Plasma fractionation by sequential extraction

Abstract

The present invention relates to a method for the purification of immunoglobulins and / or albumin from a plasma sample, the method comprising mixing a sample of plasma with medium chain fatty acids and recovering soluble immunoglobulins from the mixture.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method and system for extracting immunoglobulins (Ig), such as immunoglobulin G (IgG), from plasma.

[0002] CROSS REFERENCE TO PRIOR APPLICATION This application claims priority to Australian Provisional Application No. 2021901577, the entire contents of which are hereby incorporated by reference in their entirety. [Background technology]

[0003] 2. Background of the Invention The demand for purified proteins, such as specific antibodies, which can be used for therapeutic and / or diagnostic purposes, has increased considerably.

[0004] Human blood plasma has been industrially utilized for decades for the production of widely established and recognized plasma-protein products such as human albumin (HSA), immunoglobulins (IgG), coagulation factor concentrates (coagulation factor VIII, coagulation factor IX, prothrombin complex, etc.) and inhibitors (antithrombin, C1 inhibitor, etc.). In the course of such plasma-derived drug development, plasma fractionation methods have been established, leading to intermediate products enriched in specific protein fractions, which then serve as starting compositions for plasma-protein products. Typical processes are outlined, for example, in Non-Patent Document 1. These types of separation techniques allow the production of several therapeutic plasma-protein products from the same plasma donor pool. This is more economically advantageous than producing only one plasma-protein product from one donor pool and has therefore been adopted as the industrial standard in blood plasma fractionation.

[0005] One example of this type of fractionation process, low-temperature ethanol fractionation of plasma, was developed by EJ Cohn and his team during World War II, primarily for the purification of albumin (Non-Patent Document 2). The Cohn fractionation process involves increasing the ethanol concentration stepwise, from 0% to 40%, while decreasing the pH from neutral (pH 7) to about 4.8, resulting in the precipitation of albumin. Although Cohn fractionation has evolved over the past 70 years or so, most commercial plasma fractionation processes are based on the original process or variations of it (e.g., Kistler / Nitschmann) and use pH, ionic strength, solvent polarity and alcohol concentration to separate plasma into a series of major precipitable protein fractions (e.g., Fractions I-V in the Cohn method).

[0006] Variations of the Cohn fractionation process have been developed with the aim of improving polyvalent IgG recovery. For example, Oncley and coworkers used Cohn fraction II+III as starting material to produce active immunoglobulin serum fractions using different combinations of cold ethanol, pH, temperature and protein concentration than those described by Cohn (Oncley et al., 2003). Today, the Oncley method is the classical method used for the production of polyvalent IgG. Nevertheless, it is known that about 5% of gamma-globulins (antibody-rich portion) are co-precipitated with fraction I, and about 15% of the total gamma-globulins present in plasma are lost by the fraction II+III step (see Table III, 2003). The Kistler / Nitschmann method was intended to improve IgG recovery by reducing the ethanol content of some of the precipitation steps (precipitation B vs. fraction III). However, the increased yield comes at the expense of purity (Oncley et al., 2003).

[0007] Initially, immunoglobulin G (IgG) preparations derived from these fractionation processes were successfully used to prevent and treat a variety of infectious diseases. However, because ethanol fractionation is a relatively crude process, the IgG products contain impurities and aggregate to such an extent that they can only be administered intramuscularly. Since that time, further improvements in the purification process have led to IgG preparations suitable for intravenous (called IVIg) and subcutaneous (called SCIg) administration.

[0008] It is estimated that approximately 30 million liters of plasma were processed worldwide in 2010, providing a range of therapeutic products, including approximately 500 tonnes of albumin and 100 tonnes of IVIg. The IVIg market represents approximately 40-50% of the total plasma fractionation market (Non-Patent Document 5). Thus, as the demand for IVIg remains strong (combined with the increasing demand for SCIg), there remains a need to improve immunoglobulin recovery from plasma and related fractions. Preferably, this must be achieved in a way that ensures that the recovery of other plasma-derived therapeutic proteins is not adversely affected.

[0009] From a commercial perspective, the initial fractionation process is critical to the overall production time and costs associated with the manufacture of therapeutic proteins, particularly plasma-derived proteins, since subsequent purification steps depend on the yield and purity of the protein of interest within these initial fractions. Although several variations of the low-temperature ethanol fractionation process have been developed for plasma-derived proteins to improve protein yields with lower operating costs, higher protein yields are typically accompanied by lower purity. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Molecular Biology of Human Proteins (Schultze HE, Heremans JF;Volume I: Nature and Metabolism of Extracellular Proteins 1966, Elsevier Publishing Company;p. 236-317) [Non-Patent Document 2] Cohn EJ, et al. 1946, J. Am. Chem. Soc. 62: 459-475 [Non-Patent Document 3] Oncley et al., (1949) J. Am. Chem. Soc. 71, 541-550 [Non-Patent Document 4] Kistler & Nitschmann, (1962) Vox Sang. 7, 414-424 [Non-Patent Document 5] P. Robert, Worldwide supply and demand of plasma and plasma derived medicines (2011) J. Blood and Cancer, 3, 111-120 Summary of the Invention [Problem to be solved by the invention]

[0011] There is a need for improved methods and systems for industrial-scale production of proteins from blood-derived plasma or serum that meet stringent safety standards. Currently used end-stage technologies are relatively expensive and their yields are not optimal. It is therefore essential to develop more efficient and economical methods for the extraction and purification of proteins, such as immunoglobulins, from plasma.

[0012] The reference to any prior art in the specification is neither an affirmation nor an indication that this prior art forms part of the common knowledge in any jurisdiction, nor is it an affirmation or an indication that this prior art could reasonably be expected to be understood, considered relevant, and / or combined with other parts of the prior art by a person skilled in the art. [Means for solving the problem]

[0013] Summary of the Invention The present invention is based on the discovery that it is possible to obtain relatively pure immunoglobulin, particularly IgG, preparations directly from plasma and without the need for an ethanol fractionation step.

[0014] Furthermore, the present invention is based on the discovery that it is also possible to obtain relatively pure preparations of immunoglobulins, in particular IgG and albumin, directly from plasma and without the need for an ethanol fractionation step.

[0015] Still further, the present invention is based on the discovery that it is also possible to obtain relatively pure preparations of albumin directly from plasma and without the need for an ethanol fractionation step.

[0016] Thus, in a first aspect, the present invention provides a method for obtaining a solution of immunoglobulins, comprising: - contacting a sample of blood-derived plasma with medium chain fatty acids under conditions to allow selective precipitation of albumin from said sample; - [wherein the conditions include a pH range between about 4.6 and about 5.0]; - thereby forming a solution of immunoglobulins Includes.

[0017] Preferably, the conditions also include a conductivity of from about 5 mS / cm to about 12 mS / cm, preferably from 8 mS / cm to about 12 mS / cm.

[0018] In a second aspect, the present invention provides a method for the purification of an immunoglobulin, the method comprising: - contacting a sample of blood-derived plasma with medium chain fatty acids under conditions comprising a pH range of between about 4.6 and about 5.0 to allow selective precipitation of albumin from the sample; - thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin; - separating the soluble protein-containing components from the insoluble protein-containing components to obtain a solution of purified immunoglobulins; Includes.

[0019] The step of separating the soluble protein-containing component from the insoluble protein-containing component preferably comprises subjecting the suspension to a continuous extractive filtration. Preferably, the step of separating the soluble protein-containing component from the insoluble protein-containing component comprises: - feeding the suspension into a filtration unit including a dynamic filter element adapted to produce a first retentate and a first filtrate; - recovering the first filtrate; Includes.

[0020] The first retentate, containing the insoluble protein-containing components, is typically enriched in albumin, and the first filtrate, containing the soluble protein-containing components, is enriched in immunoglobulins.

[0021] The first filtrate enriched in immunoglobulins may be subjected to a concentration step before further processing. The concentration step may comprise a continuous concentration process, whereby the first filtrate is fed into a second filtration unit comprising a cross-flow filter element or TFF adapted to produce a second retentate enriched in immunoglobulins; and a second filtrate depleted in immunoglobulins. Optionally, the second filtrate depleted in immunoglobulins may be run back into the first tank and / or the second retentate enriched in immunoglobulins may be run back into the second tank.

[0022] Of course, the immunoglobulin-enriched solution may be subjected to further purification using standard techniques as further described herein.

[0023] In a preferred embodiment, a method for the purification of an immunoglobulin is provided, the method comprising: - contacting a sample of blood-derived plasma with medium-chain fatty acids under conditions comprising: a pH range of between about 4.6 and about 5.0, and a conductivity of between about 8 mS / cm and about 12 mS / cm, to allow selective precipitation of albumin from said sample; - thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin; - separating the soluble protein-containing components from the insoluble protein-containing components to obtain a solution of purified immunoglobulins; Including, wherein optionally the step of separating the soluble protein-containing component from the insoluble protein-containing component comprises feeding the suspension into a filtration unit comprising a dynamic filter element adapted to produce a first retentate and a first filtrate, and recovering the first filtrate.

[0024] The pH of a sample of blood-derived plasma can be adjusted directly and without substantial dilution of the sample, for example, by adding a concentrated acid (e.g., acetic acid) or, if further adjustment of the pH is required, by using a combination of an acid and a base (e.g., NaOH). Similarly, the conductivity of blood-derived plasma can be adjusted directly without substantial dilution of the sample.

[0025] In a third aspect, there is provided a method for purifying immunoglobulins from plasma, the method comprising: a) in a first tank: mixing a sample of blood-derived plasma with medium chain fatty acids under conditions comprising a pH range of between about 4.6 and about 5.0 and optionally a conductivity of between about 8 mS / cm and about 12 mS / cm to enable selective precipitation of albumin from the sample, thereby forming a suspension comprising soluble immunoglobulins and insoluble albumin; b) feeding the suspension into a first filtration unit comprising a dynamic filter element adapted to produce a first retentate comprising insoluble albumin and a first filtrate comprising soluble immunoglobulins; c) optionally diluting the suspension in the first tank by flowing a first retentate into the first tank; d) collecting the first filtrate in a second tank; and e) optionally concentrating the first filtrate; Includes.

[0026] Thus, in a fourth aspect, the present invention provides a method for obtaining a solution of immunoglobulins and a precipitate of albumin, the method comprising: - contacting a sample of blood-derived plasma with medium chain fatty acids under conditions to allow selective precipitation of albumin from said sample; - [wherein the conditions include a pH range between about 4.2 and about 5.0]; - thereby forming a solution of immunoglobulins and a precipitate of albumin. Includes.

[0027] Preferably, the conditions also include a conductivity of about 5 mS / cm to about 12 mS / cm, preferably 8 mS / cm to about 12 mS / cm. More preferably, when the blood-derived plasma is diluted, the conductivity is equal to or greater than 5 mS / cm but less than or less than about 12 mS / cm. When the blood-derived plasma is not diluted, the conductivity is equal to or greater than 8 mS / cm but less than or less than about 12 mS / cm.

[0028] In a fifth aspect, the present invention provides a method for the purification of immunoglobulins and albumins, the method comprising: - contacting a sample of blood-derived plasma with medium chain fatty acids under conditions comprising a pH range of between about 4.2 and about 5.0 to allow selective precipitation of albumin from the sample; - thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin; - separating the soluble protein-containing component from the insoluble protein-containing component to obtain a solution of purified immunoglobulins and a suspension containing albumin. Includes.

[0029] The step of separating the soluble protein-containing component from the insoluble protein-containing component preferably comprises subjecting the suspension to a continuous extraction filtration. Preferably, the step of separating the soluble protein-containing component from the insoluble protein-containing component comprises: - feeding the suspension into a filtration unit including a dynamic filter element adapted to produce a first retentate and a first filtrate; - recovering a first filtrate and a first retentate. Includes.

[0030] The first retentate, containing the insoluble protein-containing components, is typically enriched in albumin, and the first filtrate, containing the soluble protein-containing components, is enriched in immunoglobulins.

[0031] The first filtrate enriched in immunoglobulins may be subjected to a concentration step before further processing. The concentration step may include a continuous concentration process, whereby the first filtrate is fed into a second filtration unit comprising a cross-flow filter element or TFF adapted to produce a second retentate enriched in immunoglobulins; and a second filtrate depleted in immunoglobulins. Optionally, the second filtrate depleted in immunoglobulins may be run back into the first tank and / or the second retentate enriched in immunoglobulins may be run back into the second tank.

[0032] Of course, the immunoglobulin-enriched solution may be subjected to further purification using standard techniques, as further described herein.

