Composition and preparation method of plasma products with reduced levels of FcεRIβ autoantibodies

Abstract

A method for preparing therapeutic blood products from pooled plasma donors is provided. The therapeutic blood products include, for example, plasma, plasma proteins, and plasma fractions, in which safety is improved by reducing or eliminating the concentration of donor autoantibodies against FcεRIβ.

Description

Technical Field

[0001] The present invention relates to the preparation of therapeutic blood products pooled from multiple donors and an improved method for their preparation to ultimately reduce or eliminate certain hypersensitivity adverse events. The present invention relates to the improvement of the safe use of blood products such as plasma, plasma proteins, and plasma fractions that are prepared without containing autoantibodies against the high-affinity immunoglobulin epsilon receptor subunit beta (FcεRIβ) or with a significantly reduced concentration thereof.

Background Art

[0002] Overview Preparations derived from pooled human plasma, such as plasma fractions or plasma proteins, are generally well tolerated when administered to patients. However, in patients administered these preparations, hypersensitivity reactions may occur periodically in groups. These group occurrences of hypersensitivity are related to specific product lots and have led to the voluntary recall of these products from the market, such as immune globulin preparations (see, for example, fda.gov / vaccines-blood-biologics / safety-availability-biologics / voluntary-lot-withdrawals-immune-globulin-intravenous-igiv-and-immune-globulin-subcutaneous-igsc, as of March 4, 2022). The symptoms of patients who have experienced hypersensitivity events range from urticaria, itching, and facial swelling to anaphylactic-like reactions.

[0003] Autoantibodies against the human antigen FcεRIβ have been shown to be usable as markers for screening individual plasma units, donor plasma pools, or plasma-based preparations such as immunoglobulins to prevent the occurrence of these hypersensitivity reactions. Furthermore, their presence or the presence of autoantibodies can be used to prepare plasma-based preparations such as plasma fractions or plasma proteins with a significantly reduced risk of hypersensitivity adverse reactions. FcεRIβ is part of the FcεRI receptor complex found in human mast cells and basophils. FcεRIβ is involved in cell activation resulting from IgE-mediated allergic reactions. The association between FcεRIβ and IgE-mediated allergic reactions presented here is surprising because the FcεRIα segment of the FcεRI receptor complex is the site of IgE binding, and FcεRIβ has been shown to be involved only downstream of receptor binding.

[0004] Inclusion of references All publications and patent applications referenced herein are incorporated herein by reference to the same extent as any individual publication or patent application is specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawing]

[0005] [Figure 1] Figure 1 shows that the results for donor 18 significantly outperformed the buffer control and the other 17 samples for basophil sources of all five reagents. [Figure 2] Figure 2 shows that the BAT activation signal was reduced to background levels at a dilution ratio of 1:100. [Figure 3] Figure 3 shows that BAT responsiveness was low in donor 18's blood donations during the first year. [Figure 4] Figure 4 shows hypersensitivity adverse events as a function of BAT activation. [Figure 5] Figure 5 shows the BAT reactivity of IgG derived from donor 18. [Figure 6]Figure 6 shows that basophil activation by donor 18 plasma is similar whether whole blood or purified basophils are used. [Figure 7] Figure 7 shows that basophil activation, as measured by CD203c, is dose-dependently reduced by Umbrilisb. [Figure 8] Figure 8 shows basophil activation by plasma tracking of donor 18 under anti-FcεRI / anti-IgG control. [Figure 9] Figure 9 shows that the receptor did not show a response above baseline. [Figure 10] Figure 10 shows the degree of FcεRIβ signaling compared to plasma from 17 other donors and 3 control samples. [Modes for carrying out the invention]

[0006] Detailed description of the invention A. Introduction Mast cells found in tissues and basophils found in the blood are cells responsible for allergic reactions. When these cells are activated, they release mediators such as tryptase, histamine, prostaglandins, leukotrienes (LTs), and cytokines, which manifest as allergic reactions. These reactions include a variety of symptoms, from hives, itching, and facial swelling to anaphylaxis.

[0007] The primary activation mechanism of an allergic reaction begins with the binding of allergens to IgE present on FcεRIα receptors on mast cells and basophils. When an allergen binds to multiple IgE receptors simultaneously, crosslinking of FcεRI receptors occurs, initiating a signaling cascade. This results in a maximum degranulation of cells and the release of histamine and other mediators. It is well known that autoantibodies against IgE or FcεRIα are present in some individuals and can trigger crosslinking and subsequent activation of mast cells and basophils even without the interaction of specific allergens with IgE.

[0008] Individuals with autoantibodies against IgE or FcεRIα often develop chronic urticaria. The presence of autoantibodies against the FcεRIβ subunit of the FcεRI complex has not been previously reported. The FcεRIβ subunit is encoded by the MS4A2 (membrane spanning four domains, A2) gene and is a transmembrane protein. While the FcεRIβ subunit is known to be involved in mast cell and basophil signaling downstream of allergen crosslinking of the FcεRI receptor, it was not known to provide a pathway for activation itself. Furthermore, activation may be sufficient for the presence of autoantibodies against FcεRIβ from even a single donor to cause hypersensitivity adverse events in patients receiving immunoglobulin preparations produced from plasma pools of many other donors. Testing to detect donor FcεRIβ in relation to autoantibodies allows for plasma screening, thus avoiding hypersensitivity adverse events in patients. Furthermore, by measuring the amount of FcεRIβ or FcεRIβ autoantibodies in the provided plasma, improved formulations of pooled plasma, plasma fractions, or plasma proteins (e.g., intravenous IgG [IGIV] or other IgG preparations) can be manufactured.

