阅读说明：本技术 用于纯化白蛋白的方法 (Process for purifying albumin ) 是由 J·S·徐 D·米勒 于 2018-03-16 设计创作，主要内容包括：描述了一种用于从血浆中纯化白蛋白的方法。所述方法包括(a)使所述血浆与一定量的辛酸钠(NaCP)接触,所述辛酸钠(NaCP)的量取决于所述血浆中的总蛋白浓度和NaCP与总蛋白的比率；(b)在接近中性的pH值范围内加热所述血浆；以及(c)将所述白蛋白与非白蛋白相分离。所述方法提供了高收率和纯度的白蛋白溶液。(A process for the purification of albumin from plasma is described. The method comprises (a) contacting the plasma with an amount of sodium caprylate (NaCP) that is dependent on the total protein concentration and the ratio of NaCP to total protein in the plasma; (b) heating the plasma at a near neutral pH range; and (c) separating the albumin from the non-albumin phase. The process provides an albumin solution in high yield and purity.)

1. A process for purifying albumin from plasma comprising:

(a) contacting the plasma with sodium caprylate (NaCP);

wherein the amount of NaCP is from 0.1mmol to 5mmol NaCP per gram of total protein in the plasma;

(b) heating the plasma at a temperature of 60 ℃ to 70 ℃; and

(c) separating the albumin from the non-albumin phase.

2. The method of claim 1, wherein the plasma comprises total protein at a concentration of 20g/L to 150 g/L.

3. The method of claim 1 or 2, wherein the plasma is partially purified plasma, cold plasma poor, a plasma intermediate, a plasma protein solution, or a mixture thereof.

4.The method of any one of claims 1-3, wherein the amount of NaCP is from 0.2mmol to 3mmol, optionally from 0.5mmol to 2mmol, optionally from 0.8mmol to 1.3mmol of NaCP per gram of total protein.

5. The method of any one of claims 1-4, wherein the concentration of total protein is 30 to 100g/L, optionally 35 to 80g/L, optionally 45 to 65 g/L.

6. The method of any one of claims 1-5, wherein the amount of NaCP is 0.8mmol to 1.3mmol NaCP per gram of total protein, wherein the total protein has a concentration of 45g/L to 65 g/L.

7. The method of any one of claims 1-6, wherein the NaCP has a concentration of 20mM to 140mM, optionally 40mM to 100mM, optionally 50mM to 90mM, optionally 60mM to 85 mM.

8. The method of any one of claims 1-7, wherein the heating of the plasma is performed at a pH value of 6.0 to 8.0, optionally 6.5 to 7.5, optionally 6.7 to 7.3, optionally 6.7 to 6.9.

9. The method of any one of claims 1-8, wherein the conductivity of the plasma during heating is less than 9mS/cm, optionally less than 5 mS/cm.

10. The method of any one of claims 1-9, wherein the heating of the plasma is performed at a temperature of 60 ℃ to 70 ℃, optionally 62 ℃ to 65 ℃, for a time of 0.5 hours to 24 hours, optionally 2 hours to 12 hours, optionally 3 hours to 12 hours, optionally 4 hours to 6.5 hours.

11. The method according to any one of claims 1-10, wherein said heating of said plasma is performed at a pH of 6.7 to 6.9 at a temperature of 62 ℃ to 65 ℃ for a period of at least 4 hours, wherein the amount of NaCP is 0.8mmol to 1.3mmol NaCP per gram of total protein, wherein said total protein has a concentration of 45g/L to 65 g/L.

12. The method of any one of claims 1-11, wherein the concentration of total protein is adjusted by adding water.

13. The method of any one of claims 1-12, wherein separating the albumin from the non-albumin phase comprises precipitating non-albumin protein and lipid impurities.

14.The method of any one of claims 1-13, wherein separating the albumin from the non-albumin phase comprises cooling the plasma to below 30 ℃.

15. The method of any one of claims 1-14, wherein separating the albumin from the non-albumin phase comprises filtration by depth filtration, Tangential Flow Filtration (TFF) using a cartridge, or hollow fiber TFF, preferably filtration by hollow fiber TFF.

16. The method of any one of claims 1-15, wherein separating the albumin from the non-albumin phase comprises adjusting the pH of the plasma to 4.8 to 5.4, optionally 5.1 to 5.3.