[0033] In a preferred embodiment, a method for the purification of immunoglobulins and albumins is provided, the method comprising: - contacting a sample of blood-derived plasma with medium chain fatty acids under conditions comprising a pH range of between about 4.2 and about 5.0 and a conductivity of between about 8 mS / cm and about 12 mS / cm to allow selective precipitation of albumin from the sample; - thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin; - separating the soluble protein-containing component from the insoluble protein-containing component to obtain a solution of purified immunoglobulins and a suspension containing albumin; Including, wherein optionally the step of separating the soluble protein-containing component from the insoluble protein-containing component comprises feeding the suspension into a filtration unit comprising a dynamic filter element adapted to produce a first retentate and a first filtrate, and recovering the first filtrate.

[0034] The pH of a sample of blood-derived plasma can be adjusted directly and without substantial dilution of the sample, for example, by adding a concentrated acid (e.g., acetic acid) or, if further adjustment of the pH is required, by using a combination of an acid and a base (e.g., NaOH). Similarly, the conductivity of blood-derived plasma can be adjusted directly without substantial dilution of the sample.

[0035] In a sixth aspect, there is provided a method for purifying immunoglobulins and albumin from plasma, the method comprising: a) in a first tank: mixing a sample of blood-derived plasma with medium chain fatty acids under conditions comprising a pH range of between about 4.2 and about 5.0 and optionally a conductivity of between about 8 mS / cm and about 12 mS / cm to enable selective precipitation of albumin from the sample, thereby forming a suspension comprising soluble immunoglobulins and insoluble albumin; b) feeding the suspension into a first filtration unit comprising a dynamic filter element adapted to produce a first retentate comprising insoluble albumin and a first filtrate comprising soluble immunoglobulins; c) optionally diluting the suspension in the first tank by flowing a first retentate into the first tank; d) collecting the first filtrate in a second tank; and e) optionally concentrating the first filtrate; Includes.

[0036] Thus, in a seventh aspect, the present invention provides a method for obtaining a precipitate of albumin, the method comprising: - contacting a sample of blood-derived plasma with medium chain fatty acids under conditions to allow selective precipitation of albumin from said sample; - [wherein the conditions include a pH range of between about 4.15 and about 4.25]; - thereby precipitating albumin Includes.

[0037] Preferably, the conditions also include a conductivity of about 5 mS / cm to about 12 mS / cm, preferably 8 mS / cm to about 12 mS / cm. More preferably, when the blood-derived plasma is diluted, the conductivity is equal to or greater than 5 mS / cm, but less than 12 mS / cm or less than about 12 mS / cm. When the blood-derived plasma is not diluted, the conductivity is equal to or greater than 8 mS / cm, but less than 12 mS / cm or less than about 12 mS / cm.

[0038] In an eighth aspect, the present invention provides a method for the purification of albumin, the method comprising: - contacting a sample of blood-derived plasma with medium chain fatty acids under conditions comprising a pH range of between about 4.15 and about 4.25 to allow selective precipitation of albumin from the sample; - thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin; - Separating the soluble protein-containing component from the insoluble protein-containing component to obtain a precipitate or suspension containing albumin. Includes.

[0039] The step of separating the soluble protein-containing component from the insoluble protein-containing component preferably comprises subjecting the suspension to a continuous extraction filtration. Preferably, the step of separating the soluble protein-containing component from the insoluble protein-containing component comprises: - feeding the suspension into a filtration unit including a dynamic filter element adapted to produce a first retentate and a first filtrate; - Recovering the first retentate. Includes.

[0040] The first retentate, containing the insoluble protein-containing components, is typically enriched in albumin, and the first filtrate, containing the soluble protein-containing components, is enriched in immunoglobulins.

[0041] Of course, the first retentate, enriched in albumin, may be subjected to further purification using standard techniques as further described herein.

[0042] In a preferred embodiment, a method for the purification of albumin is provided, the method comprising: - contacting a sample of blood-derived plasma with medium-chain fatty acids under conditions comprising: a pH range of between about 4.15 and about 4.25 and a conductivity of between about 8 mS / cm and about 12 mS / cm to allow selective precipitation of albumin from said sample; - thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin; - separating the soluble protein-containing component from the insoluble protein-containing component to obtain a suspension containing albumin; Including, wherein optionally the step of separating the soluble protein-containing component from the insoluble protein-containing component comprises feeding the suspension into a filtration unit comprising a dynamic filter element adapted to produce a first retentate and a first filtrate, and recovering the first retentate.

[0043] The pH of a sample of blood-derived plasma may be adjusted directly and without substantial dilution of the sample, for example by adding concentrated acid (e.g., acetic acid) or, if further pH adjustment is required, by adding a combination of acid and base (e.g., NaOH). Similarly, the conductivity of blood-derived plasma may be adjusted directly and without substantial dilution of the sample.

[0044] In a ninth aspect, there is provided a method for purifying albumin from plasma, the method comprising: a) in a first tank: mixing a sample of blood-derived plasma with medium chain fatty acids under conditions comprising a pH range of between about 4.15 and about 4.25 and optionally a conductivity of between about 8 mS / cm and about 12 mS / cm to enable selective precipitation of albumin from the sample, thereby forming a suspension comprising soluble immunoglobulins and insoluble albumin; b) feeding the suspension into a first filtration unit comprising a dynamic filter element adapted to produce a first retentate comprising insoluble albumin and a first filtrate comprising soluble immunoglobulins; c) optionally diluting the suspension in the first tank by flowing a first retentate into the first tank; d) Recovering the first retentate. Includes.

[0045] In any embodiment, the residual suspension or first retentate in the first tank contains insoluble albumin complexed with medium chain fatty acids. To break the bonds between albumin and medium chain fatty acids, the pH of the residual suspension or first retentate may be adjusted to 6.4-6.7, preferably 6.8-7.2. In one embodiment, the pH may be adjusted using 1 M sodium hydroxide. Optionally, the first retentate is then mixed until the pH of the solubilized albumin is stable (typically about 30-60 minutes). This solution may then be fed to a further filtration unit (e.g., a second system including a further filtration unit, e.g., a further filtration unit equivalent to 5 shown in FIG. 16) that includes a dynamic filter element to produce an albumin-depleted retentate and an albumin-enriched filtrate.

[0046] In one embodiment, the albumin-enriched filtrate is then continuously concentrated, preferably using a TFF membrane, to at or about 20-45 g / L of protein to form a concentrated albumin solution (e.g., a second system including an additional filtration unit, e.g., an additional filtration unit equivalent to 8 shown in FIG. 16).

[0047] The concentrated albumin solution may then be heated in the range of 60-65° C. for a period of typically 90 minutes. Without being bound by any theory, it is believed that various proteins other than albumin are denatured at this stage.

[0048] After heating the concentrated albumin solution, the pH of the solution can be adjusted to or about 4.20, typically using 1M hydrochloric acid. At the same time, the concentrated albumin solution can be cooled to 4°C, which forms a precipitate of the proteins that were denatured during the heating step described above. The precipitate can then be removed by filtration, whereby the filtrate contains purified albumin (typically with an albumin purity equal to or greater than 95%, 96%, 97%, or 98% of the total protein, and an albumin yield equal to or greater than 85%, 86%, 87%, 88%, 89%, or 90%).

[0049] In any embodiment of the invention, the dynamic filter element in the first filtration unit adapted to produce a filtrate (permeate) enriched in soluble proteins (immunoglobulins) is a dynamic cross-flow filter element.

[0050] In any embodiment of the invention, the dynamic filter element in the second filtration unit adapted to produce an immunoglobulin-enriched retentate is a dynamic cross-flow filter element.

[0051] In any embodiment of the invention, the dynamic filter element in the further filtration unit adapted to produce an albumin-enriched filtrate is a dynamic cross-flow filter element.

[0052] In a preferred embodiment, the dynamic crossflow filter element is a rotating crossflow filter element. More preferably, the rotating crossflow filter element comprises a filter disc. Typically, the filter disc is attached to a shaft member. In one embodiment, the rotating crossflow filter element comprises at least one filter disc and at least one shaft member.

[0053] According to a preferred embodiment of any aspect of the invention, the filter disc membrane is a ceramic membrane. More preferably, the ceramic membrane has a pore size in the range of equal to or greater than 5 nm and equal to or less than 2 μm. In certain embodiments, the ceramic membrane has a pore size of about 0.2 μm to 2 μm. In certain embodiments, the ceramic filter membrane has an average pore size in the range of equal to or greater than 5 nm and equal to or less than 200 nm (0.2 μm). In certain embodiments, the ceramic filter membrane has an average pore size in the range of equal to or greater than 50 nm and equal to or less than 100 nm. Such filter discs are supplied by Kerafol and Flowserve.

[0054] Of course, the blood-derived plasma sample may include any plasma sample derived from blood, preferably human blood. In certain embodiments, the blood-derived plasma sample includes fresh plasma, cryo-poor plasma, or cryo-rich plasma. The plasma may be obtained from multiple donations and / or subjects and may be pooled. The plasma may be hyperimmune plasma.

[0055] Preferably, the plasma sample does not contain a filter aid and / or has not been subjected to an alcohol or other fractionation process.

[0056] In any of the first, second or third aspects, the pH of the plasma sample is adjusted to a pH range of about 4.6 to about 5.0 prior to contact and / or mixing with the medium chain fatty acid. For example, the pH of the plasma sample may be adjusted to about pH 4.6, about 4.7, about 4.8, about 4.9, about 5.0. The pH of the plasma sample may be adjusted to pH 4.6, pH 4.7, pH 4.8, pH 4.9 or pH 5.0.

[0057] In any of the fourth, fifth or sixth aspects, the pH of the plasma sample is adjusted to a pH range of about 4.2 to about 5.0 prior to contact and / or mixing with the medium chain fatty acid. For example, the pH of the plasma sample may be adjusted to about pH 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9 or about 5.0. The pH of the plasma sample may be adjusted to pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9 or pH 5.0.

[0058] In any of the seventh, eighth or ninth aspects, the pH of the plasma sample is adjusted to a pH range of about 4.15 to about 4.25 before contacting and / or mixing with the medium chain fatty acid. For example, the pH of the plasma sample may be adjusted to about pH 4.15, about 4.16, about 4.17, about 4.18, about 4.19, about 4.20, about 4.21, about 4.22, about 4.23, about 4.24 or about 4.25. The pH of the plasma sample may be adjusted to pH 4.15, pH 4.16, pH 4.17, pH 4.18, pH 4.19, pH 4.20, pH 4.21, pH 4.22, pH 4.23, pH 4.24 or pH 4.25.

[0059] In any embodiment, the pH of the plasma sample may be adjusted without substantially diluting the plasma prior to contacting with the medium chain fatty acids. Such pH adjustment may be accomplished by adding a concentrated acid, such as acetic acid, or a combination of an acid and a base (e.g., NaOH) if further adjustment of the pH is required. If necessary, the conductivity of the resulting solution is adjusted to achieve a desired conductivity of about 8 mS / cm to about 12 mS / cm.

[0060] In certain embodiments of any aspect of the present invention, the plasma sample may be diluted with a buffer prior to mixing with the medium chain fatty acid. The dilution of the plasma may be about 1:0.5, about 1:0.75, about 1:1, about 1:1.25, about 1:1.5, about 1:1.75, or about 1:2 dilution. In situations where the plasma is diluted, the conductivity may be about 5 mS / cm to about 12 mS / cm.

[0061] The buffer in which the plasma is diluted can be any buffer suitable for diluting plasma, such as an acetate buffer (e.g., sodium acetate). The acetate buffer can include sodium acetate trihydrate and glacial acetic acid. The concentration of the buffer can be 60 mM, 80 mM, 100 mM, or 0.22 M, and can have a pH of 4.1 or about 4.1; 4.2 or about 4.2; 4.3 or about 4.3; 4.4 or about 4.4; 4.5 or about 4.5; 4.6 or about 4.6; 4.7 or about 4.7; 4.8 or about 4.8. Alternatively, a phosphate-acetate buffer can be used. The phosphate-acetate buffer can include 10 mM phosphate (e.g., sodium phosphate) and 10 mM acetate (e.g., sodium acetate). The pH of this buffer can be about 4.3 to about 4.4. If the conductivity of the resulting solution needs to be adjusted to achieve a desired conductivity of about 5 to about 12 mS / cm, preferably about 8 to about 12 mS / cm, this can be accomplished, for example, by adding a more concentrated acetate buffer (e.g., 3.5 M acetate buffer containing sodium acetate trihydrate and glacial acetic acid, pH 5).