[0009] B. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the invention, but some possible preferred methods and materials are described below. All publications referenced herein are incorporated herein by reference to disclose and describe methods and / or materials relating to the methods and / or materials related to which those publications are cited. In the event of any conflict, it should be understood that this disclosure takes precedence over the disclosure of the incorporated publication.

[0010] When used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context indicates otherwise. For example, “cell” includes multiple such cells, and “peptide” includes one or more peptides and their equivalents, such as polypeptides known to those skilled in the art.

[0011] When describing the methods of the present invention, the terms “host,” “subject,” “individual,” and “patient” are used interchangeably and refer to any mammal that requires such treatment according to the disclosed methods. Such mammals include, for example, humans, sheep, cattle, horses, pigs, dogs, cats, non-human primates, mice, and rats. In certain embodiments, the subject is a non-human mammal. In some embodiments, the subject is a farm animal. In other embodiments, the subject is a pet. In some embodiments, the subject is a mammal. In certain examples, the subject is a human. Other subjects may include domestic pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., disease animal models, etc.), and non-human primates (e.g., chimpanzees and monkeys). Thus, the subjects of the present invention include, but are not limited to, mammals, e.g., humans and other primates, e.g., chimpanzees, other apes and monkey species, and in certain embodiments, the subject is a human. Furthermore, the term "subject" means that it includes any person or organism of any age, weight, or other physical characteristics, and the subject may be an adult, a child, an infant, or a newborn.

[0012] As used herein, “treatment” means either (i) prevention of a disease or disorder, or (ii) reduction or elimination of the symptoms of a disease or disorder. Treatment may be prophylactic (before the onset of the disease) or therapeutic (after the onset of the disease). Its effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or partially or completely cures the disease and / or adverse effects caused by the disease. Accordingly, as used herein, the term “treatment” encompasses all treatments of a disease or other pathological condition, including (a) preventing the onset of the condition in a subject; (b) suppressing the condition, i.e., preventing its onset; or (c) alleviating the condition, i.e., causing regression of the condition. Treatments may produce a variety of different physical effects, such as, for example, regulation of gene expression, rejuvenation of tissue or organ, or reduction of inflammation. Therapeutic agents may be administered before, during, or after the onset of a pathological condition. Targeted therapies may be administered during the symptomatic stage of the condition, and in some cases, after the symptomatic stage.

[0013] Blood products containing plasma components"Blood products containing plasma components" means any blood-derived product containing plasma (e.g., whole blood, plasma or fractions thereof). The term "plasma" is used in its traditional sense to refer to the straw-yellow / pale-yellow liquid component of blood, which consists of approximately 92% water, 7% proteins such as albumin, gamma globulin, antihemophilic factors, and other coagulation factors, and 1% mineral salts, sugars, fats, hormones, and vitamins. Non-limiting examples of plasma-containing blood products suitable for use in the methods of the subject include whole blood treated with anticoagulants (e.g., EDTA, citrate, oxalate, heparin, etc.), blood products produced by filtering whole blood to remove leukocytes ("leukocyte-removed"), plasma obtained by plasmapheresis, blood products consisting of plasma obtained by apheresis, blood products consisting of fresh frozen plasma, blood products consisting of essentially purified plasma, and blood products consisting of essentially plasma fractions. In some cases, the plasma product used is a non-whole blood plasma product, meaning that the product is not whole blood, and is lacking, for example, one or more components found in whole blood, such as red blood cells and white blood cells, to the extent that these components are present in whole blood. In some cases, the plasma product is substantially, if not completely, cell-free, in which case the cell content may be 5% by volume or less, for example, 1% or less, 0.5% or less, and in some cases, the cell-free plasma fraction is a composition that is completely devoid of cells, i.e., cell-free.

[0014] Collection of blood products containing plasma components Embodiments of the methods described herein involve the use or preparation of blood products containing plasma components that may be derived from donors, including human volunteers. The term “human-derived” may refer to such products. Methods for collecting plasma containing donor-derived blood products are well known in the art (see, for example, the AABB Technical Manual, (Mark A. Fung, et al., eds., 18th ed. 2014), incorporated herein by reference).

[0015] In one embodiment, the offering is obtained by venipuncture. In another embodiment, venipuncture is performed only once. In another embodiment, volume replacement with physiological saline is not employed. In a preferred embodiment, a plasmapheresis step is used to obtain a blood product containing plasma components. Plasmapheresis may involve returning cellular components to the donor and removing a weight-adjusted amount of plasma. In a preferred embodiment, sodium citrate is used during plasmapheresis to prevent cellular coagulation. The amount of plasma collected from the donor is preferably between 690 and 880 mL after citrate administration, and preferably matched to the donor's body weight. In another embodiment, the acquisition of a blood product containing plasma components is performed by total plasma exchange.

