ and collecting FV precipitate;

(9) FV precipitation refining: dissolving the FV precipitate by using 2-4 times of volume of 0-5 ℃ injection water, and keeping the temperature of the solution at-2 to-3 ℃ to obtain FV precipitate solution;

(10) adsorption of A50: adding pretreated A50 gel into the FV precipitation solution, stirring and adsorbing for 4-5 hours to obtain suspension adsorbed by A50 gel; the addition amount of the pretreated A50 gel is 3-5 g per kilogram of protein;

the method for pretreating the A50 gel comprises the following steps: adding 250ml of water for injection into each gram of gel, expanding for 15 minutes, filtering with a 300um filter bag net, and treating the collected gel at 121 ℃ and 0.21Mpa for 30 minutes to obtain pretreated A50 gel;

(11) adsorption by an active carbon filter plate: filtering the suspension adsorbed by the A50 gel by using an activated carbon filter plate, collecting clear filtrate, and adjusting the pH to 7.0-7.5 to obtain clear filtrate with the pH adjusted;

(12) preparing ultrafiltration dialysis: concentrating the clarified filtrate with the adjusted pH value by using a 10KD ultrafiltration membrane to obtain a first concentrated solution; dialyzing with dialysate with a volume not less than 8 times of the volume and a constant volume, and concentrating to obtain a second concentrated solution; adding sodium caprylate into the second concentrated solution to prepare a stock solution with the protein content of 100g/L or 200g/L, the sodium caprylate content of 0.16mmol/g protein and the pH value of 6.4-7.4;

(13) inactivation of pasteur virus: the pasteurization temperature of the stock solution is 60 +/-0.5 ℃, and the pasteurization time is more than 10 hours;

(14) and (5) sterilizing, subpackaging and checking to obtain the human serum albumin.

2. The method for preparing human serum albumin with PKA content effectively reduced according to claim 1, characterized in that in step (1), 1.0mol/L of phosphate buffer is added to the supernatant FI + II + III/FII + III to control the ionic strength at 0.10, so as to obtain the supernatant with adjusted ionic strength.

3. The method of claim 1, wherein the pH of the supernatant of step (2) is adjusted to 6.0 with acetate buffer to obtain a pH-adjusted supernatant.

4. The method according to claim 1, wherein the volume fraction of 95% ethanol with a temperature below-15 ℃ is added to the supernatant of the adjusted pH in step (3) until the volume of the ethanol reaches 40% of the volume of the mixed solution, and the temperature of the mixed solution is adjusted to-6 ℃ to obtain the tempered mixed solution.

5. The process for preparing human serum albumin with reduced PKA content effectively according to claim 1, characterized in that the filter aid used in the step (6) is CELITE503 diatomaceous earth filter aid.

6. The method of claim 5, wherein the clear filtrate is subjected to pH adjustment to 4.8, ethanol volume to 40% of the volume of the clear filtrate, temperature to-9 ℃, and standing for 6 hours or more in step (7) to obtain a precipitate suspension.

7. The method for preparing human serum albumin with PKA content effectively reduced according to claim 1, characterized in that the centrifugation process parameters in said step (8) are as follows: the centrifuge speed was 15000 rpm.

8. The method for preparing human serum albumin with reduced PKA content effectively according to claim 1, characterized in that in said step (9), 3 times of volume of 2 ℃ water for injection is used for dissolving said FV precipitate, and the temperature of the solution is kept at-2.5 ℃ to obtain FV precipitate solution.

9. The method for preparing human serum albumin capable of effectively reducing PKA content according to claim 1, characterized in that in said step (12), said clarified filtrate with adjusted pH is concentrated with a 10KD ultrafiltration membrane until the protein content is 14% -16%, so as to obtain a first concentrated solution; dialyzing with dialysate with a constant volume not less than 8 times of the volume of the dialysate to obtain a second concentrated solution; and adding sodium caprylate into the second concentrated solution to prepare a stock solution with the protein content of 100g/L or 200g/L, the sodium caprylate content of 0.16mmol/g protein and the pH value of 6.4-7.4.

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