[0062] Typically, the buffer is used not only to dilute the plasma but also to facilitate pH adjustment of the plasma sample. Thus, in any embodiment of the first, second or third aspect of the present invention, the pH of the diluted plasma sample is about pH 4.6 to about pH 5.0. For example, the pH of the diluted plasma sample may be about pH 4.6, about pH 4.7, about pH 4.8, about pH 4.9, about pH 5.0. The pH of the diluted plasma sample may be pH 4.6, pH 4.7, pH 4.8, pH 4.9 or pH 5.0. Thus, in any embodiment of the fourth, fifth or sixth aspect of the present invention, the pH of the diluted plasma sample is about pH 4.2 to about pH 5.0. For example, the pH of the diluted plasma sample may be about pH 4.2, about pH 4.3, about pH 4.4, about pH 4.5, about pH 4.6, about pH 4.7, about pH 4.8, about pH 4.9 or about pH 5.0. The pH of the diluted plasma sample may be pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9 or pH 5.0. Thus, in any embodiment of the seventh, eighth or ninth aspect of the present invention, the pH of the diluted plasma sample is about pH 4.15 to about 4.25. For example, the pH of the diluted plasma sample may be about pH 4.15, about 4.16, about 4.17, about 4.18, about 4.19, about 4.20, about 4.21, about 4.22, about 4.23, about 4.24 or about 4.25. The pH of the diluted plasma sample may be pH 4.15, pH 4.16, pH 4.17, pH 4.18, pH 4.19, pH 4.20, pH 4.21, pH 4.22, pH 4.23, pH 4.24 or pH 4.25.

[0063] In any aspect or embodiment, the conditions may include a conductivity of about 5 mS / cm to about 12 mS / cm, preferably 8 mS / cm to about 12 mS / cm. More preferably, when the blood-derived plasma is diluted, the conductivity is equal to or greater than 5 mS / cm, but less than 12 mS / cm or less than about 12 mS / cm. When the blood-derived plasma is not diluted, the conductivity is equal to or greater than 8 mS / cm, but less than 12 mS / cm or less than about 12 mS / cm.

[0064] For example, the conductivity of the diluted or undiluted plasma sample is about 8 to about 12 mS / cm. Thus, in any embodiment, the conductivity of the diluted or undiluted plasma sample is about 8 mS / cm, about 9 mS / cm, about 10 mS / cm, about 11 mS / cm, or about 12 mS / cm. In any embodiment, the conductivity of the diluted or undiluted plasma sample is 8 mS / cm, 9 mS / cm, 10 mS / cm, 11 mS / cm, or 12 mS / cm. In any embodiment, the conductivity of the diluted plasma is equal to or greater than 5 mS / cm, 6 mS / cm, or 7 mS / cm. Typically, the conductivity is measured at room temperature, and preferably, the conductivity is measured between 18 and 25°C.

[0065] In any embodiment, the medium chain fatty acid has the general structure CH3(CH2) n The fatty acid may be selected from fatty acids containing COOH, where the fatty acid is a C4-C10 carboxylic acid. The fatty acid may be saturated or unsaturated. More preferably, the fatty acid comprises enanthic acid (heptanoic acid), caprylic acid (octanoic acid), octenoic acid, pelargonic acid (nonanoic acid), nonenoic acid, or capric acid (decanoic acid). Most preferably, the fatty acid is caprylic acid (octanoic acid). Also contemplated as reagents are salts or esters (e.g., caprylate) of any of the fatty acids described herein.

[0066] In any embodiment of the first, second and third aspect, the amount of fatty acid, preferably caprylic acid (octanoic acid), mixed with the plasma sample is about 0.30g / g total protein (in the plasma sample), about 0.35g / g total protein, about 0.40g / g total protein, about 0.45g / g total protein or about 0.50g / g total protein. Preferably, the amount of fatty acid, preferably caprylic acid (octanoic acid), is about 0.300g / g total protein, or about 0.325g / g total protein, or about 0.350g / g total protein, or about 0.375g / g total protein, or about 0.400g / g total protein, or about 0.425g / g total protein, or about 0.450g / g total protein. More preferably, the amount of fatty acid, preferably caprylic (octanoic) acid, is at least about 0.350 g / g total protein. Preferably, the amount of fatty acid, preferably caprylic (octanoic) acid, ranges from about 0.38 g / g total protein to about 0.50 g / g total protein. More preferably, the amount of fatty acid, preferably caprylic (octanoic) acid, is about 0.38g / g total protein, about 0.39g / g total protein, about 0.40g / g total protein, about 0.41g / g total protein, about 0.42g / g total protein, about 0.43g / g total protein, about 0.44g / g total protein, about 0.45g / g total protein, about 0.46g / g total protein, about 0.47g / g total protein, about 0.48g / g total protein, about 0.49g / g total protein, or about 0.50g / g total protein.

[0067] In any embodiment of the first, second and third aspect, the amount of fatty acid, preferably caprylic acid (octanoic acid), is 0.30g / g total protein (in plasma samples), 0.35g / g total protein, 0.40g / g total protein, 0.45g / g total protein or 0.50g / g total protein. Preferably, the amount of fatty acid, preferably caprylic acid (octanoic acid), is 0.300g / g total protein, or 0.325g / g total protein, or 0.350g / g total protein, or 0.375g / g total protein, or 0.400g / g total protein, or 0.425g / g total protein, or 0.450g / g total protein. More preferably, the amount of fatty acid, preferably caprylic acid (octanoic acid), is at least 0.350g / g total protein. Preferably, the amount of fatty acid, preferably caprylic (octanoic) acid, is in the range of 0.38g / g total protein to 0.50g / g total protein. More preferably, the amount of fatty acid, preferably caprylic (octanoic) acid, is 0.38g / g total protein, 0.39g / g total protein, 0.40g / g total protein, 0.41g / g total protein, 0.42g / g total protein, 0.43g / g total protein, 0.44g / g total protein, 0.45g / g total protein, 0.46g / g total protein, 0.47g / g total protein, 0.48g / g total protein, 0.49g / g total protein, or 0.50g / g total protein.

[0068] In any embodiment of the fourth, fifth and sixth aspects, the amount of fatty acid, preferably caprylic acid (octanoic acid), mixed with the plasma sample is about 0.35g / g total protein (in the plasma sample), about 0.40g / g total protein, about 0.45g / g total protein, about 0.50g / g total protein or about 0.55g / g total protein. Preferably, the amount of fatty acid, preferably caprylic acid (octanoic acid), is about 0.35g / g total protein, about 0.36g / g total protein, about 0.37g / g total protein, about 0.38g / g total protein, about 0.39g / g total protein, about 0.40g / g total protein, about 0.41g / g total protein, about 0.42g / g total protein, about 0.43g / g total protein, about 0.45g / g total protein, about 0.46g / g total protein, about 0.47g / g total protein, about 0.48g / g total protein, about 0.49g / g total protein, about 0.50g / g total protein, about 0.55g / g total protein. 4g, about 0.45g per gram total protein, about 0.46g per gram total protein, about 0.47g per gram total protein, about 0.48g per gram total protein, about 0.49g per gram total protein, about 0.50g per gram total protein, about 0.51g per gram total protein, about 0.52g per gram total protein, about 0.53g per gram total protein, about 0.54g per gram total protein, or about 0.55g per gram total protein.

[0069] In any embodiment of the fourth, fifth and sixth aspect, the amount of fatty acid, preferably caprylic acid (octanoic acid), mixed with the plasma sample is 0.36g / g total protein, 0.37g / g total protein, 0.38g / g total protein, 0.39g / g total protein, 0.40g / g total protein, 0.41g / g total protein, 0.42g / g total protein, 0.43g / g total protein, 0.44g / g total protein, 0.45g / g total protein, 0.46g / g total protein, 0.47g / g total protein, 0.48g / g total protein, 0.49g / g total protein, 0.50g / g total protein, 0.51g / g total protein, 0.52g / g total protein, 0.53g / g total protein, 0.54g / g total protein, 0.55g / g total protein, 0.56g / g total protein, 0.57g / g total protein, 0.58g / g total protein, 0.59g / g total protein, 0.60g / g total protein, 0.61g / g total protein, 0.62g / g total protein, 0.63g / g total protein, 0.64g / g total protein, 0.65g / g total protein, 0.66g / g total protein, 0.67g / g total protein, 0.68g / g total protein, 0.69g / g total protein, 0.70g / g total protein, 0.71g / g total protein, 0.72g / g total protein, 0.73g / g total protein, 0.74g / g total protein, 0.75g / g total protein, 0.76g / g total protein, 0.77g / g total protein, 0.78g / g total protein, 0 0.44g, 0.45g per g total protein, 0.46g per g total protein, 0.47g per g total protein, 0.48g per g total protein, 0.49g per g total protein, 0.50g per g total protein, 0.51g per g total protein, 0.52g per g total protein, 0.53g per g total protein, 0.54g per g total protein, or 0.55g per g total protein.

[0070] In any embodiment of the seventh, eighth or ninth aspect, the amount of fatty acid, preferably caprylic (octanoic) acid, mixed with the plasma sample is equal to or greater than about 0.35 g / g total protein. Preferably, the amount of fatty acid, preferably caprylic (octanoic) acid, mixed with the plasma sample is equal to or greater than about 0.35 g / g total protein, but less than or less than about 1.1 g / g total protein. The amount of fatty acid, preferably caprylic acid (octanoic acid), mixed with the plasma sample is about 0.35g / g total protein, about 0.36g / g total protein, about 0.37g / g total protein, about 0.38g / g total protein, about 0.39g / g total protein, about 0.40g / g total protein, about 0.41g / g total protein, about 0.42g / g total protein, about 0.43g / g total protein, about 0.44g / g total protein, about 0.45g / g total protein, about 0.46g / g total protein, about 0.47g / g total protein, about 0.48g / g total protein, about 0.49g / g total protein, about 0.50g / g total protein, about 0.51g / g total protein, about 0.52g / g total protein, about 0.53g / g total protein, about 0.54g / g total protein, about 0.55g / g total protein, about 0.56g / g total protein, about 0.57g / g total protein, about 0.58g / g total protein, about 0.59g / g total protein, about 0.60g / g total protein, about 0.61g / g total protein, about 0.62g / g total protein, about 0.63g / g total protein, about 0.64g / g total protein, about 0.65g / g total protein, about 0.66g / g total protein, about 0.67g / g total protein, about 0.68g / g total protein, about 0.69g / g total protein, about 0.70g / g total protein, about 0.71g / g total protein, about 0.72g / g total protein, about 0.73g / g total protein, about 0.74g / g total protein, about 0.75g Approximately 0.43g, approximately 0.44g per g total protein, approximately 0.45g per g total protein, approximately 0.46g per g total protein, approximately 0.47g per g total protein, approximately 0.48g per g total protein, approximately 0.49g per g total protein, approximately 0.50g per g total protein, approximately 0.51g per g total protein, approximately 0.52g per g total protein, approximately 0.53g per g total protein, approximately 0.54g per g total protein, approximately 0.55g per g total protein, approximately 0.56g per g total protein, approximately 0.57g per g total protein, approximately 0.58g per g total protein, approximately 0.59g per g total protein, approximately 0.60g per g total protein, approximately 0.61g per g total protein, approximately 0.62g per g total protein, approximately 0.63g per g total protein, approximately 0.64g per g total protein, approximately 0.65g per g total protein, approximately 0.66g per g total protein, approximately 0.67g per g total protein, approximately 0.68g per g total protein, approximately 0.69g per g total protein, approximately 0.70g per g total protein, approximately 0.71g per g total protein, approximately 0.72g per g total protein, approximately 0.73g per g total protein, approximately 0.74g per g total protein, approximately 0.75g per g total protein, approximately 0.76g per g total protein, approximately 0.77g per g total protein, approximately 0.78g per g total protein, approximately 0.79g per g total protein, approximately 0.80g per g total protein, approximately 0.81g per g total protein, approximately 0.82g per g total protein, approximately 0.83g per g total protein, approximately 0.84g per g total protein, approximately 0.85g per g total protein 0.54g per g total protein, 0.55g per g total protein, 0.56g per g total protein, 0.57g per g total protein, 0.58g per g total protein, 0.59g per g total protein, 0.60g per g total protein, 0.61g per g total protein, 0.62g per g total protein, 0.63g per g total protein, 0.64g per g total protein, Approximately 0.65g per g protein, approximately 0.66g per g total protein, approximately 0.67g per g total protein, approximately 0.68g per g total protein, approximately 0.69g per g total protein, approximately 0.70g per g total protein, approximately 0.71g per g total protein, approximately 0.72g per g total protein, approximately 0.73g per g total protein, approximately 0.74g per g total protein, approximately 0.75g, 0.76g per g total protein, 0.77g per g total protein, 0.78g per g total protein, 0.79g per g total protein, 0.80g per g total protein, 0.81g per g total protein, 0.82g per g total protein, 0.83g per g total protein, 0.84g per g total protein, 0.85g per g total protein, 0.86g per g total protein, 0.87g per g total protein, 0.88g per g total protein, 0.89g per g total protein, 0.90g per g total protein, 0.91g per g total protein, 0.92g per g total protein, The amount of glycerol per gram of protein may be about 0.93g per gram, about 0.94g per gram total protein, about 0.95g per gram total protein, about 0.96g per gram total protein, about 0.97g per gram total protein, about 0.98g per gram total protein, about 0.99g per gram total protein, about 1.0g per gram total protein, about 1.01g per gram total protein, about 1.02g per gram total protein, about 1.03g per gram total protein, about 1.04g per gram total protein, about 1.05g per gram total protein, about 1.06g per gram total protein, about 1.07g per gram total protein, about 1.08g per gram total protein, about 1.09g per gram total protein, or about 1.1g per gram total protein.