17. The method of claim 16, wherein adjusting the pH of the plasma is performed by adding an acid.

18. The method of claim 17, wherein the acid comprises an organic acid and/or an inorganic acid.

19. The method of claim 18, wherein the organic acid is selected from the group consisting of citric acid, acetic acid, and trifluoroacetic acid.

20. The method of claim 18 or 19, wherein the organic acid is acetic acid.

21. The method of claim 18, wherein the mineral acid is selected from the group consisting of hydrochloric acid, sulfuric acid, and phosphoric acid.

22. The method of any one of claims 15-21, wherein separating albumin from non-albumin comprises filtering at a feed flow rate of 1 to 10 liters per minute per square meter, optionally 2 to 10 liters per minute per square meter, optionally 3 to 10 liters per minute per square meter, optionally 8 to 9 liters per minute per square meter.

23. The method of any one of claims 1-22, wherein the method is capable of linear scaling.

24. An albumin solution recovered by the method of any one of claims 1-23, comprising an albumin content of at least 90% (w/w) protein, optionally at least 92% (w/w) protein, optionally at least 94% (w/w) protein, optionally at least 96% (w/w) protein, optionally at least 98% (w/w) protein, optionally at least 99% (w/w) protein.

Technical Field

The present disclosure relates to a novel method for purifying albumin from plasma. The method comprises contacting plasma with an amount of sodium caprylate (NaCP) that is dependent on the total protein concentration and the ratio of NaCP to total protein in the plasma; pasteurizing the plasma at a near neutral pH range; and separating the albumin from the non-albumin phase. The process provides an albumin solution in high yield and purity.

Background

Human and animal blood is a source of molecules with therapeutic properties. Many of these molecules are proteins that can be found in plasma or serum. These proteins have been the target of specific isolation with the aim of purifying and standardizing molecules for use as human therapeutics. For example, albumin, immunoglobulin G, factor VIII and alpha-1-proteinase inhibitor are all available as isolated therapeutic products.

One of the conventionally used methods for fractionation of plasma or serum proteins is described in U.S. patent No. 2,390,074, which discloses a method for large-scale fractionation of plasma or serum proteins by precipitation with ethanol and adjusting the temperature, pH, ionic strength and time to control the precipitation of certain proteins from human plasma. The fractionation process comprises stepwise addition of ethanol to the plasma feedstock to obtain several fractions and corresponding supernatants containing different enriched protein solutions.

The ethanol precipitation method has the following disadvantages: some proteins tend to denature in the presence of ethanol, leading to reduced yields of the protein to be separated and contamination with aggregates that need to be removed before an acceptable therapeutic product can be obtained. Furthermore, the precipitated proteins require re-solubilization for further processing, which can result in significant levels of insoluble protein and lipid material, which in turn can interfere with the purification of the final product, resulting in further reduced yields.

In addition, Human Serum Albumin (HSA) has been purified from plasma, in particular, by various fractionation methods. Albumin is the most abundant protein in human plasma, accounting for up to 60% of serum proteins. Therapeutic albumin was formulated by following the pharmaceutical guidelines. According to regulatory guidelines, the preparation contains N-acetyl tryptophan and caprylate followed by pasteurization at 60 ℃. + -. 0.5 ℃ for 10.5 hours. + -. 0.5 hours for viral inactivation. The amounts of these two reagents as stabilizers were determined based on the amount of HSA, set to 0.08mmol ± 0.016mmol per gram of HSA protein.

During pasteurization, the integrity of HSA is maintained by the presence of the caprylate/tryptophan regimen. The octanoate ion is believed to reduce the rate of reversible partial unfolding by binding to HSA, thereby inhibiting a step in the pathway leading to irreversible precipitation. Faroongsarng and Kongprasertkit (2014) observed that the denaturation temperature of HSA increased from 67.23 ℃ to 72.85 ℃ in the presence of 0.08 mmoleNaCP per gram of HSA.

The basic concepts and prior art in the field of albumin separation have been described in, for example, U.S. patent No. 2,705,230, U.S. patent No. 3,992,367, U.S. patent No. 4,156,681, U.S. patent No. 5,118,794, U.S. patent No. 6,022,954, and U.S. patent No. 6,504,011. These documents are incorporated herein by reference.