[0016] C. Plasma fraction During World War II, there was a need for a stable plasma expander that could be used on the battlefield where soldiers lost large amounts of blood. As a result, methods for preparing lyophilized plasma were developed. However, the use of lyophilized plasma in combat situations was difficult because it required sterile water for reconstitution. As an alternative, Dr. E.J. Cohn proposed using albumin to prepare a readily usable, stable solution, which could be quickly introduced for treating shock (see Johan, Current Approaches to the Preparation of Plasma Fractions in (Biotechnology of Blood) 165 (Jack Goldstein ed., 1st ed. 1991)). Dr. Cohn's method for purifying plasma fractions utilized denaturing cold ethanol and achieved separation by changing the pH and temperature.

[0017] One embodiment of the method described herein involves administering a plasma fraction to a subject. Fractionation is a method for separating a specific protein subset from plasma. Fractionation techniques are known in the art and are based on a process developed by Cohn et al. in the 1940s (see E. Cohn, Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. 68 J Am Chem Soc 459 (1946), incorporated herein by reference). This method involves several steps, each involving a specific ethanol concentration, as well as shifts in pH, temperature, and osmotic pressure, resulting in selective protein precipitation. The precipitate is also separated by centrifugation or sedimentation. The original “Cohn fractionation method” involved separating proteins via precipitate into five fractions called fraction I, fraction II+III, fraction IV-1, fraction IV-4, and fraction V. Albumin was initially identified as the endpoint (fraction V) product of this method.According to embodiments of the present invention, each fraction, filtrate (or sometimes referred to as effluent or wastewater from a previous separation step) contains, or may contain, therapeutically useful protein fractions (see, incorporated herein by reference: Thierry Burnouf, Modern Plasma Fractionation, 21(2) Transfusion Medicine Reviews 101 (2007); Adil Denizli, Plasma fractionation: conventional and chromatographic methods for albumin purification, 4 J. Biol. & Chem. 315, (2011); Gjessing EC, et al., J. Biol. & Chem. (174):682-96 (1948); and T. Brodniewicz-Proba, Human Plasma Fractionation and the Impact of New Technologies on the Use and Quality of Plasma-derived Products, 5 Blood Reviews 245). (1991), and see U.S. Patents No. 3,869,431, 5110,907, 5219,995, 7531,513, and 8772,461). The above experimental parameters can be adjusted to obtain a specific protein fraction.

[0018] More recently, fractionation has become even more complex. This recent increase in complexity has occurred due to the introduction of chromatography, which has enabled the separation of new proteins from existing fractions such as cryoprecipitate, cryo-poor plasma, and Cohn fractions; the improvement in IgG recovery by integrating chromatography and ethanol fractionation methods; and the ability to reduce / inactivate / remove viruses (ibid.). Anion exchange chromatography can be used to capture proteins at physiological pH and ionic strength. This maintains the functional activity of the protein and / or protein fraction. Heparin and monoclonal antibodies are also used in affinity chromatography. Additionally, fractionation using gel filtration, fractionation by salts, and fractionation by polyethylene glycol are also used (Hosseini M Iran J Biotech, 14(4): 213-20 (2016), which is incorporated herein by reference). One skilled in the art will recognize that the above parameters and techniques can be adjusted to specifically obtain the desired plasma protein-containing fraction.

[0019] Plasma fractionation can also be based on ammonium sulfate (see, for example, Odunuga OO, Biochem Compounds, 1:3 (2013); Wingfield PT, Curr Protoc Protein Sci, Appx. 3 (2001), which are incorporated herein by reference). In addition to obtaining specific blood fractions, ammonium sulfate-based fractionation has been employed to reduce abundant proteins from plasma (Saha S, et al., J. Proteomics Bioinform, 5(8) (2012), which is incorporated herein by reference).

[0020] In one embodiment of the present invention, plasma is fractionated in an industrial environment. Frozen plasma is thawed at 1°C to 4°C. Continuous cold centrifugation is applied to the thawed plasma to isolate cold precipitates. The recovered cold precipitates are cryopreserved at -30°C or lower. Cryoprecipitate-poor (“cryo-poor”) plasma is immediately processed (e.g., by primary chromatography) for the capture of labile coagulation factors such as factor IX complex and its components, as well as protease inhibitors such as antithrombin and C1 esterase inhibitor. In subsequent steps, continuous centrifugation and separation of precipitates can be performed. Such techniques are known to those skilled in the art and are described, for example, in U.S. Patent Nos. 4,624,780, 5,219,995, 5,288,853, and U.S. Patent Application Nos. 2014 / 0343255 and 2015 / 0343025, the disclosures of which are hereby incorporated by reference in their entirety.

[0021] In one embodiment of the present invention, the plasma fraction may include a plasma fraction containing substantial concentrations of albumin. In another embodiment of the present invention, the plasma fraction may include a plasma fraction containing substantial concentrations of IgG or intravenous immunoglobulin (IGIV) (e.g., Gamunex-C®). In another embodiment of the present invention, the plasma fraction may include an IGIV plasma fraction such as Gamunex-C® from which immunoglobulin (IgG) has been substantially depleted by a method well known to those skilled in the art, such as protein A-mediated depletion (see, for example, Keshishian, H., et al., Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields Novel Candidates for Early Myocardial Injury, Molecular & Cellular Proteomics, 14 at 2375-93 (2015)). In an additional embodiment, the plasma fraction may be depleted of substantially all coagulation factors to reduce the risk of thrombosis and maintain the efficacy of the fraction. For example, the plasma fraction may be a plasma fraction such as that described in U.S. Patent No. 62 / 376,529 filed on August 18, 2016, the disclosure thereof is incorporated herein by reference in its entirety.