[0071] In any embodiment of the fourth, fifth and sixth aspect, the amount of fatty acid, preferably caprylic acid (octanoic acid), mixed with the plasma sample is 0.35g / g total protein, 0.36g / g total protein, 0.37g / g total protein, 0.38g / g total protein, 0.39g / g total protein, 0.40g / g total protein, 0.41g / g total protein, 0.42g / g total protein, 0.43g / g total protein, 0.44g / g total protein, 0.45g / g total protein. , 0.46g per g total protein, 0.47g per g total protein, 0.48g per g total protein, 0.49g per g total protein, 0.50g per g total protein, 0.51g per g total protein, 0.52g per g total protein, 0.53g per g total protein, 0.54g per g total protein, 0.55g per g total protein, 0.56g per g total protein, 0.57g per g total protein, 0.58g per g total protein, 0.59g per g total protein, 0.60g per g total protein, 0.61g per g total protein, 0.62g per g total protein, 0.63g per g total protein, 0.64g per g total protein, 0.65g per g total protein, 0.66g per g total protein, 0.67g per g total protein, 0.68g per g total protein, 0.69g per g total protein, 0.70g per g total protein, 0.71g per g total protein, 0.72g per g total protein, 0.73g per g total protein, 0.74g per g total protein, 0.75g per g protein, 0.76g per g total protein, 0.77g per g total protein, 0.78g per g total protein, 0.79g per g total protein, 0.80g per g total protein, 0.81g per g total protein, 0.82g per g total protein, 0.83g per g total protein, 0.84g per g total protein, 0.85g per g total protein, 0.86g per g total protein, 0.87g per g total protein, 0.88g per g total protein, 0.89g, 0.90g per g total protein, 0.91g per g total protein, 0.92g per g total protein, 0.93g per g total protein, 0.94g per g total protein, 0.95g per g total protein, 0.96g per g total protein, 0.97g per g total protein, 0.98g per g total protein, 0.99g per g total protein, 1.0g per g total protein, 1.01g per g total protein, 1.02g per g total protein, 1.03g per g total protein, 1.04g per g total protein, 1.05g per g total protein, 1.06g per g total protein, 1.07g per g total protein, 1.08g per g total protein, 1.09g per g total protein, or 1.1g per g total protein.

[0072] In a preferred embodiment, the step of contacting the plasma sample with the medium chain fatty acid comprises mixing the plasma sample and the fatty acid to obtain a homogenous emulsion of the medium chain fatty acid and the plasma sample, in a preferred embodiment, the mixing is vigorous mixing to allow for the formation of a homogenous emulsion.

[0073] Preferably, the plasma sample and the medium chain fatty acid, preferably caprylic acid (octanoic acid), are mixed for a period of at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, or at least about 50 minutes or more.

[0074] Preferably, after the mixing step, there is an incubation period prior to the step of separating the soluble protein-containing components (soluble immunoglobulins) from the insoluble protein-containing components (insoluble albumin), which incubation period is preferably at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 130 minutes, at least about 140 minutes, at least about 150 minutes or more.

[0075] In any embodiment of any aspect of the invention, unless otherwise specified, the method steps are carried out at a temperature between about 18° C. and about 37° C., preferably between about 18° C. and about 24° C. In any embodiment, the temperature is about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., or about 24° C. In any embodiment, the temperature is 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., or 24° C.

[0076] The immunoglobulin purified according to any of the aspects of the present invention preferably comprises immunoglobulin G (IgG), preferably human immunoglobulin G (IgG). The immunoglobulin may comprise any one of the IgG subclasses IgG1, IgG2, IgG3 or IgG4, preferably wherein the relative distribution of IgG subclasses is similar or substantially the same as the distribution of IgG subclasses typically observed in plasma. Optionally, IgG1 is present in the composition in an amount of about 60% to about 70% of the total immunoglobulin, IgG2 is present in about 25% to about 35% of the total immunoglobulin, IgG3 is present in about 2% to about 3% of the total immunoglobulin, and IgG4 is present in about 0.5% to about 1.5% of the total immunoglobulin.

[0077] In any embodiment of any aspect of the invention, the immunoglobulin solution, or the concentrated immunoglobulins, is subjected to further processing to further purify the immunoglobulins. Preferably, the further processing does not include a further step of successive filter extraction.

[0078] In certain embodiments, the immunoglobulins are subjected to further processing such as low pH treatment, chromatography steps (including anion exchange chromatography and / or immunoaffinity chromatography), viral filtration and inactivation steps, concentration, and formulation such that the final product can be administered, for example, to the human body. The final product may be used in the treatment of immune conditions, certain autoimmune diseases, and certain neurological diseases. These conditions include rheumatoid arthritis, systemic lupus erythematosus (SLE), antiphospholipid syndrome, immune thrombocytopenia (ITP), Kawasaki disease, Guillain-Barre syndrome (GBS), multiple sclerosis (MS), chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy (MMN), myasthenia gravis (MG), bullous disease, scleroderma, dermatomyositis, polymyositis, Alzheimer's disease, Parkinson's disease, Alzheimer's disease associated with Down's syndrome, cerebral amyloid angiopathy, dementia with Lewy bodies, frontotemporal lobar degeneration or vascular dementia. Additionally, the final IVIg and SCIg products may be used in other medical procedures such as cell and organ transplantation.

[0079] In any embodiment of any aspect of the invention, the purified immunoglobulin solution contains one or more of the following impurities: IgA, IgM, albumin, alpha-2 macroglobulin, alpha-1 antitrypsin, lipids, and lipoproteins.

[0080] In any embodiment, the insoluble protein-containing component of the suspension (i.e., obtained from contacting the plasma sample with medium chain fatty acids) is retained after the step of separating the soluble protein-containing component from the insoluble protein-containing component.

[0081] Of course, the insoluble protein-containing component can then be used for the purpose of obtaining a purified fraction of albumin, for example as described herein.

[0082] For example, in the context of the third, sixth or ninth aspects of the invention, once the suspension obtained from mixing plasma and medium chain fatty acids has been fed through a first filtration unit, any remaining suspension, and / or the first retentate may be further processed to obtain purified albumin.

[0083] Thus, in any embodiment of the invention, the method comprises: a) adjusting the pH of the insoluble protein-containing component containing albumin to a pH between about 6.4 and about 7.2 (preferably about 6.8 and about 7.2) to obtain solubilized albumin; b) optionally subjecting the solubilized albumin to further processing steps to remove impurities therefrom; c) recovering purified albumin from the solubilized albumin. Further includes:

[0084] In the context of the third, sixth and ninth aspects of the invention, the insoluble albumin may be residual insoluble protein remaining in the first tank and / or may further comprise the first retentate.

[0085] Adjustment of the pH of the insoluble protein-containing component may be done directly without substantial dilution of the insoluble protein-containing component. Such pH adjustment may be accomplished by adding a concentrated acid, such as acetic acid, or a combination of an acid and a base (e.g., NaOH) if further adjustment of the pH is required. For example, the insoluble protein-containing component (e.g., the residual suspension in the first tank) may be pH adjusted to 6.4-7.2 (preferably 6.8-7.2) using 1 M sodium hydroxide or phosphate buffer (pH 7.1-7.4). Also contemplated is a combination of sodium hydroxide and phosphate buffer at 0.12 M. If necessary, the conductivity of the resulting solution is adjusted to achieve a desired conductivity of about 8 mS / cm to about 15 mS / cm.

[0086] Optionally, adjusting the pH of the insoluble protein-containing component comprises first contacting the insoluble protein-containing component (insoluble albumin) with a buffer having a pH between 7.1 and 7.4 and optionally a conductivity of about 8 to about 15 mS / cm to form a further suspension, and adjusting the pH of this further suspension to a pH of at least about 6.4, preferably neutral, more preferably between about 6.4 to about 7.2, or about 6.4 to about 6.7, or about 6.8 to about 7.2, thereby obtaining solubilized albumin.

[0087] Any buffer capable of breaking the bond between albumin and medium chain fatty acids to liberate the fatty acids from albumin, thereby rendering the albumin soluble, is suitable for use in this step. For example, the buffer may have a pH of about 7 and a conductivity of about 8 to about 15 mS / cm. This step may be carried out by adding the buffer to a tank containing the suspension and stirring (e.g., for 5 minutes) until the pH changes to about 7, preferably about 7.2 (particularly at least about 6.4, more preferably between about 6.4 and about 6.7, between about 6.4 and about 7.2, and most preferably between about 6.8 and about 7.2). An example of a suitable buffer is a phosphate buffer. Phosphate buffers may include NaH2PO4.2H2O and NaH2PO4.12H2O. The buffer may have a concentration of 0.12 M and a pH of 7.3±0.2. Alternatively, the pH adjustment may be carried out directly using a base such as NaOH, as described above.

[0088] The solubilized albumin may be subjected to further processing steps to remove impurities.

[0089] In one embodiment of any aspect of the invention, further processing of the solubilized albumin comprises: - providing solubilized albumin into a filtration unit including a dynamic filter element adapted to produce an albumin-depleted retentate and an albumin-enriched filtrate; - Recovering the albumin-enriched filtrate. include.

[0090] The albumin-enriched filtrate may optionally be subjected to a concentration step prior to further processing. The concentration step may comprise a continuous concentration process, whereby the filtrate is fed into a second filtration unit comprising a cross-flow filter element adapted to produce an albumin-enriched retentate; and an albumin-depleted filtrate.

[0091] According to this embodiment of the invention, the method for the further processing of solubilized albumin thus comprises: d) providing solubilized albumin to a first tank: e) feeding the solution of solubilized albumin into a first filtration unit comprising a dynamic filter element adapted to produce an albumin-depleted retentate and a filtrate enriched in soluble albumin; f) optionally diluting the solution in the first tank by flowing retentate into the first tank; g) recovering the albumin-enriched filtrate in a second tank; and h) Optionally, concentrate the filtrate. Includes.

[0092] In any embodiment, step h) of concentrating the filtrate comprises subjecting the filtrate to a continuous concentration process in a second filtration unit comprising a dynamic filter element or TFF adapted to produce an albumin-enriched retentate and an albumin-depleted filtrate. Optionally, the albumin-depleted filtrate may be passed back into the first tank and / or the albumin-enriched retentate may be passed back into the second tank.

[0093] Preferably, the dynamic filter element in the first filtration unit adapted to produce an albumin-enriched filtrate (permeate) is a dynamic cross-flow filter element. Preferably, the dynamic filter element in the second filtration unit adapted to concentrate the albumin solution and produce an albumin-enriched retentate is a dynamic cross-flow filter element or TFF.

[0094] In alternative embodiments, the solubilized albumin fraction may be subjected to alcohol precipitation and / or chromatography as commonly known in the art to further purify the albumin. Depending on the nature of contaminants present in the albumin, various purification schemes may be employed. For example, the albumin may be subjected to well-known fractionation processes such as ethanol fractionation to yield Supernatant I, Supernatant II+III, Supernatant-IV-1, Supernatant-IV-4, or Fraction V. The albumin may be further pH adjusted, UF / Diafiltered, and pasteurized as per the AlbuRx manufacturing process. Other methods for producing purified albumin are well known and include subjecting the albumin to ion exchange chromatography followed by gel filtration chromatography and pasteurization as done in the Albumex manufacturing process. Suitable albumin purification processes are discussed in Matejtschuk, P. et al. (2000) British Journal of Anaesthesia 85(6);887-95, and Australian Public Assessment Report for Albumin (human) (2017) Therapeutic Goods Administration, pp. 8-9 (available online at https: / / www.tga.gov.au / sites / default / files / auspar-albumin-human-170502.pdf).