Us patent No. 6,022,954 relates to a process for the preparation of purified albumin from a physiological solution, such as plasma or plasma fractions, of a human or animal. This document teaches a process for degreasing using an anionic detergent and two chromatographic separation stages using ion exchange resins. The degreasing treatment consists of the following steps: the albumin solution from the first chromatography stage is contacted with the detergent at 60 ℃ and in the presence of a small amount of NaCP as a stabilizer for about 1 hour. The pasteurization treatment consisted of the following steps: the solution is heated at over 60 ℃ for at least 10 hours.

Us patent No. 5,118,794 relates to a method for stabilizing human albumin during heat treatment in a container comprising the addition of a surfactant. This document discloses a method comprising pasteurising the solution at pH 7.0 with a small amount of NaCP in the amount of 0.08-0.64 mmol NaCP per gram of albumin. The document also discloses a final pasteurization of the product at 60 ℃ for 10 hours.

In us patent No. 3,992,367, the process of albumin purification includes a heating step in which the solution consists of 15% to 30% by weight NaCP at a pH of 4.8 to 5.25. In U.S. patent No. 2,705,230, albumin isolation involves a pasteurization heat treatment, which is performed at pH 8 using 50mM and 500mM NaCP.

Us patent No. 4,156,681 relates to a method for extracting serum albumin from blood, comprising the steps of: separating the plasma from the solid components of the blood; separating the dissolved non-albumin fraction from the plasma; and adding an albumin stabilizer and treating such fluid with a lower aliphatic alcohol. The method comprises adding a small amount of NaCP to a large volume of plasma for albumin purification and heat treatment at a pH of 4.5 to 7.5.

Us patent No. 6,504,011 relates to a process for the purification of recombinantly produced serum albumin by incubation with an anion exchange adsorbent followed by affinity chromatography using a hydrophobic solid phase and a water-soluble lipid anion as desorbent in the aqueous phase. This document teaches purification of albumin by chromatography without a pasteurization stage and without the use of NaCP in the elution buffer to recover the albumin.

Previous reports did not help to understand the complex relationship between total protein concentration in plasma and the ratio of NaCP to total protein during pasteurization to purify albumin. The high yield and purity achieved by the disclosed process provides the advantage of maximizing the quality and quantity of albumin recovered.

Disclosure of Invention

The present disclosure describes an improved process for purifying albumin from plasma.

Accordingly, the present disclosure provides a method for purifying albumin from plasma comprising:

(a) contacting the plasma with sodium caprylate (NaCP);

wherein the amount of NaCP is from 0.1mmol to 5mmol NaCP per gram of total protein in said plasma;

(b) heating the plasma at a temperature of 60 ℃ to 70 ℃; and

(c) the albumin is separated from the non-albumin phase.

In one embodiment, the plasma is concentrated to a total protein concentration of 20 to 150g/L, preferably 45 to 65g/L, prior to step (a).

In another embodiment, the amount of NaCP is from 0.2mmol to 3mmol, optionally from 0.5mmol to 2mmol, optionally from 0.8mmol to 1.3mmol of NaCP per gram of total protein.

In another embodiment, the NaCP is added at a concentration of 20mM to 140mM, optionally 40mM to 100mM, optionally 50mM to 90mM, optionally 60mM to 85 mM.

In another embodiment, the heating of the plasma in step (b) is performed at a pH value of 6.0 to 8.0, optionally 6.5 to 7.5, optionally 6.7 to 7.3, optionally 6.7 to 6.9.

In another embodiment, the conductivity of the plasma is less than 9mS/cm, optionally less than 5 mS/cm.

In another embodiment, the heating of the plasma is performed at a temperature of 60 ℃ to 70 ℃, optionally 62 ℃ to 65 ℃, for a time of 0.5 hours to 24 hours, optionally 2 hours to 12 hours, optionally 3 hours to 12 hours, optionally 4 hours to 6.5 hours.

In another embodiment, separating the albumin from the non-albumin phase in step (c) comprises precipitating non-albumin protein and lipid impurities.

In another embodiment, separating the albumin from the non-albumin phase comprises cooling the plasma to less than 30 ℃.