[0022] D. Coagulation factor removal fraction In another embodiment of the present invention, a plasma fraction from which substantially all coagulation factors have been removed is used to reduce the risk of thrombosis and maintain the efficacy of the fraction. Conveniently, the blood product is derived from a young donor or a pool of young donors, and IgM can be removed to provide an ABO-matched young blood product. Currently, transfused plasma is ABO-matched because the presence of naturally occurring antibodies against A and B antigens can cause transfusion reactions. IgM appears to be involved in transfusion reactions when a patient is administered plasma that is not ABO-matched. Removing IgM from a blood product or fraction helps eliminate transfusion reactions in the recipient of the blood product and plasma fraction of the present invention.

[0023] E. Protein-enriched plasma protein preparation treatment Additional embodiments of the present invention include plasma protein fractions (PPF), human albumin solution (HAS), effluent I, and specific fractions such as effluents II / III, effluent IV-1, effluent IV-4, and effluent V, all of which are substantially free of coagulation factors. Hereinafter, such plasma fractions will be referred to as "protein-enriched plasma protein preparations." For example, in one embodiment of the present invention, a protein-enriched plasma protein preparation consisting of 82% albumin and 18% α, β, and γ globulin and other plasma proteins can be used. In another embodiment of the present invention, a protein-enriched plasma protein preparation consisting of 81% albumin and 19% α, β, and γ globulin and / or other plasma proteins can be used. In yet another embodiment of the present invention, a protein-enriched plasma protein preparation consisting of 80% albumin and 20% α, β, and γ globulin and / or other plasma proteins can be used. In further embodiments of the present invention, a protein-enriched plasma protein preparation comprising 70-79% albumin and corresponding 21-30% α, β, and γ globulins and other plasma proteins can be used. In further embodiments of the present invention, a protein-enriched plasma protein preparation comprising 60-69% albumin and corresponding 31-40% α, β, and γ globulins and other plasma proteins can be used. In further embodiments of the present invention, a protein-enriched plasma protein preparation comprising 50-59% albumin and corresponding 41-50% α, β, and γ globulins and other plasma proteins can be used. In further embodiments of the present invention, a protein-enriched plasma protein preparation comprising 40-49% albumin and corresponding 51-60% α, β, and γ globulins and other plasma proteins can be used. In further embodiments of the present invention, a protein-enriched plasma protein preparation comprising 30-39% albumin and corresponding 61-70% α, β, and γ globulins and other plasma proteins can be used.In further embodiments of the present invention, a protein-enriched plasma protein preparation comprising 20-29% albumin and corresponding 71-80% α, β, and γ globulins and other plasma proteins can be used. In further embodiments of the present invention, a protein-enriched plasma protein preparation comprising 10-19% albumin and corresponding 81-90% α, β, and γ globulins and other plasma proteins can be used. In further embodiments of the present invention, a protein-enriched plasma protein preparation comprising 1-9% albumin and corresponding 91-99% α, β, and γ globulins and other plasma proteins can be used. In further embodiments of the present invention, a protein-enriched plasma protein preparation comprising 0% albumin and 100% α, β, and γ globulins and other plasma proteins can be used. The above embodiments of the present invention may also have a total γ globulin concentration of 1-5%.

[0024] "Plasma protein" or "plasma protein preparation" refers to concentrated protein derived from plasma fractionation or other related technologies described above. Embodiments of the present invention include the preparation of these preparations with reduced potential for hypersensitivity adverse events. Examples of such preparations, but not limited to, include intravenous IgG or plasma-derived IgG, prolastin-C (α-1 antitrypsin), albumin, and other preparations of antithrombin III.

[0025] The specific concentration of proteins in plasma fractions can be determined using techniques well known to those skilled in the art. Examples of such techniques include, but are not limited to, electrophoresis, mass spectrometry, ELISA analysis, and Western blotting.

[0026] F. Preparation of plasma fractions Methods for preparing PPF and other plasma fractions are well known to those skilled in the art. In embodiments of the present invention, blood to be used for the preparation of the human plasma protein fraction is collected in a flask to which citrate or the anticoagulant glucose citrate solution (or other anticoagulant) is added for coagulation inhibition, and fractions I, II+III, IV, and PPF can be further separated according to the method disclosed by Hink et al. (see Hink, JH, Jr., et al., Preparation and Properties of a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2(174) (1957), incorporated herein by reference). According to this method, the mixture can be recovered at 2-8°C. The plasma can then be separated by centrifugation at 7°C, removed, and stored at -20°C. The plasma can then preferably be removed from -20°C storage and thawed at 37°C within 8 hours to be fractionated.

[0027] Plasma can be separated from fraction I using 8% ethanol at a pH of 7.2, a temperature of -2 to -2.5°C, and a protein concentration of 5.1 to 5.6%. Cold 53.3% ethanol (176 mL / L plasma) and acetate buffer (200 mL of 4M sodium acetate and 230 mL of glacial acetic acid saturated with H2O to 1 L) can be added using a jet at a rate of, for example, 450 mL / min while lowering the plasma temperature to -2°C. Fraction I can be separated from the effluent (effluent I) and removed by ultracentrifugation. Fibrinogen can be obtained from fraction I according to methods well known to those skilled in the art.