[0095] As used herein, unless the context requires otherwise, the term "comprise" and variations of this term, such as "comprising", "comprises" and "comprised", are not intended to exclude additional additives, components, integers or steps.

[0096] Further aspects of the invention and further embodiments of the aspects described in the previous paragraphs will become apparent from the following description, given by way of example only, and with reference to the accompanying drawings, in which:

[0097] BRIEF DESCRIPTION OF THE DRAWINGS The following drawings are not necessarily drawn to scale, emphasis instead being placed generally on illustrating the principles of various embodiments. In the following description, various embodiments of the present invention are described with reference to the following drawings: [Brief description of the drawings]

[0098] [Figure 1] FIG. 1: Distribution of IgG subclasses in blood-derived plasma and in the clarified filtrate containing purified immunoglobulins obtained according to the method of the invention. [Diagram 2] Figure 2: Protease composition of blood derived plasma, diluted pooled plasma, 0, 1, 2 and 3 hours (T0, T1, T2 and T3, respectively) incubation with octanoic acid, and UF / DF filtrate containing purified immunoglobulins produced according to the method of the invention. Protease levels are shown as nkat / L. [Diagram 3] FIG. 3: Impurities detected in plasma and in the clarified filtrate containing purified immunoglobulin obtained according to the method of the invention. [Figure 4] FIG. 4: Prekallikrein activator (PKA), factor IX (FIX) and factor XI(a) (FXI(a)) activities (IU / mL) in plasma and in clarified filtrate containing purified immunoglobulins obtained according to the method of the invention. [Diagram 5] FIG. 5: Concentration of albumin (g / L) in blood-derived plasma and in the clarified filtrate containing purified immunoglobulins obtained according to the method of the invention. [Figure 6] Figure 6: Protein composition of plasma and of the clarified filtrate containing purified immunoglobulins obtained according to the method of the invention. The relative percentages of gamma-globulins, alpha- / beta-globulins and albumin are shown. [Figure 7] Figure 7: IgG yield (g per L plasma) at different amounts of octanoic acid and different pH values ​​at constant ionic strength. [Figure 8]Figure 8: Albumin yield (g per L plasma) at different amounts of octanoic acid and different pH values ​​at constant ionic strength. [Figure 9] Figure 9: Yields of IgG, albumin, IgA and IgM (g per L plasma) at constant pH and varying ionic strength. [Figure 10] Figure 10: IgG yield (g / L plasma) of clarified and concentrated octanoic acid filtrate at different pH and different ionic strengths. [Figure 11] Figure 11: Albumin yield (g per L plasma) of clarified and concentrated octanoic acid filtrate at different pH and different ionic strengths. [Figure 12] Figure 12: IgA yield (g / L plasma) of clarified and concentrated octanoic acid filtrate at different pH and different ionic strengths. [Figure 13] Figure 13: IgM yield (g / L plasma) of clarified and concentrated octanoic acid filtrate at different pH and different ionic strengths. [Figure 14] Figure 14: Product related impurities (g per L of plasma). [Figure 15] Figure 15: Impurity profile of heat-treated albumin (g per L of plasma). [Figure 16] Figure 16: Schematic flow chart overview of the continuous extraction filtration system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0099] FIG. 16 illustrates a schematic flow chart overview of the system 100 and the method according to one preferred embodiment of the present invention. Plasma is placed in tank 1, pH and conductivity are adjusted by adding buffers / acids etc. Then OA is added and the OA suspension is incubated in tank 1. The OA suspension can be fed to the first filtration unit 5 via pump 2 and flow control valve 3 in pipe 12, several types of pumps can be used (e.g. piston pump; rotary pump; centrifugal pump and membrane pump). The first filtration unit 5 comprises a rotating hollow shaft on which a filter disc is attached (filtrate flows from the outside to the inside of the hollow shaft). The first filtration unit 5 is further equipped with a height adjustable scraper to keep the filter cake thickness constant and thus achieve a constant filtrate flow. The desired filtration pressure is controlled and regulated by an overflow valve (unfiltered suspension outlet). The filter discs used can be ceramic membranes, depth filter layers and sintered porous metal filter discs. Once the container of the first filtration unit 5 is filled with the suspension, continuous pressure extraction and separation can be started. The first filtration unit 5, which may comprise a pressure unit / container, is provided with suitable internal settings and conditions to simultaneously increase the extraction efficiency and the filtration process. The extraction efficiency is increased by turbulent mixing in the unit 5 without the need for a mixer. Nevertheless, it can be envisaged that an additional mixer may be provided to assist the extraction process by creating turbulence. Furthermore, higher final dilution ratios disclosed in the present invention, for example 1:≧30, also increase the extraction efficiency and result in high protein (e.g. IgG) yield. Of course, any other higher final dilution ratios (higher than 70) can also be envisaged.

[0100] The first filtrate flows through a flowmeter 6 installed in a pipe (or channel) 14 and is collected in a second tank 7. The unfiltered suspension (e.g., the first retentate) flows back through a regulated outlet 3 installed in a pipe 13 in the tank 1. When a defined volume is reached in the second tank 7, the UF 8 concentration process can be started in the second filtration unit. The first filtrate in the second tank 7 flows through a pipe 15 into an ultrafiltration (UF) system 8 (e.g., a tangential flow filtration (TFF) using a TFF membrane). The transmembrane pressure is set so that the permeate flow rate 17 is the same or approximately the same as the flow rate of the first filtrate in the pipe 14. The permeate (or second filtrate) of the UF system 8 flows back through a pipe (or line or channel) 17 to the first tank 1, while the retentate (or second retentate) of the UF 8 system (= concentrated proteins) flows back through a pipe 16 to the second tank 7.

[0101] According to the invention, the first processing unit 5 comprises one or more rotating filter discs containing one or more first filter elements for turbulent mixing of the contents of the first processing unit 5 to produce a first retentate and a first permeate / filtrate. The first retentate can be sent back to the first tank 1 via a control valve 3 through a channel 13, while the first permeate / filtrate can be fed to the second tank 7 via another channel 14. The first filter element can be a filtration membrane based on a ceramic material with a pore diameter of about between 5 nm and 5000 nm, preferably between 20 nm and 100 nm or more preferably between 30 nm and 80 nm. It can be expected that inorganic membranes or any other suitable membranes can also provide similar effects to ceramic-based membranes. The first filtration unit 5 can be provided with a pressure control device 4, such as a manometer, to regulate the pressure inside. Similarly, a flow meter 6 can be installed in the system of the invention to measure the flow rate of the suspension or solution.

[0102] Detailed Description of the Preferred Embodiments Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the invention is not limited to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the invention as defined by the claims.

[0103] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is not in any way limited to the methods and materials described. It is to be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0104] For purposes of interpreting this specification, terms used in the singular also include the plural and vice versa.

[0105] The present invention relates to a system and method for the efficient purification of immunoglobulins, preferably IgG, from plasma. The present invention advantageously allows the use of plasma as a starting material and allows the extraction of immunoglobulins from plasma in a single step, without the need for alcohol-based precipitation methods.

[0106] A further advantage of the present invention is the ability to simultaneously purify albumin from the starting plasma sample, thereby providing a method for rapidly obtaining substantially pure preparations of both immunoglobulins and albumin from plasma via a single precipitation and filtration step.

[0107] A particular benefit of the method approach of the present invention is the absence of any filter aids, which advantageously ensures that protease activity is reduced early in the process, ensuring maximum protein recovery and yield. Further advantages arise, in part, from the application of a single, continuous extraction filtration method to separate the immunoglobulin-containing and albumin-containing components of plasma. The continuous extraction method facilitates downstream processing, for example, by reducing the total amount of reagents required to precipitate impurities or non-target proteins, using conditions that minimize loss of the protein of interest.

[0108] definition The term "soluble protein-containing component" is intended to refer to the water-soluble component of the aqueous phase resulting after mixing of a sample of blood-derived plasma with medium-chain fatty acids according to the method of the present invention. Typically, the soluble protein-containing component is highly enriched in immunoglobulins and other proteins that remain soluble after mixing of the plasma with the fatty acids.

[0109] The term "insoluble protein-containing component" is intended to refer to the solid phase water-insoluble component resulting after mixing a sample of blood-derived plasma with medium-chain fatty acids according to the method of the present invention. Typically, the insoluble protein-containing component contains precipitated proteins, primarily albumin, but also other contaminating proteins that are denatured after mixing with the plasma fatty acids.

[0110] The method of the present invention allows for the selective precipitation of albumin from a sample of blood-derived plasma. In a preferred embodiment, the precipitation results in the precipitation of at least 50% of the albumin in the sample. More preferably, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% or more of the albumin in the sample is precipitated.

[0111] By "high yield" is meant that the yield of the protein of interest, such as immunoglobulin G or albumin (as well as other proteins and immunoglobulins), is at least 80%, at least 85%, at least 95%, preferably at least 96%, more preferably at least 98%, and most preferably greater than 98% of the amount of protein in the soluble or insoluble protein-containing component.

[0112] The concentration of immunoglobulins and / or albumins in a sample can be measured by any means known to those skilled in the art. Of course, the method used to measure immunoglobulins or albumins can depend on the nature of the sample. For example, of course, if the sample is an albumin-containing precipitate, the precipitate (or the sample) may need to be dissolved in a suitable buffer before measurement. Examples of suitable assays for measuring proteins of interest include high pressure liquid chromatography (HPLC; e.g., size-exclusion HPLC), enzyme-linked immunosorbent assay (ELISA), and quantitative immunoturbidimetry.

[0113] The pH and / or conductivity of any sample may be measured by methods known in the art. Typically, the pH and / or conductivity are measured at room temperature, preferably, the pH and / or conductivity are measured between 18-25° C. The unit of measurement for conductivity is millisiemens per centimeter (mS / cm) and may be measured using a standard conductivity meter. The conductivity of a solution may be altered by changing the concentration of ions therein. For example, one may change the concentration of a buffering agent and / or the concentration of a salt (e.g., NaCl or KCl) in the solution to achieve a desired conductivity. Preferably, the salt concentration is modified to achieve a desired conductivity, as described in the Examples below or elsewhere herein.

[0114] With reference to a numerical value given for a percentage, pH, amount or time or other reference, "about" or "approximately" means the inclusion of that numerical value within 10% of the specified value.

[0115] Large-scale or industrial-scale processes or systems Larger scale or industrial scale in the context of the present invention refers to manufacturing procedures based on at least 200 L of starting material, such as human plasma, preferably at least 500 L, and even more preferably at least 2000 L. For example, typical commercial plasma donor pool sizes used in industrial scale protein production range from 2500 L to 6000 L of plasma per batch. In certain embodiments of the present invention, precipitate is obtained from 2500 L to 6000 L of plasma. Some commercial manufacturing processes are also capable of using larger plasma donor pool sizes, including up to 7500 L, 10000 L, and / or 15000 L of plasma.

[0116] The methods and systems of the present invention can also be used as stand-alone systems and / or methods for smaller manufacturing scale applications (where the starting material may be less than 200 L) as well as for larger industrial scale applications.

[0117] Starting materials The method of the invention advantageously allows to use blood-derived plasma as starting material for the extraction of immunoglobulins and albumin. The plasma can be fresh plasma, "normal" plasma, "hyperimmune" plasma, cryo-poor plasma (also called cryo-supernatant) or cryo-rich plasma. Optionally, the plasma has been treated to remove components such as C1 inhibitor, PCC (prothrombin complex concentrate) and / or AT-III. Plasma can be obtained from multiple transfusions and / or individuals and can be pooled.

[0118] The term "cryosupernatant" (also called cryoprecipitate-depleted plasma, cryoprecipitate-depleted plasma, and the like) refers to plasma (derived from either whole blood donations or plasma exchange) from which the cryoprecipitate has been removed. Cryoprecipitation is the first step in most plasma protein fractionation methods currently used for large-scale production of plasma protein therapeutics. The method generally involves pooling frozen plasma that is thawed under controlled conditions (e.g., at or below 6°C) and then collecting the precipitate by either filtration or centrifugation. The supernatant fraction, known to those skilled in the art as "cryosupernatant," is usually retained for use. The resulting cryoprecipitate-depleted plasma has reduced levels of Factor VIII (FVIII), von Willebrand Factor (VWF), Factor XIII (FXIII), fibronectin, and fibrinogen. Cryosupernatant provides a common raw material used to manufacture a range of therapeutic proteins including alpha 1-antitrypsin (AAT), apolipoprotein AI (APO), antithrombin III (ATIII), prothrombin complex containing clotting factors (II, VII, IX and X), albumin (ALB) and immunoglobulins such as immunoglobulin G (IgG).