In another embodiment, the plasma is filtered in step (c), optionally via filtration using a Pall Seitz K700P depth filter, using a cartridge for Tangential Flow Filtration (TFF) or hollow fiber TFF, preferably hollow fiber TFF, in the presence of a Harborlite Filter aid (2% w/v H900, 2% w/v H1900).

In another embodiment, separating the albumin from the non-albumin phase comprises adjusting the pH of the plasma to 4.8 to 5.4, optionally 5.1 to 5.3.

In another embodiment, the adjustment of the pH of the plasma is performed by adding an acid.

In another embodiment, the method for purifying albumin may be linearly scalable.

Further, the present disclosure includes a recovered albumin solution having an albumin content of at least 90% (w/w) protein, optionally at least 92% (w/w) protein, optionally at least 94% (w/w) protein, optionally at least 96% (w/w) protein, optionally at least 98% (w/w) protein, optionally at least 99% (w/w) protein.

Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Drawings

The present disclosure will now be described with respect to the accompanying drawings, in which:

fig. 1 shows a graphical representation of the purity and recovery of HSA after each purification step.

Figure 2 shows a size exclusion chromatogram of a heated sample.

FIG. 3 shows a non-reduced SDS-PAGE gel.

Fig. 4 shows the purity of HSA as a function of pasteurization time.

Figure 5 shows a size exclusion chromatogram of a PAS2 sample at each pasteurization time.

FIG. 6 shows a non-reduced SDS-PAGE gel.

Fig. 7 shows a graphical representation of the purity and recovery of HSA after each purification step.

Figure 8 shows a size exclusion chromatogram of a PAS1 sample after each treatment step.

FIG. 9 shows a non-reduced and reduced SDS-PAGE gel.

Fig. 10 shows the purity of HSA after each treatment.

Figure 11 shows a superimposed view of a size exclusion chromatogram.

Fig. 12 shows a graphical representation of the purity and recovery of HSA after each purification step.

Figure 13 shows a size exclusion chromatogram of a sample after each purification step.

FIG. 14 shows an SDS-PAGE gel, an 8% Invitrogen gel (200 V.times.22 min).

Fig. 15 shows a western blot of HSA.

Figure 16 shows a size exclusion chromatogram of a sample after each purification step.

Figure 17 shows a size exclusion chromatogram of a sample.

FIG. 18 shows a Western blot, 7.5% Invitrogen gel (200V × 22 min) under non-reducing conditions.

FIG. 19 shows an SDS-PAGE gel, a 7.5% Invitrogen gel (200 V.times.22 min.).

Figure 20 shows size exclusion chromatograms of samples according to different conductivities.

Figure 21 shows size exclusion chromatograms of samples pH adjusted with different acids.

Figure 22 shows a graph of purity and recovery of samples according to different conductivities.

Figure 23 shows the purity and dimer formation profiles for samples of different acids used for pH adjustment.

FIG. 24 shows reduced SGS-PAGE gels.

Figure 25 shows a graph of purity, dimer and aggregate formation from different samples including the PAS3 sample.

FIG. 26 shows SDS-PAGE, 7.5% gels and under reducing conditions.

Figure 27 shows the purity, dimer and aggregate formation profiles of the samples.

Fig. 28 shows non-reduced and reduced gels of the samples.

Figure 29 shows SEC HPLC overlay view.

Figure 30 shows SEC HPLC overlay view.

FIG. 31 shows the purity of HSA recovered on SDS-PAGE gels.

Fig. 32 shows a contour plot illustrating the effect of total protein concentration ([ TP ]) and NaCP to total protein ratio on HSA recovery during pasteurization.

Fig. 33 shows a contour plot illustrating the effect of total protein concentration ([ TP ]) and NaCP to total protein ratio on HSA purity during pasteurization.

Fig. 34 shows a Pareto chart (Pareto chart) of normalized effect of HSA recovery in relation to (a) total protein concentration ([ TP ]) and (B) NaCP to total protein ratio in the material during pasteurization.

Fig. 35 shows a pareto plot of the normalized effect of HSA purity as a function of (a) total protein concentration ([ TP ]) and (B) NaCP to total protein ratio in the material during pasteurization.

FIG. 36 shows a superimposed contour plot of HSA recovery and purity, indicating the optimal working range for the parameter of interest; the non-feasible ranges are shown in grey.