[0028] Fractions II and III can be separated from effluent I by adjusting the effluent to 21% ethanol at pH 6.8, temperature -6°C, and protein concentration 4.3%. While the temperature of effluent I is lowered to -6°C, cold 95% ethanol (176 mL / L of effluent I) containing 10 M acetic acid used for pH adjustment can be added using a jet at a rate of, for example, 500 mL / min. The resulting precipitate (fractions II and III) can be removed by centrifugation at -6°C. Gamma globulin can be obtained from fractions II and III using methods well known to those skilled in the art.

[0029] Fraction IV-1 can be separated from effluent II+III ("effluent II / III") by adjusting the effluent to 19% ethanol at pH 5.2, temperature -6°C, and protein concentration 3%. H2O and 10M acetic acid used for pH adjustment can be added using a jet while maintaining effluent II / III at -6°C for 6 hours. The precipitated fraction IV-1 can be separated from the effluent by standing at -6°C for 6 hours and then centrifugation at the same temperature. A stable plasma protein fraction can be recovered from effluent IV-1 by adjusting the ethanol concentration to 30% at pH 4.65, temperature -7°C, and protein concentration 2.5%. This can be achieved by adjusting the pH of effluent IV-1 with cold acid-alcohol (2 parts 2M acetic acid and 1 part 95% ethanol). Add 170 mL of cold ethanol (95%) per liter of adjusted waste IV-1 while maintaining the temperature at -7°C. The precipitated protein is allowed to stand for 36 hours and then removed by centrifugation at -7°C. The paste / precipitate of fraction IV-4 can also be obtained using the Cohn fractionation method and can be resuspended. In fact, fraction IV-4 and its preparation method have been previously described (Schopfer LM, et al., PLoS ONE, 14(1):e0209795 (2018) is incorporated herein by reference in its entirety) (Schopfer LM, et al., PLoS ONE, 14(1):e0209795 (2018) is incorporated herein by reference in its entirety, and Bertolini J, Goss N, Curlin J eds., PRODUCTION OF PLASMA PROTEINS FOR THERAPEUTIC USE, 16.4: 231:232 (2013)).

[0030] The recovered protein (stable plasma protein fraction) can be dried to remove alcohol and H2O (e.g., by freeze-drying). The resulting dried powder can be dissolved in sterile distilled water using, for example, 15 liters of water per 1 kg of powder, and the solution can be adjusted to pH 7.0 with 1 M NaOH. By adding sterile distilled water containing sodium acetyltryptophanate, sodium caprylate, and NaCl, the final concentration can be adjusted to 0.004 M acetyltryptophanate, 0.004 M caprylate, and 0.112 M sodium, a final concentration of 5% protein can be obtained. Finally, this solution can be filtered at 10°C to obtain a clear solution, which can then be heat-treated at 60°C for at least 10 hours to inactivate the pathogen.

[0031] The methods described above for preparing plasma fractions and plasma protein fractions (PPFs) are illustrative and represent only a limited number of embodiments of the present invention. Those skilled in the art will recognize that these methods are varied. For example, pH, temperature, and ethanol concentration, among other things, can be adjusted to produce different forms of the plasma fractions and plasma protein fractions in different embodiments and methods of the present invention. In another example, further embodiments of the present invention intend to use nanofiltration for the removal / inactivation of pathogens from the plasma fractions and plasma protein fractions.

[0032] G. Preparation method Embodiments of the present invention include methods for preparing plasma preparations in which the likelihood or incidence of hypersensitivity reactions is reduced. Further embodiments include, but are not limited to, the preparation of plasma preparations such as IgG (intravenous administration), albumin, antithrombin III, and prolastin-C (α-1 antitrypsin). Further embodiments include plasma preparations such as whole blood or plasma from a single donor, or plasma preparations pooled from multiple donors. Further embodiments include plasma preparations derived from a single donor center or geographical area.

[0033] Further embodiments of the present invention include obtaining plasma from multiple donors for the purpose of creating pooled plasma from multiple donors. In another embodiment, the plasma from each donor is tested for the presence of a predetermined concentration of autoantibodies against FcεRIβ receptors or for the amount of FcεRIβ receptors themselves to determine whether the donor is "positive" for the risk of causing hypersensitivity adverse events. The testing of the plasma from each donor may also be in the form of detecting autoantibodies against FcεRIβ receptors or detecting FcεRIβ receptors themselves, thereby identifying positive donors. Embodiments of the present invention include preventing the plasma of positive donors from being pooled with the plasma of other donors.

[0034] Further embodiments of the present invention include testing a pool of plasma from multiple donors. Further embodiments determine or detect the concentration of autoantibodies against the FcεRIβ receptor in a pool of donors if a predetermined concentration of autoantibodies in the plasma from one donor having autoantibodies against the FcεRIβ receptor is sufficient to cause a hypersensitivity adverse reaction in the pooled plasma. In another embodiment, the use of such "contaminated" pooled plasma is excluded from use in the preparation of plasma products or proteins.

[0035] Another embodiment includes subsequently determining whether the degree of dilution of one donor in the pooled blood or plasma of other donors is sufficient to detect the risk of hypersensitivity adverse events. Another embodiment of the present invention involves dilution degrees ranging from 1:1000 to 1:10,000. A further embodiment of the present invention excludes the entire plasma pool from use in patients.

[0036] A further embodiment of the present invention involves pooling the plasma of donors whose plasma was not excluded from the pool. Plasma preparations or proteins can then be prepared from these pools.