[0119] The term "cryo-rich plasma" refers to plasma (either from whole blood donation or plasma exchange) that has been frozen and then thawed, but from which the cryoprecipitate has not been removed.

[0120] If the plasma was frozen for shipment from the collection site, the frozen plasma is thawed and then collected in a storage tank prior to centrifugation. Cryoprecipitate is removed by continuous centrifugation. Cryo-depleted plasma can be pumped into a stainless steel fractionation tank and sampled for in-process control.

[0121] The plasma may be hyperimmune plasma, whether pooled from more than one individual, from hundreds of individuals, or obtained from a single individual, for example, plasma may be obtained from the blood of individuals who have mounted an immune response to an infection and from the blood of recovered (and therefore otherwise healthy) individuals.

[0122] Dynamic Filter Elements In any aspect of the invention, the dynamic filter element filtration unit adapted for separating soluble immunoglobulins from insoluble albumin is a dynamic cross-flow filter element. In a preferred embodiment, the dynamic cross-flow filter element is a rotating cross-flow filter element. More preferably, the rotating cross-flow filter element comprises a filter disc. The filter disc is typically mounted on a shaft member. In one embodiment, the rotating cross-flow filter element comprises at least one filter disc and at least one shaft member.

[0123] According to a preferred embodiment of any aspect of the invention, the filter disc membrane is a ceramic membrane. More preferably, the ceramic membrane has a pore size ranging from greater than or equal to 5 nm to less than or equal to 2 μm. In certain embodiments, the ceramic membrane has a pore size of about 0.2 μm to 2 μm. In certain embodiments, the ceramic filter membrane has an average pore size ranging from greater than or equal to 5 nm to less than or equal to 200 nm (0.2 μm). In certain embodiments, the ceramic filter membrane has an average pore size ranging from greater than or equal to 50 nm to less than or equal to 100 nm. Such filter discs are supplied by Kerafol and Flowserve.

[0124] Of course, multiple filter disk membranes may be included in a dynamic filter element filtration unit adapted to separate soluble immunoglobulins from insoluble albumin. As such, the method of the present invention contemplates the use of 1, 2, 3, 4, 5, 6 or more filter disk membranes to separate soluble immunoglobulins from insoluble albumin. The multiple filter disk membranes may have the same or different pore sizes.

[0125] The filtration unit in a preferred embodiment includes a pressure vessel. The suspension from the first tank can be continuously fed into the pressure vessel through an inlet port. Uniform distribution of the suspension in the vessel can be achieved using a distribution manifold. Thus, in a particular embodiment, the pressure vessel includes a distribution manifold. In some embodiments, the first filtration unit includes a rotating cross-flow filter element. Preferably, the filter element includes more than one filter disk equally spaced along at least one hollow central collection shaft. The filter disks can be arranged either horizontally or vertically. In the case of a horizontal orientation, they are spaced along a vertically oriented hollow collection shaft. The collection shaft and the disks are rotatable. The suspension in the pressure vessel can then permeate the outer membrane of the rotating filter disk to pass through and into the hollow central portion of the disk, which is then directed into the central collection shaft. Typically, the filtrate (e.g., containing partially purified immunoglobulins) can then be removed from the shaft portion of the first filtration unit through a flanged port. The retentate (containing insoluble components) remaining in the pressure housing can be fed from the vessel through an outlet port. Typically, the retentate is recycled to the first tank to dilute the suspension. In this manner, the retentate from the first filtration unit can be utilized to dilute the suspension in the first tank.

[0126] Dynamic cross-flow filtration, such as rotary filtration, provides maximum filtration efficiency. The cross-flow effect (tangential flow cleaning of the filter surface) occurs by rotating the filter discs and not by pumping over a fixed membrane as used in conventional (static) cross-flow filtration systems. The extreme cross-flow velocities that occur at the surface of the rotating filter discs ensure highly efficient cleaning of the filter surface, while consuming a very low amount of energy compared to conventional cross-flow technologies.

[0127] Dynamic filter elements can also be used to perform continuous concentration processes. Such dynamic filter elements typically contain one or more ultrafiltration or diafiltration membranes.

[0128] The cross-flow filter element for carrying out the continuous concentration process can include a dynamic ultrafiltration filter device. Alternatively, the process includes a static ultrafiltration device, such as a tangential flow filtration (TFF).

[0129] In preferred embodiments of the invention, the dynamic cross-flow filter element or ultrafiltration filter device for carrying out the concentration process comprises a membrane with a molecular weight cut-off less than the molecular weight of the protein of interest (e.g., immunoglobulin G in the case of step e) of the third aspect of the invention). The molecular cut-off may be one-third or less of the molecular weight of the protein of interest (e.g., for a protein having a molecular weight of about 150 kDa, the membrane may have a cut-off of about 30-50 kDa). In these embodiments, the membrane cut-off is selected to retain the protein of interest during the concentration process. As a general guideline, a nominal membrane cut-off of one-third or less of the molecular weight of the protein of interest may be selected to ensure that the protein is retained in the retentate.

[0130] In an alternative embodiment, the dynamic cross-flow filter element or static ultrafiltration element for carrying out the concentration process comprises a membrane with a molecular weight cut-off greater than the molecular weight of the protein of interest, in such an embodiment, the nominal membrane cut-off is selected to ensure that the protein of interest passes through the membrane and is collected in the filtrate rather than the retentate.

[0131] In embodiments in which the cross-flow filter element is dynamic, preferably the element is a rotating cross-flow filter element adapted to carry out a continuous concentration process.

[0132] According to a further preferred embodiment, the filtration element for carrying out the continuous concentration process comprises a filtration membrane having an average pore size between 5 nm and 5000 nm, preferably between 5 nm and 2000 nm, between 5 nm and 1000 nm, between 5 nm and 500 nm, between 5 nm and 200 nm, between 7 nm and 1000 nm, more preferably between 7 nm and 500 nm, even more preferably between 7 nm and 100 nm, and most preferably between 7 nm and 80 nm. Of course, the average pore size may be other combinations of the ranges indicated above. Filter manufacturers often assign terms such as nominal or average pore size grades to commercial filters, which usually indicate the fulfillment of certain retention criteria for particles or microorganisms rather than the actual geometric dimensions of the pores.

[0133] In one embodiment, the filtration element that performs the continuous concentration process is a dynamic flow filtration or tangential flow filtration (TFF) using a TFF membrane.

[0134] In certain embodiments, the rotating cross-flow filter element for carrying out the continuous concentration process comprises a filter disk (e.g., a ceramic disk). In some embodiments, the filter disk comprises a membrane having an average pore size of a microfilter. In other embodiments, the filter disk comprises a membrane having an average pore size of an ultrafiltration membrane. In further embodiments, the filter disk comprises a membrane having an average pore size of a diafilter. In one embodiment, the average pore size of the filter disk membrane ranges from an average pore size of greater than or equal to 5 nm to an average pore size of less than or equal to 2 μm. In certain embodiments, the average pore size of the filter disk membrane ranges from an average pore size of greater than or equal to 50 nm to an average pore size of less than or equal to 500 nm (i.e., 0.5 μm). In some embodiments, the filter disk membrane has an average pore size ranging from greater than or equal to 50 nm to less than or equal to 100 nm, or from greater than or equal to 60 nm to less than or equal to 90 nm, or from greater than or equal to 60 nm to less than or equal to 80 nm. In some embodiments, the filter disk membrane has an average pore size of 60 nm or 80 nm.

[0135] In a particularly preferred embodiment, the rotating cross-flow filter element for carrying out a continuous concentration process comprises a plurality of ceramic disks having a pore size suitable for ultrafiltration and / or diafiltration. For example, the element preferably comprises at least one ceramic membrane having a pore size of 3 nm. Alternatively, the element preferably comprises at least one ceramic membrane having a pore size of 5 nm. Alternatively, the element preferably comprises at least one ceramic membrane having a pore size of 7 nm. Alternatively, the element preferably comprises at least one ceramic membrane having a pore size of 30 nm. The element may comprise a plurality of ceramic disks having different pore sizes, including those having a pore size of 3 nm and 5 nm. The element may comprise a plurality of ceramic disks having different pore sizes, including those having a pore size of 5 nm and 7 nm. The element may comprise a plurality of ceramic disks having different pore sizes, including those having a pore size of 3 nm and 30 nm. The element may comprise a plurality of ceramic disks having different pore sizes, including those having a pore size of 3 nm, 5 nm, 7 nm and 30 nm.

[0136] According to yet a further preferred embodiment, the filtration element for carrying out the continuous concentration process comprises an ultrafiltration device comprising a membrane in the form of a polymer membrane such as polyethersulfone or regenerated cellulose.

[0137] Dynamic cross-flow filtration, such as rotary filtration, provides maximum filtration efficiency. The cross-flow effect (tangential flow cleaning of the filter surface) occurs by rotating the filter discs and not by pumping over a fixed membrane as used in conventional (static) cross-flow filtration systems. The extreme cross-flow velocities that occur at the surface of the rotating filter discs ensure highly efficient cleaning of the filter surface, while consuming a very low amount of energy compared to conventional cross-flow technologies.

[0138] In the dynamic cross-flow filtration units and systems of the invention, the rotating ceramic filter discs are typically assembled in a pressurized housing. The design of the discs shows internal drainage channels. The filtrate is transported from the outside to the inside of the discs. The rotation of the discs creates shear forces on the membrane surface. With this technique, the build-up of filter cake is avoided, resulting in a high filtration flux. Some of the main parameters of rotary filtration are the rotation speed for rotating the ceramic filter discs, the solids content (concentration of liquid resulting from the removal of filtrate) and the transmembrane pressure. The transmembrane pressure is typically between 0.1 and 2.5 bar, preferably between 0.2 and 2.4 bar, more preferably between 0.4 and 2.0 bar, 0.5 and 1.8 bar, 0.6 and 1.6 bar, 0.6 and 1.5 bar, 0.7 and 1.5 bar, most preferably between 0.8 and 1.5 bar. According to another embodiment, a pressure of up to 2 bar, preferably between 0.1 and 2.0 bar, or about 1.5 bar, 1.0 bar or 0.5 bar is applied to the filter unit.

[0139] Temperature has an effect on the viscosity of the protein solution and therefore on the flux during filtration through the membrane. The starting suspension to be used in the method of the invention preferably has a temperature in the range of 0° C. to the temperature at which the protein of interest denatures. The temperature is typically in the range of about 18° C. to about 40° C. In a particular embodiment, the temperature is in the range of about 18° C. to about 35° C. According to one preferred embodiment, the temperature of the suspension tank (i.e. the tank containing the fatty acid, preferably caprylic (octanoic) acid, for mixing with the plasma sample) is at a temperature between about 18° C. and about 40° C. More preferably, the fatty acid, preferably caprylic (octanoic) acid, is mixed with the plasma in the second suspension tank at a temperature between about 18° C. and about 24° C., optionally at about 21° C., about 22° C. or about 23° C.

[0140] According to a further preferred embodiment, the temperature of the filter unit is preferably controlled between 2° C. and 25° C., more preferably at about 18° C. to about 24° C. Such a temperature ensures optimal extraction and separation processes while maintaining the bioreactivity of the protein of interest throughout the process.

[0141] Filtration is carried out at a transmembrane pressure that is equal to or lower than the membrane can withstand, depending on the material of the membrane to be used herein, for example at a pressure of about 0.2 to about 3 bar. The transmembrane pressure is typically 0.1 to 2.5 bar, preferably 0.2 to 2.4 bar, more preferably 0.4 to 2.0 bar, 0.5 to 1.8 bar, 0.6 to 1.6 bar, 0.6 to 1.5 bar, 0.7 to 1.5 bar, most preferably 0.8 to 1.5 bar. According to another embodiment, a pressure of up to 2 bar, preferably between 0.1 and 2.0 bar, or about 1.5 bar, 1.0 bar or 0.5 bar, is applied to the filtration unit.