Detailed Description

HSA is purified from plasma and partially fractionated plasma by using NaCP at a near neutral pH, at a concentration dependent on the total protein concentration in the plasma and the amount of NaCP per gram of total protein, followed by separation of the albumin and non-albumin phases by cooling and lowering the pH of the pasteurized plasma. The purification process can be performed without the use of ethanol or extensive processing steps. The finally recovered HSA has a high yield and an extremely high purity.

Accordingly, the present disclosure provides a method for purifying albumin from plasma comprising:

(a) contacting the plasma with sodium caprylate (NaCP);

wherein the amount of NaCP is from 0.1mmol to 5mmol NaCP per gram of total protein in said plasma;

(b) heating the plasma at a temperature of 60 ℃ to 70 ℃; and

(c) the albumin is separated from the non-albumin phase.

Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to apply to all embodiments and aspects of the disclosure described herein to which they apply, as will be understood by those of skill in the art.

In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. As used herein, the term "consisting of … …" and its derivatives are intended to be inclusive terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers, and/or steps. The term "consisting essentially of … …" as used herein is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps, as well as those that do not materially affect one or more of the basic and novel characteristics of the features, elements, components, groups, integers, and/or steps.

As used herein, the singular forms "a/an" and "the" include plural referents unless the content clearly dictates otherwise. In embodiments comprising an "additional" or "second" component, the second component, as used herein, is different from the other components or the first component. The "third" component is different from the other components, the first component, and the second component, and further enumerated or "additional" components are similarly different.

In the absence of any contrary indication, reference to "%" content throughout this specification should be understood to mean% w/v (weight/volume).

The term "purified" and its derivatives as used herein means purified from other common components present in plasma, fractionated plasma, cold plasma poor, plasma intermediates, plasma protein solutions or mixtures thereof. For example, purified albumin is purified from a fraction of fractionated plasma, cold plasma poor, plasma intermediates, plasma protein solutions, or mixtures thereof, from other proteins, nucleic acids, lipids, and small metabolites present therein. The purified albumin is at least 60% pure, optionally at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or even 100% pure by weight, and free of other proteins, nucleic acids, lipids or small metabolites present in the plasma, partially fractionated plasma, cold plasma poor, plasma intermediates, plasma protein solutions or mixtures thereof.

The term "plasma" and its derivatives as used herein includes partially fractionated plasma, cold plasma poor, plasma intermediates, plasma protein solutions or mixtures thereof. It is obvious to the person skilled in the art how to obtain partially fractionated plasma, cold plasma poor, plasma intermediates, plasma protein solutions or mixtures thereof. For example, a portion of the fractionated plasma may be passed through

EK1 filter (0.45 μm-0.65 μm).

The methods of the present disclosure can be used in principle to purify any known albumin, such as albumin derived from humans, non-human primates, sheep, goats, bovines, donkeys, canines, felines, rabbits, rodents, hamsters, guinea pigs, and birds.

The total protein in the plasma can be concentrated prior to addition of NaCP. Concentration can be performed using techniques known in the art, including the addition of water or any suitable buffer known to those skilled in the art. NaCP is preferably added to plasma at a pH range of 6.6 to 7.2, most preferably at a pH range of 6.7 to 6.9.

It is important to regulate total protein in plasma. In particular, the total protein in plasma should have a concentration of 20g/L to 150g/L, such as at least 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L, 55g/L, 60g/L, 65g/L or 70g/L, and a concentration of at most 45g/L, 50g/L, 55g/L, 60g/L, 65g/L, 70g/L, 75g/L, 80g/L, 85g/L, 90g/L, 95g/L, 100g/L, 105g/L, 110g/L, 115g/L, 120g/L, 125g/L, 130g/L, 135g/L, 140g/L, 145g/L, or 150 g/L. In a specific embodiment, the total protein has a concentration of 35g/L to 80g/L or 45g/L to 65 g/L.