[0037] Other embodiments of the present invention may include the identification and use of antigens other than FcεRIβ receptors, such as FcεRIα receptors, to determine whether a particular donor or donor pool should be excluded. This allows for the preparation of plasma preparations or proteins with a reduced risk of causing hypersensitivity adverse reactions.

[0038] Further embodiments of the present invention include examining the donor's plasma using a basophil activation test (BAT) or a histamine release assay (HRA) against the donor's basophils or mast cells. The level of BAT or HRA compared to other donors is used to determine whether there is an unacceptable risk of hypersensitivity adverse reactions.

[0039] Techniques for identifying autoantibodies against antigens have been previously described. Chemiluminescence techniques have been used to identify autoantibodies in the diagnosis of various autoimmune diseases (Maler M et al., Immunopharmacology and Immunotoxicology, 38:1(14-20) (2015)). Autoantibodies in the plasma of cancer patients have been identified using microtiter arrays of antigens (Bassaro L et al., Cancer Genomics & Proteomics, 14(427-35) (2017)). Multiplex chip-based assays have been used to detect autoantibodies in the serum of rheumatoid arthritis patients (Hansson M et al., Arthritis Res & Therapy, 14:R201 (2012)). IgE autoantibodies associated with hypersensitivity reactions have also been described (Badloe et al., Clin and Translational Allergy, 10(34) (2020)).

[0040] H. Reagents, instruments, and kits Furthermore, reagents, apparatus, and kits for carrying out one or more of the above methods are also provided. The reagents, apparatus, and kits may vary.

[0041] The reagents and equipment covered include those described above regarding the preparation method of plasma products.

[0042] The kit may also include blood collection bags, tubes, needles, centrifuge tubes, etc. In yet another embodiment, the kit as described herein includes two or more containers of plasma products, such as plasma protein fractions, e.g., three or more, four or more, five or more, e.g., six or more containers of plasma products. In some examples, the number of separate containers of plasma products in the kit may be nine or more, twelve or more, fifteen or more, eighteen or more, twenty or more, twenty or more, or thirty or more, e.g., thirty or more, e.g., four or more. Each container may have associated identification information that includes various data about the plasma product contained therein, which may include one or more of the age of the plasma product donor, details of the processing of the plasma product, e.g., whether the plasma product has been processed to remove proteins of average molecular weight or higher (e.g., as described above), details of the blood type, etc. In some examples, each container in the kit contains identification information about the plasma it contains, and this identification information includes information about the donor center of the plasma product, for example, the identification information provides confirmation data originating from the donor center of the plasma product to help test and / or identify a potential donor or plasma lot for the risk of hypersensitivity adverse events due to the presence of FcεRIβ receptor or its autoantibodies. The identification information can be printed on any convenient component of the container, such as a label or RFID chip. The identification information may be human-readable or computer-readable, as desired. The containers may have any convenient configuration. The volume of the containers may vary, but in some examples, the volume is in the range of 10 mL to 5000 mL, e.g., 25 mL to 2500 mL, e.g., 50 mL to 1000 mL, e.g., 100 mL to 500 mL. The containers may be rigid or flexible and can be made from any convenient material, e.g., polymer materials including medical-grade plastic materials. In some examples, the containers are in the form of a bag or pouch. In addition to the container, such a kit may further include, for example, a dispensing device as described above. The components of such a kit may be provided in any suitable packaging, such as a box or similar structure, configured to hold the container and other kit components.

[0043] In addition to the components described above, the kit further includes instructions for carrying out the method. These instructions may be present in the kit in various forms, and one or more of these forms may be present in the kit. One possible form in which these instructions may be present is as information printed on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, the kit's packaging, or packaging inserts. Another possible means is a computer-readable medium on which the information is recorded, such as a diskette, CD, or portable flash drive. Yet another possible means is a website address that can be used to access the information at an isolated site via the internet. Any convenient means may be present in the kit. [Examples]

[0044] I. Experimental Examples 1. Example 1 - Manufacturing and Quality Investigation An increased rate of hypersensitivity adverse events (Hypertension, Itching, and Rash (Groin, Chest, Legs, and other areas)) was observed in some lots of the IgG plasma protein intravenous formulation. Further adverse events included dyspnea, lip swelling, bronchospasm, and laryngeal edema. These symptoms were consistent with a mast cell response and resolved rapidly with antihistamine treatment. When such hypersensitivity reactions are observed, lots of the formulation are typically recalled from the market, often from multiple manufacturers. In this case, a significant number were recalled.

[0045] Several possible causes of the increased hypersensitivity reactions were investigated. No significant manufacturing deviations were found in the recalled lots, and no inconsistencies were found after extensive verification of raw materials, manufacturing processes, and in-process and final container inspections. Quality control inspections met specifications, and no significant differences were found between the recalled lots and comparison lots.

[0046] No significant differences were found between the retrieved lot and the comparison lot during testing. The tests included those shown in Table 1.

[0047] [Table 1]

[0048] 2. Example 2 - Donor Investigation An investigation was conducted into the association of lots recalled from the market showing an increase in hypersensitivity adverse events with a common donor center. The results revealed that these lots originated from a single donor center. A thorough review of the center's processes, operations, products, and quality systems was conducted. No identifiable issues related to or contributing to the hypersensitivity adverse events were found.