[0142] According to another embodiment, the continuous extraction process in a filtration unit adapted to separate impurities / precipitates from the first and second suspensions is further assisted by adjusting the flow rate and / or residence time of the suspension or solution entering the filter unit, and / or the flow rate of the retentate / raffinate containing the impurities / precipitates, and / or the flow rate of the first permeate / filtrate enriched in the protein of interest. For example, in one embodiment, the linear velocity of the suspension or solution entering the pressure vessel (filtration process unit) can be about 0.27-1.66 m / s. In another example, the linear velocity of the retentate containing the impurities / precipitates can be 0.25-1.33 m / s. In another example, the linear velocity of the permeate filtrate enriched in the protein of interest can be 0.03-0.33 m / s. Multiplying the linear velocity by the cross-sectional area gives the volumetric flow rate. Additionally, turbulent flow can occur in the first process unit as a result of the speed of the rotating filter disc, where the velocity (sometimes referred to as tangential velocity) can be between about 1-7 m / s. According to one embodiment of the present invention, the speed of the rotating disc filter is between 1 and 10 m / s. In a preferred embodiment of the present invention, the speed of the rotating disc filter is between 5 and 7 m / s. More preferably, the speed of the rotating disc filter is 7 m / s at 60 Hertz (800 rpm). The rotation speed of the rotating cloth filter element is between about 600 rpm (50 Hz) and about 1600 rpm (100 Hz), preferably between about 800 rpm (60 Hz) and about 1200 rpm (80 Hz), preferably about 800 rpm (60 Hz), about 1000 rpm (70 Hz) or about 1200 rpm (80 Hz). As used herein, the rotation speed in Hz is intended to refer to the speed of the motor. This can be related to the speed in rpm using an appropriate calibration curve.

[0143] This method allows for a continuous extraction and separation process to be realized to maximize recovery of the protein of interest from the starting precipitate / material (i.e., blood-derived plasma). As a result of the extraction process, almost all of the protein of interest is extracted and recovered in a subsequent step. This method also allows for the liquid or diluent, e.g., buffer or water, to be recirculated in a closed system, thus reducing the footprint (i.e., large tank volume) while maintaining the amount of liquid throughout the process.

[0144] In yet a further embodiment, the invention includes a step of backwashing in conjunction with dynamic cross-flow filtration to flush out contaminants that may have built up in the system. Typically, the methods and systems of the invention include alternating between filtration and backwashing at intervals such that filtration is temporarily stopped (i.e., the feed pump is turned off) while backwashing occurs, where the flow of liquid into the filtration system is reversed.

[0145] Of course, the frequency, duration and flow rate of backwashing may be adjusted to maximize filtration efficiency and the duration of filtration before backwashing is required.

[0146] In certain preferred embodiments, the frequency of backwashing (and therefore the duration of filtration) is determined based on the total protein concentration or the number of impurities present in the starting material. As a result, of course, backwashing is required more frequently when filtering a suspension containing plasma and fatty acids, compared to a later step of further purification, in which a solubilized albumin preparation (with relatively low impurities) is filtered. In other words, as the total protein concentration and turbidity of the filtrate decrease, the frequency of backwashing intervals also decreases (i.e., the period between backwashing increases, and filtration can continue for a longer period before backwashing is required).

[0147] Protein concentration and turbidity of the filtrate may be monitored by a variety of methods known in the art. In certain embodiments, the methods and systems of the invention include the use of an in-line detection unit that allows for measurement of protein concentration and / or turbidity as the filtrate enters and / or exits the filtration unit. In further embodiments, a dual wavelength photometer may be used to facilitate simultaneous assessment of protein concentration (e.g., by detecting the absorbance of the solution at a wavelength suitable for detecting protein concentration, e.g., in the range of 260-280 nm, preferably about 280 nm) and solution turbidity (e.g., by detecting the absorbance of the solution at a wavelength suitable for detecting light scattering caused by the presence of particulate matter, e.g., in the range of 400 nm-900 nm, preferably about 600 nm-about 880 nm). Dual wavelength photometer devices for use in conjunction with chromatography and filtration units are well known in the art.

[0148] In certain examples, the backwash frequency is at intervals of 15 seconds, 30 seconds, 45 seconds, 60 seconds, 75 seconds, 90 seconds, 105 seconds, 120 seconds, 135 seconds, 150 seconds, 200 seconds, 230 seconds, 260 seconds, 300 seconds, 330 seconds, 360 seconds, 400 seconds, 1000 seconds, 2000 seconds, 3000 seconds, 4000 seconds or more.

[0149] Of course, the duration of the backwash interval will vary depending on the filtration area and number of disks requiring backwashing. The larger the filtration area, the larger the volume of backwash buffer required will typically be, and the duration of the backwash will also depend on the flow rate during the backwash. Those skilled in the art will be well able to determine the appropriate duration, frequency, and flow rate for backwashing depending on the size of the system and the number of disks used. In certain instances, the duration of the backwashing is about 5 seconds, about 10 seconds, about 15 seconds, about 30 seconds, about 45 seconds, about 60 seconds, or more.

[0150] Of course, for practical reasons (and to maximize filtration efficiency), the duration of the backwashing interval is typically shorter than the duration of the filtration interval, and in certain embodiments, the duration of the backwashing interval is at least one-quarter, one-eighth, one-tenth, one-sixteenth, or less, of the filtration interval.

[0151] It will further be appreciated that the flow rate used during backwashing may be the same as or different from the flow rate used for filtration. In certain embodiments, the flow rate during dynamic filtration is in the range of about 15-100 L / hr, preferably about 20-50 L / hr (about 200 ml / min to about 1 L / min, preferably about 300-900 ml / min, more preferably about 300-600 ml / min). Preferably, the backwashing flow rate is lower than the flow rate used for filtration, such that in certain embodiments, the backwashing flow rate is in the range of about 100-400 times slower than the flow rate used for filtration.

[0152] In certain embodiments, the backwashing is performed with the same buffer as that contained in the first or second suspension. Alternatively, the backwashing can be performed using the permeate obtained during the concentration process (e.g., when ultrafiltration coupled with dynamic cross-flow filtration is used to concentrate the filtrate obtained from dynamic cross-flow filtration).

[0153] Protein Recovery and Further Processing The concentration of protein in a sample (e.g., the supernatant or a preparation thereof subsequently purified) can be measured by any means known to those skilled in the art. Examples of suitable assays include high pressure liquid chromatography (HPLC; e.g., size-exclusion HPLC), enzyme-linked immunosorbent assay (ELISA), nephelometry, and immunoturbidimetry. Such techniques can be used to assess the purity of a sample (e.g., to identify the presence of undesired protein contaminants, including proteases). In addition, gel electrophoresis such as SDS-PAGE with staining and densitometry can be used to assess the purity of a sample and to detect the presence of contaminating proteins. Reducing agents such as dithiothreitol can be used with SDS-PAGE to cleave any disulfide-linked polymers.

[0154] In any embodiment, the temperature at which the conductivity of the solution is measured can be between about 4° C. and about 37° C., preferably where the temperature is between about 18° C. and about 25° C., or between about 20° C. and about 25° C. (room temperature).

[0155] The ultrafiltered product containing immunoglobulins (i.e. after successive extractions and subsequent concentration) may be subsequently subjected to further processing such as chromatography steps, viral inactivation steps, concentration and formulation so that the final product can be administered, for example, to the human body. The final product may be used in the treatment of immune conditions, particularly autoimmune diseases and certain neurological diseases. These conditions include rheumatoid arthritis, systemic lupus erythematosus (SLE), antiphospholipid syndrome, immune thrombocytopenia (ITP), Kawasaki disease, Guillain-Barre syndrome (GBS), multiple sclerosis (MS), chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy (MMN), myasthenia gravis (MG), bullous disease, scleroderma, dermatomyositis, polymyositis, Alzheimer's disease, Parkinson's disease, Alzheimer's disease associated with Down's syndrome, cerebral amyloid angiopathy, dementia with Lewy bodies, frontotemporal lobar degeneration or vascular dementia. Additionally, the final IVIg and SCIg products may be used in other medical procedures such as cell and organ transplantation. EXAMPLES

[0156] Example 1: Isolation of purified IgG Four independent experiments were performed in which IgG was purified from plasma according to the method of the present invention.

[0157] In all four experiments, fresh plasma was diluted 1:2 with phosphate-acetate buffer (1 part plasma to 2 parts buffer), where the pH of the diluted plasma was about 4.6 to about 5.0 and the conductivity was 8 to 12 mS / cm. The diluted plasma was transferred to a tank (suspension tank).

[0158] Octanoic acid was added to the diluted plasma in the suspension tank in an amount of at least 0.35 g per g of total protein in the dilution buffer with vigorous mixing over a period of 20-60 minutes to produce a plasma / octanoic acid emulsion. This emulsion was further incubated at about 22°C for 60-240 minutes. Without wishing to be bound by theory, it is believed that the temperature, slow addition, and vigorous mixing contribute to a thorough distribution of octanoic acid into the diluted plasma. This in turn is believed to result in more efficient mixing of the octanoic acid with the albumin, resulting in more albumin coming into contact with the octanoic acid and a higher yield of precipitate. The end result is a more efficient separation of soluble immunoglobulins from the insoluble albumin-octanoic acid complex.

[0159] The resulting suspension was then fed to a continuous extraction filter unit, from which the retentate was recycled back into the suspension tank, and from which the filtrate (containing the immunoglobulins) was fed to a second tank. The filtrate collected in the second tank was then fed into a second unit containing an ultrafiltration and diafiltration system. The retentate of the UF / DF system was run back into the second tank, while the filtrate was run back to the first tank.

[0160] The solution was diluted to a protein concentration of 20 g / L and the pH was adjusted to about pH 4.0 in the presence of polysorbate 80 (P80). The solution was subjected to further clarification depth filtration. The resulting filtrate (referred to herein as "clarified filtrate") was further evaluated to determine various product properties, as further described below.

[0161] [Table 1]

[0162] These results demonstrate that the method of the present invention provides a highly efficient approach for isolating high levels of IgG directly from plasma samples. The yield of IgG is high and the recovery of IgG is also consistent with other commercial manufacturing processes for the isolation of IgG. Immunoglobulin levels were measured by immunoturbidimetry.

[0163] The IgG subclass distribution was determined by immunoturbidimetry, and the results shown in FIG. 1 indicate that the IgG subclass distribution in purified immunoglobulin preparations is similar to that seen in plasma.

[0164] Example 2: Determination of proteases and other contaminants in IgG preparations The concentration of proteases and the extent of protease activity in the clarified filtrate were determined using standard methods. Briefly, a chromogenic substrate-based assay was used. Serine protease activity was measured by the ability of the protein concentrate to cleave the chromogenic substrate lle-Pro-Arg-pNA (S-2288). During this reaction, p-nitroaniline (pNA) is released, which is measured in a photometer at 405 nm. Serine protease activity was measured under conditions of pH 8.4 and 37°C. Kallikrein-like activity was measured by cleavage of the chromogenic substrate HD-Pro-Phe-Arg-pNA (S-2302). During this reaction, p-nitroaniline (pNA) is released, which is measured in a photometer in kinetic mode at 405 nm.

[0165] As shown in FIG. 2, the method of the present invention effectively eliminated protease contamination.

[0166] Other impurities such as IgA, IgM, alpha 1-antitrypsin were determined using standard techniques. Impurities such as IgA, IgM and ceruloplasmin were found to be present at lower but still detectable levels. Other impurities were determined to be below the detection limit (alpha 1-antitrypsin, alpha 2-macroglobulin, haptoglobin, hemopexin, fibrinogen, fibronectin, cholestrin, transferrin, triglycerides and phospholipids). The results are shown in Figure 3. Protein impurities were determined by immunoturbidimetry, whereas cholestrin triglyceride and phospholipid levels were determined using enzymatic assays.

[0167] The amounts of prekallikrein activator (PKA), factor IX, and factor XI(a) were also determined. The results are shown in Figure 4. PKA was measured with a chromogenic substrate (as discussed above), FIX was measured by ELISA, and FXI(a) was measured using an activated partial thromboplastin time (aPTT) assay.

[0168] The amount of albumin (g / L) in the starting plasma material was compared to the amount of albumin present in the IgG preparation described in Example 1 (i.e., the clarified filtrate). The total albumin present in the plasma was about 32.2 g / L. The amount of albumin in the IgG preparation of Example 1 was less than 0.341 g / L, consistent with the typical amount of albumin found in commercial grade IgG preparations. The results are shown in Figure 5.

[0169] Further results, shown in Figure 6, indicate that the immunoglobulin preparation produced in Example 1 contains low levels of contaminating alpha / beta-globulins and albumin and is highly enriched in gamma globulins. Gamma globulin and alpha- / beta-globulin levels were determined using cellulose acetate / agarose electrophoresis.

[0170] Example 3: Isolation of purified albumin After completion of Example 1, the remaining suspension in the first tank was mixed with phosphate buffer (pH 7.1-7.4) and the pH was adjusted to between 6.4-6.7. The resulting solution contained soluble albumin. Alternatively, after completion of Example 1, the remaining suspension in the first tank was adjusted to between pH 6.4-7.2 (preferably 6.8-7.2) with 1M sodium hydroxide. A combination of sodium hydroxide and phosphate buffer at 0.12M is also contemplated.