Adjusting the amount of NaCP used is an important aspect of the process. In particular, the amount of NaCP is between 0.1mmol and 5mmol NaCP per gram of total protein in the plasma, such as at least 0.1mmol, 0.2mmol, 0.3mmol, 0.4mmol, 0.5mmol, 0.6mmol, 0.65mmol, 0.66mmol, 0.67mmol, 0.68mmol, 0.69mmol, 0.7mmol, 0.75mmol, 0.8mmol, 0.85mmol, 0.9mmol, 0.95mmol, 1mmol, 1.1mmol, 1.05mmol, 1.15mmol, 1.2mmol, 1.3mmol or 1.4mmol NaCP per gram of total protein in the plasma, and at most 0.9mmol, 0.95mmol, 1mmol, 1.05mmol, 1.1mmol, 1.15mmol, 1.2mmol, 1.3mmol, 1.4mmol, 1.5mmol, 1.6mmol, 1.8mmol, 2.5mmol, 2mmol, 5mmol or 3mmol NaCP per gram of total protein in the plasma. In a specific embodiment, the amount of NaCP is from 0.2mmol to 3mmol, from 0.5mmol to 2mmol, or from 0.8mmol to 1.3mmol of NaCP per gram of total protein in the plasma. It will be apparent to those skilled in the art that the values may be converted and expressed in terms of weight, molar amounts, concentrations including molar concentrations, or combinations thereof.

In another embodiment, the NaCP is added at a concentration of 20mM to 140mM, such as at least 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 80mM, or 85mM, and at most 65mM, 70mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, or 140 mM. In a specific embodiment, the NaCP has a concentration of 40mM to 100mM, 50mM to 90mM, or 60mM to 85 mM.

After addition of NaCP, the plasma is heat treated or pasteurized. The pasteurisation or heating of the plasma is carried out at a temperature of 60 ℃ to 70 ℃, such as at least 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃ or 67 ℃, and at most 64 ℃, 65 ℃, 66 ℃, 67 ℃,68 ℃, 69 ℃ or 70 ℃. In a particular embodiment, the pasteurisation or heating process of the plasma is carried out at a temperature of 60 ℃ to 65 ℃ or 62 ℃ to 65 ℃. It is obvious to the person skilled in the art how the temperature can be adjusted, for example by using a pasteuriser.

The pasteurisation or heating process of the plasma is preferably performed at a pH value of 6.0 to 8.0, such as at least 6.0, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0 and at most 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.5 or 8.0. In a particular embodiment, the pasteurisation or heating process of the plasma is performed at a pH value of 6.5 to 7.5, 6.7 to 7.3 or 6.7 to 6.9.

The conductivity of the plasma may also be an important factor in the method. In one embodiment, the conductivity is less than 9mS/cm, 8mS/cm, 7mS/cm, 6mS/cm, 5mS/cm, 4mS/cm, 3mS/cm, 2mS/cm, or 1 mS/cm. The conductivity of plasma can be measured by a conductivity (EC) meter.

The pasteurisation or heating process of the plasma is preferably carried out for 0.5 hours to 24 hours, such as at least 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours, and at most 5 hours, 6 hours, 6.5 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 20 hours or 24 hours. In a particular embodiment, the process of pasteurising or heating the plasma is carried out for a period of time ranging from 0.5 hours to 12 hours, from 2 hours to 12 hours, from 3 hours to 12 hours or from 4 hours to 6.5 hours.

In a particular embodiment, the process of pasteurising or heating the plasma is carried out at a temperature of 62 ℃ to 65 ℃ for a period of 4 hours to 6.5 hours.

In a specific embodiment, the process of pasteurization or heating of the plasma is carried out at a pH value of 6.7 to 6.9 at a temperature of 62 ℃ to 65 ℃ for a period of at least 4 hours, wherein the amount of NaCP is 0.8mmol to 1.3mmol NaCP per gram of total protein, wherein the total protein has a concentration of 45g/L to 65 g/L.

In step (c), albumin is separated from non-albumin proteins in the plasma. Separating the albumin from the non-albumin phase includes precipitating non-albumin protein and lipid impurities. In a particular embodiment, separating the albumin from the non-albumin phase comprises cooling the pasteurized plasma to below 30 ℃.

In another embodiment, separating the albumin from the non-albumin phase comprises adjusting the pH of the plasma to 4.8 to 5.4, such as 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, or 5.4. In a particular embodiment, separating the albumin from the non-albumin phase comprises adjusting the pH to 4.9 to 5.3, optionally to 5.1 to 5.3. It will be apparent to those skilled in the art that the separation step is carried out using a filter aid, including a filter aid, such as

P1000、

P300 or a mixture thereof is added to the pasteurized plasma.