[0049] Subsequently, a common donor analysis was conducted at this center. As additional lots were identified and recovered, the analysis progressed, and it was found that 18 donors with a high rate of hypersensitivity reactions could be identified in all recovered lots. These donors withdrew from further plasma donation, and their plasma was analyzed. After the plasma of these 18 donors was no longer included in any lot, no hypersensitivity reports from any lot from this common donor center were reported.

[0050] The donation history and medical information of 18 general donors were analyzed, but nothing noteworthy was found. All had extensive donation histories and no significant medication history. There was nothing noteworthy in their medical history itself. Some had general allergies, but nothing noteworthy. Electrophoretic serum protein profiles were all normal. No anti-Gal (IgE or IgG) was detected when samples were submitted for diagnostic testing. Some donors showed elevated IgE levels, indicating chronic allergies.

[0051] One of the 18 common donors ("Donor 18") tested positive for both the basophil activation assay (BAT) and histamine release assay (HRA). The plasma from the other 17 donors tested negative for both BAT and HRA.

[0052] 3. Example 3 - Characterization and in vitro methods applied to donor batch investigation (a) 2D-DIGE and LC-MS / MS Two-dimensional difference gel electrophoresis was performed on sets of three batches of normal plasma and three batches of hypersensitivity plasma, detecting approximately 600 proteins per spot. Advanced imaging software and statistical analysis were used to analyze the identified spots. No specific spots or intensities were detected in the hypersensitivity batches compared to the normal batches.

[0053] Proteins not bound to protein G (non-IgG proteins) were purified and trypsin-digested. These were then analyzed using a combination of liquid chromatography and tandem mass spectrometry (LC-MS / MS), with three batches of normal plasma and three batches of hypersensitivity plasma analyzed as a set. Proteins were obtained from sets of five batches of normal plasma and five batches of hypersensitivity plasma. Tandem MS enabled peptide identification through matching with databases (human and non-human). Approximately 30 impurity (non-IgG) proteins were detected in the samples, but there were no significant differences in these impurities between the recovery batches and the comparison batches.

[0054] (b) Characterization of aggregates in formulation batches Advanced characterization methods for aggregate and sub-visible particle analysis were performed in both standard and hypersensitivity batches. This was due to historical issues with aggregate-related adverse events in older formulations. These techniques allowed for the investigation of a wide range of molecular / particle sizes.

[0055] In size exclusion chromatography using multi-angle light scattering detection and batch-mode testing without chromatography, no batch differences were observed with the combination of the Dawn HELEOS II detector and OptiLab rEX refractometer. Similarly, no batch differences were observed in nanoparticle tracking analysis (NTA) using the Stokes-Einstein equation to calculate hydrodynamic diameter, and with the Malvern NanoSight LM10 instrument. No differences were observed between batch types in resonant mass measurement (RMM) using the Malvern-Archimedes instrument. Furthermore, image analysis of particles in the diameter range of approximately 1 μm to mm using microflow imaging (MFI) revealed no batch differences.

[0056] 4. Characteristics of autoantibodies involved in chronic urticaria Autoimmune anti-FcεRIα and anti-IgE have both been found to be involved in the causes of chronic urticaria and exhibit symptoms consistent with many hypersensitivity reactions observed in patients who discontinued intravenous IgG batch administration. Anti-FcεRIα and anti-IgE can be tested in the diagnostic laboratory or by commercially available enzyme-linked immunosorbent assay (ELISA), and both methods were used. Some anti-FcεRIα signals were observed in formulation lots, but there was no difference between hypersensitive and normal formulation lots, and no anti-IgE detectable in the normal batches was found in the hypersensitive batches.

[0057] 5. Functional methods for basophil activation (a) Reactivity of donor plasma pool and IgG final product Two in vitro assays, a histamine release assay (HRA) and a basophil activation assay (BAT), were performed to test basophil activation in plasma pools and IgG final product batches. The HRA was performed as follows: Fresh human whole blood was used as the source of basophils. Samples from the donor plasma pool or IgG final product batches were incubated with fresh whole blood samples in LDN-free buffer. Acetylation was used to protect released histamine, and histamine was detected by ELISA (Immunotech). Anti-IgE and N-formylmethionyl-leucyl-phenylalanine (fMLP) were used as positive controls for histamine release.

[0058] In the BAT (Batch Attack) study, fresh human whole blood was used as the source of basophils, which were incubated with samples from donor plasma pools or final IgG formulation batches. Cell surface expression of CD63 or CD203c on basophils was measured by flow cytometry using the Buhlmann Lab Flow CAST Basophil Activation Test. Anti-FcεRIα and N-formylmethionyl-leucyl-phenylalanine (fMLP) were used as positive controls.

[0059] Positive controls in both assays showed activation via independent activation pathways. Anti-FcεRIα and anti-IgE were associated with the FcεRI pathway, while fMLP was associated with the G protein-coupled receptor pathway. Ultimately, no signals were detected in either the plasma pool or the final IgG formulation batch in either assay.

[0060] (b) Responsiveness of 18 specific donors BAT and HRA were performed on 18 general donors (listed above) identified as having a high incidence of hypersensitivity reactions from the recovered lots. The plasma from these donors was mixed with basophils from five different healthy donors.

[0061] Figure 1 shows that the results for donor 18 significantly outperformed the buffer control and the other 17 samples for basophil sources in all five reagents. Due to this consistent response with BAT / HRA, the plasma of donor 18 was further characterized.

[0062] (c) Plasma dilution system of donor 18 by BAT A series of plasma dilutions were prepared from donor 18 at dilutions ranging from 1:0 (undiluted) to 1:2000 using buffer. These dilutions were incubated with fresh reagent blood (basophil source), and basophil activation (BAT) was examined using CD63 and CD203C.

[0063] Figure 2 shows that the BAT activation signal decreased to background levels at a dilution ratio of 1:100. Therefore, appropriate dilution ratios for detecting a response from a single donor in the pool or final formulation would be 1:1000 to 1:10,000.

[0064] (d) BAT time course study of plasma from donor 18 Plasma samples were retained from donor 18 from 22 plasma donations over a blood donation history of approximately 3.5 years. Figure 3 shows that BAT responsiveness was low in donor 18's first year of donations. However, activation dramatically increased around the time when adverse events of drug hypersensitivity began to increase. Following this dramatic increase, there was some variability in the activation signal.

[0065] (e) Relationship between BAT response and hypersensitivity events in donor 18 Figure 4 shows hypersensitivity adverse events as a function of BAT activation. Hypersensitivity adverse events were normalized to the relative quality of donor 18 plasma in the final formulation. Response fitting using four-parameter logistic fitting shows a latency period followed by a sharp increase in BAT. This is consistent with the threshold relationship, where hypersensitivity occurs when the threshold is reached.

[0066] (f) BAT reactivity of IgG from donor 18 Figure 5 shows the BAT reactivity of IgG derived from donor 18. Plasma from donor 18 and control plasma were fractionated using Capture Select IgG affinity resin. To confirm the composition of these fractions, the fractions were examined by SDS-PAGE, and then these fractions were examined by BAT using both CD63 and CD203c markers. SDS-PAGE confirmed the acquisition of IgG and IgG-depleted fractions, and basophil activation remained mainly in the IgG fraction from donor 18, while it was significantly reduced in the IgG-depleted fraction. This suggests that the observed basophil activation was due to IgG.

[0067] (g) Reactivity of donor 18 with purified basophils Basophils were fractionated from whole blood using a commercially available reagent (EasySep Human Basophil Isolation Kit) based on affinity. These purified basophils were used to examine the plasma of donor 18 by BAT. Figure 6 shows that basophil activation by donor 18's plasma was similar whether using whole blood or purified basophils. This suggests that there is direct activation of basophils by donor 18's plasma, and no activation by intermediate or cascade reactions.

[0068] (h) Plasma of donor 18 and FcεRI signaling inhibition Plasma from donor 18 was examined by applying Umbrilisb (Ukoniqu®), a selective phosphoinositide-3-kinase delta (PI3Kδ) inhibitor. Umbrilisb blocks the downstream activation signal of FcεRI. Three different concentrations of Umbrilisb were used.

[0069] Figure 7 shows that basophil activation, as measured by CD203c, is dose-dependently reduced by umbrilisb. Activation by anti-FcεRI-positive controls is also locked by umbrilisb, but activation by fMLP, which is activated via a different pathway, is not blocked.

[0070] (i) Activation of reagent basophils by plasma from donor 18 Figure 8 shows the activation of basophils by donor 18 plasma tracks with anti-FcεRI / anti-IgG controls. The positive controls, anti-FcεRI and anti-IgE, are activated via the FcεRI pathway. fMLP is a peptide activated via the G protein-coupled receptor pathway. Both positive controls were examined for each assay. All reagent basophils that showed activation in the positive controls via the FcεRI pathway also showed activation by donor 18 plasma. However, two reagent basophils showed activation only via the fMLP G protein-coupled receptor pathway, and donor 18 did not induce activation of these two reagent basophils. These results are consistent with donor 18 plasma inducing basophil activation via the FcεRI pathway, rather than the fMLP pathway.

[0071] (j) Testing of plasma from 18 general donors using human antigen microarrays Plasma from 18 different donors and three control plasmas were examined using a HuProt® microarray. This microarray contains approximately 21,000 human antigens. Binding to numerous mast cell receptor proteins was determined. These proteins included FcεRI, FcεR2, MRGPRX2, and complement C3a and C5a receptors. Donor 18 showed above-baseline binding to FcεRIβ (also known as MS4A2), while the other donors did not show above-baseline responses to these receptors, as reported in Figure 9. Figure 10 shows the degree of FcεRIβ signaling compared to the plasmas of the other 17 donors and three control plasmas.

Claims

1. A method for preparing plasma products with a reduced risk of adverse hypersensitivity reactions, Obtaining plasma from multiple donors; The plasma from each donor is tested for the presence of autoantibodies against the antigen at a predetermined concentration; If an individual donor's plasma contains at least a predetermined concentration of autoantibodies, that donor's plasma shall be excluded from the plasma pool; and Pooling plasma from donors whose plasma was not excluded. Methods that include...

2. The method according to claim 1, wherein the antigen is FcεRIβ.

3. A method for preparing plasma products with a reduced risk of adverse hypersensitivity reactions, Obtaining plasma from multiple donors; To detect the presence of autoantibodies against the antigen in the plasma from each donor; If the autoantibodies are detected in the plasma of an individual donor, the plasma of that donor shall be excluded from the plasma pool; and Pooling plasma from donors whose plasma was not excluded. Methods that include...

4. The method according to claim 3, wherein the antigen is FcεRIβ.

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