[0171] The solution was further treated to achieve a total protein concentration of about 0.2 g / L to less than 1.0 g / L (or about 0.2 g / L to about 0.5 g / L).

[0172] Briefly, a solution of albumin was fed to a continuous extraction filter unit, from which the retentate was recycled back into a tank, and from which the filtrate (containing albumin) was fed into a second tank. The filtrate collected in the second tank was fed into a second unit containing an ultrafiltration and diafiltration system. The retentate of the UF / DF system was run back into the second tank, while the filtrate was run back into the first tank.

[0173] The retentate of UF / DF containing concentrated albumin was completed reaching the desired protein concentration, and this sample is referred to as "Post-CE Crude Albumin" in the tables below.

[0174] [Table 2]

[0175] Example 4: Evaluation of parameters Multiple independent experiments were performed to determine the optimal parameters under which IgG or albumin could be purified from plasma according to the methods of the present invention.

[0176] In all experiments, fresh plasma was diluted 1:3 with phosphate-acetate buffer (1 part plasma to 2 parts buffer), where the pH of the diluted plasma was approximately 4.6 to approximately 5.0 and the conductivity was 8 to 12 mS / cm.

[0177] Laboratory-scale experiments were performed to evaluate the effect of parameters such as ionic strength, pH, dilution ratio, dilution type (and / or diluted acetic acid) on impurity removal, IgG and albumin recovery at different OA concentrations.

[0178] Yield and impurities at constant ionic strength and at various pH and OA amounts One kilogram of pooled plasma was diluted with 100 mM sodium acetate, pH 4.0 (ratio: 1:3). The diluted plasma was divided into three equal parts and the pH was adjusted to the desired pH (4.2; 4.5 and 4.8) by dropwise addition of concentrated acetic acid with thorough mixing. Total protein concentration was determined by measuring absorbance at A280. The conductivity of each aliquot was adjusted to within 8.5 + / - 10 mS / cm using acetate buffer.

[0179] Octanoic acid was added to the diluted plasma in an amount of at least 0.35 g per g total protein over a period of 20-40 min with vigorous mixing to obtain plasma / octanoic acid emulsions at final concentrations of 0.5, 0.75 or 1.0 g per g total protein, respectively. The emulsions were stirred for an additional 60-180 min and then incubated with Celpure 100 at 5 g per kg solution for 15 min. Filtration was then performed using a CH9 filter bed.

[0180] Subsequent washes were performed at 20% of the starting volume using dilution buffer pre-adjusted to the same pH and conductivity as the experiment was performed.

[0181] The clarified protein solution was then ultra / diafiltered to 15-20 g / L. The pH of the solution was adjusted to 4.00 ± 0.20 during diafiltration. The protein solution was then incubated at 37°C for 9 ± 1 h, followed by a pH shift to 5.80 ± 0.10. After a subsequent depth filtration step, the solution was loaded onto a strong anion exchange column and the purified IgG was collected in the flow-through and the pH was adjusted to 4.80 ± 0.10.

[0182] [Table 3]

[0183] [Table 4]

[0184] [Table 5]

[0185] result At low pH (4.2) and low concentration of octanoic acid (0.5 g per g protein), almost all of the albumin precipitated compared to pH 4.8 (Figure 8). At pH 4.8, a significant amount of albumin is still present in the clarified and diafiltered protein solution after octanoic acid treatment. IgG yields are higher at pH 4.8 compared to pH 4.2 (Figure 7).

[0186] Lower Conductivity and Higher Octanoic Acid The effect of lower conductivities (2–5 mS / cm) at high octanoic acid concentrations (e.g., 1.0 g per g protein) at pH 4.20 was investigated.

[0187] The precipitating ability of octanoic acid at a concentration of 1 g per g protein and pH 4.2 was investigated at various ionic strengths: 3; 4; 5 and 6.5 mS / cm.

[0188] [Table 6]

[0189] result The results (Table 6 and Figure 9) clearly show that low conductivity (< 5 mS / cm) results in lower IgG yields compared to higher conductivity (> 6 mS / cm). The albumin content in the clarified concentrated octanoic acid filtrate is comparable to that at higher conductivity.

[0190] Higher octanoic acid and different pH A concentration of 0.55g octanoic acid per g protein was investigated at various pH (4.2, 4.5, and 4.8) and various ionic strengths (5; 6; 7 and 8 mS / cm) - see Tables 7-10 below, and Figures 10-13.

[0191] [Table 7]

[0192] [Table 8]

[0193] [Table 9]

[0194] [Table 10]

[0195] result The data show that the IgG yield at pH 4.8 is higher than at pH 4.2 and 4.5 regardless of the octanoic acid concentration (Table 7 and Figure 10). The residual amounts of albumin (Table 8 and Figure 11), IgA (Table 9 and Figure 12) and IgM (Table 10 and Figure 13) in the clarified concentrated IgG solution are relatively low and can be easily removed in further processing steps (e.g., main chromatographic purification) downstream of this octanoic acid step. Thus, the IgG solution can be further purified continuously in the current process.

[0196] At pH 4.2, especially at higher conductivity for albumin precipitation, the IgG yield is still slightly lower than at pH 4.8, but still very acceptable. The data also show that at appropriate octanoic acid concentrations (e.g., in the range of 0.50-0.55 g / g protein), significant amounts of albumin can be precipitated by octanoic acid, so that albumin can be recovered in high yield and purity.

[0197] Example 5: Continuous filtration In this experiment, the starting plasma pool was diluted with acetate or phosphate / acetate buffers with different ionic strengths (60 mM, 80 mM or 100 mM) and pH values ​​(4.0, 4.2, 4.5, 4.8 or 5.0).

[0198] One part plasma was diluted with two parts buffer using an impeller mixer.

[0199] The diluted plasma was transferred to a tank (suspension tank). Octanoic acid was added to the diluted plasma in the suspension tank over a period of 20-40 minutes in the amounts of 0.50 g OA / g protein, 0.75 g OA / g protein, and 1.00 g OA / g protein with vigorous mixing to obtain plasma / octanoic acid emulsions.

[0200] The resulting suspension was then fed to a continuous extraction filter unit, from which the retentate was recycled back into the suspension tank, and from which the filtrate (containing the immunoglobulins) was fed into a second tank. The filtrate collected in the second tank was then fed into a second unit containing an ultrafiltration and diafiltration system. The retentate of the UF / DF system was run back into the second tank, while the filtrate was run back into the first tank.

[0201] The transmembrane pressure (TMP) is adjusted to ensure that the permeate (filtrate) flow from the second unit is equal to the filtrate flow from the first unit, ensuring a constant volume in the first tank during the extraction process.

[0202] The filtration unit is stopped when the protein concentration in the filtrate falls below a defined threshold, thereby reaching a final dilution ratio > 1:X.

[0203] The dilution ratio (1:X) can be variable depending on the initial protein of the plasma pool, the amount of OA used, and the desired protein concentration threshold concentration that the remaining OA suspension reaches. The approximate average final dilution ratio is ≧1:20. In certain embodiments, the final dilution ratio was ≧1:12 and ≧1:15, or higher (e.g., 1:≧30).

[0204] The concentration step is complete when the protein concentration reaches about 25 to about 30 g / L (i.e., UF retentate). During this final concentration, the permeate is flushed away and discarded.

[0205] Diafiltration is then initiated. The concentrated protein solution is then diafiltered against 10X volume with WFI, and during diafiltration the pH is slowly lowered with 0.2 M hydrochloric acid (HCl) such that the pH is adjusted to 4.0±0.2 before the end of diafiltration.

[0206] The diafiltered solution was diluted to a protein concentration of 20±2 g / L and the pH was adjusted to about pH 4.0±0.2. This solution was subjected to further clarification depth filtration. The resulting filtrate (referred to herein as the "clarified filtrate") was further evaluated to determine various product attributes as further described below in Example 6.

[0207] Example 6: Continuous filtration 2.6 L cryo-rich plasma was diluted with 1.26 liters of 0.2 M acetic acid to a pH of 4.8 and a conductivity of 7.5 mS / cm.

[0208] Octanoic acid (0.447 g per g protein) was added slowly over a period of 60 minutes with vigorous stirring to form an octanoic acid suspension with a pH of 4.76 and a conductivity of 7.72. The octanoic acid suspension was incubated at 20° C. for 3.25 hours and then transferred to the feed tank of the continuous extraction system of Example 1. The continuous extraction system was treated with 100 mM sodium acetate buffer (pH 4.8) to replenish, as was the backflash tank.

[0209] The octanoic acid suspension was recirculated for a period of 15 minutes without filtration, then filtration was started. The backflush time was 15 seconds and the filtration time was 5 minutes. After about 5 minutes, the TFF system (an exemplary system shown in FIG. 16) was started and recirculation was carried out as above for about 4 hours.

[0210] When the protein concentration in the feed tank reached 0.2–0.5 g / L, the permeate from the TFF system was poured off and discarded.

[0211] The solution was diluted to a protein concentration of 20 g / L and the pH was adjusted to about 4.0 in the presence of polysorbate 80 (P80). The solution was subjected to further clarification depth filtration.

[0212] 50 mg DEAE A-50 per g protein was added to the clarified filtrate solution, the pH was adjusted to 5.8 using Tris base, and the solution was stirred for 60 minutes, after which the protein solution was loaded onto a strong anion exchanger. The flow-through was collected and the pH was adjusted to 4.8 using 0.2 M HCl. The resulting flow-through was further evaluated to determine various product attributes as further described below.

[0213] [Table 11]

[0214] All product related impurities are below the limit of quantification (see Figure 14).

[0215] Example 7: Further experiments investigating parameters In a series of experiments (referred to as Examples 9-15 below), several parameters were investigated that have an effect on the formation of insoluble albumin-octanoic acid complexes. These parameters include: pH, ionic strength, OA concentration, dilution factor, and total recirculation volume (final dilution ratio). These parameters result in a more efficient separation of soluble immunoglobulins from insoluble albumin-octanoic acid complexes.

[0216] The following table (Table 12) contains the experimental test conditions.

[0217] [Table 12]

[0218] The results show very good agreement with the laboratory scale experiments. Table 13 shows the IgG yield and the major impurities and residual albumin content in the clarified concentrated OA filtrate.

[0219] The data show consistent IgG yields with an average of 88.6% (range: 84.2-93.0%). The average residual albumin in the clarified solutions is less than 0.2 g albumin per liter of plasma, except for Example 15. This is expected due to the higher IgM and lower OA concentrations in the plasma pool.

[0220] [Table 13]

[0221] After recovery of the soluble protein-containing components (immunoglobulin G and others, such as IgA and IgM), any remaining suspension and / or the first retentate can be further processed to obtain purified albumin.

[0222] Example 8: Further processing of albumin The remaining suspension in the first retentate tank contains insoluble albumin-OA complexes. The remaining suspension contains less than 0.00035 g / L IgG, less than 0.0002 g / L IgA, and 0.0002 g / L IgM.

[0223] The pH of the remaining suspension was adjusted to 6.4-6.7, preferably 6.8-7.2, using 1 M sodium hydroxide to disrupt the bonds between albumin and OA. After mixing, the pH of the solubilized albumin was stabilized (30-60 min). This solution was fed into a continuous extraction system to generate an albumin-depleted retentate and an albumin-enriched filtrate. Using a TFF membrane (an exemplary system is shown in FIG. 16), the filtrate was continuously concentrated to 20-45 g protein per L.

[0224] The concentrated albumin solution was then heated at a temperature in the range of 60-65° C. for a period of 90 minutes.

[0225] The pH of the solution was adjusted to 4.20 using 1 M hydrochloric acid and then the concentrated albumin solution was cooled to 4° C. A precipitate was formed which dissolved at the above mentioned pH during the disruption of the albumin-OA complex.

[0226] The precipitate was removed by filtration. The protein in the filtrate consisted mainly of albumin (>98% purity with 90% albumin yield). Table 14 and Figure 15 show the impurity profile of heat treated albumin.

[0227] [Table 14]

[0228] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of which different combinations constitute various alternative aspects of the invention.

Claims

**Claim 1** - contacting a sample of blood-derived plasma with a medium-chain fatty acid under conditions that permit selective precipitation of albumin from the sample, - [wherein the conditions include a pH range between about 4.6 and about 5.0]; - thereby forming a solution of immunoglobulins A method for obtaining a solution of immunoglobulins, preferably immunoglobulin G (IgG), comprising.