In one aspect, separating albumin from non-albumin can be performed by a depth filter, Tangential Flow Filtration (TFF) using a cartridge, or hollow fiber TFF. Hollow fiber cross-flow filters are open channel devices that are ideal for use with turbid liquids and can handle solids concentrations up to 40% (v/v). Hollow fiber filters are less sensitive to changes in particle/colloid/cell or liquid levels that are viscous or become viscous after concentration. These filters are often selected when it is important to recover the product from the filter (e.g., a vaccine process). Hollow fiber filters are ideal for recovering product from the filter because they have an unobstructed and unobstructed internal flow path. They can be used with different flow path geometries (nominal fiber diameter from 0.5mm to 1.75mm Inner Diameter (ID), nominal fiber length options being 30cm, 60cm or 110cm length). Those skilled in the art will appreciate that variations of hollow fiber TFF may optionally be used to separate albumin from non-albumin.

Cartridges are most commonly used for concentrating and percolating "clear" low viscosity fluids. The use of a mesh-type turbulence promoter enhances the depolarization of solutes at the membrane surface, thereby increasing sample flux. The cartridge can have up to 2 times the flux of a hollow fiber filter under the same process conditions, pore size and filtration area. However, due to the "plugging"/pressure drop of the screen spacer, the cartridge is not suitable for containing particles, colloidal suspensions or solutions with high viscosity.

In one embodiment, separating albumin from non-albumin comprises filtration or clarification by depth filter, Tangential Flow Filtration (TFF) using filter cassettes, or hollow fiber TFF, preferably hollow fiber TFF. In one embodiment, the separation of albumin from non-albumin phase by depth filter, TFF using filter cassettes or hollow fiber TFF is performed at a pH value of 4.8 to 5.4, optionally 5.1 to 5.3. In another embodiment, the separation of albumin from non-albumin by filtration or clarification is performed at a feed flow rate of 1, 2,3, 4 or 5 to 6, 7, 8, 9 or 10 liters/min per square meter, optionally 1 to 10, optionally 2 to 10, optionally 3 to 10, optionally 8 to 9, optionally 8 to 9 liters/min per square meter. In another embodiment, the separation of albumin from non-albumin by filtration or clarification is performed at an initial volume reduction concentration rate of 1-fold to 1.2-fold, preferably 1.2-fold, and 3-fold to 4-fold dialysis volume, preferably 4-fold dialysis volume. In one embodiment, separating albumin from non-albumin comprises filtering or clarifying using hollow fiber TFF at a pH of 4.8 to 5.4, optionally 5.1 to 5.3, at a feed flow rate of 3 to 10 liters per minute per square meter, optionally 8 to 9 liters per minute per square meter. In a specific embodiment, separating albumin from non-albumin comprises filtering or clarifying with hollow fiber TFF at a pH of 4.8 to 5.4, optionally 5.1 to 5.3, at a feed flow rate of 3 to 10 liters/min per square meter, optionally 8 to 9 liters/min per square meter, at an initial volume reduction of 1 to 1.2 times, optionally 1.2 times, concentration and 3 to 4 dialysis volumes, optionally 4 dialysis volumes.

The adjustment of the pH of the plasma can be carried out by adding an acid, such as an organic acid and/or an inorganic acid. In a particular embodiment, the organic acid is selected from citric acid, acetic acid and trifluoroacetic acid. In another specific embodiment, the organic acid is acetic acid. In another embodiment, the mineral acid is selected from the group consisting of hydrochloric acid, sulfuric acid, and phosphoric acid.

Plasma can be filtered through a PallSeitz K700P depth filter in the presence of Harborlite Filter aid (2% w/v H900, 2% w/v H1900). It will be apparent to those skilled in the art how to assess the purity of the resulting albumin solution. For example, size exclusion chromatography-HPLC (SEC-HPLC) or capillary electrophoresis-SDS (CE-SDS) analysis can be performed to assess the purity of albumin.

The method for purifying albumin recovers at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of albumin from plasma.

In another embodiment, the method for purifying albumin may be linearly scalable.

Further, the present disclosure includes albumin solutions having an albumin content of at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% (w/w) of the protein obtained by the methods disclosed herein.

The following non-limiting examples illustrate the disclosure: