Compositions and methods of preparation of blood plasma products with reduced levels of fceriss autoantibodies

Abstract

Methods of preparation of therapeutic blood products from pooled plasma donors are described. The therapeutic blood products include, for example, blood plasma, blood plasma proteins, and blood plasma fractions with improved safety through the reduction in concentration or elimination of donor autoantibodies to FcεRIβ.

Description

[0001] COMPOSITIONS AND METHODS OF PREPARATION OF BLOOD PLASMA PRODUCTS WITH REDUCED LEVELS OF FcsRip AUTOANTIBODIES

[0002] BACKGROUND

[0003] This invention pertains to the preparation of therapeutic blood products pooled from multiple donors and improved methods of their preparation so as ultimately reduce or eliminate certain hypersensitivity adverse events. The invention relates to improvements in the safe use of blood products, such as blood plasma, blood plasma proteins, and blood plasma fractions prepared free of or with significantly reduced concentrations of autoantibodies to the high affinity immunoglobulin epsilon receptor subunit beta (FcsRip).

[0004] SUMMARY

[0005] Products derived from pooled human plasma such as plasma fractions or plasma proteins are generally well-tolerated when administered to patients. However, periodically clusters of hypersensitivity reactions can occur in the patients receiving these products. These hypersensitivity clusters have been associated with specific product lots, leading to voluntary market withdrawals of these products, such as immune globulin products. (See, e.g., fda.gov / vaccines-blood-biologics / safety-availability- biologics / voluntary-lot-withdrawals-immune-globulin-intravenous-igiv-and-immune- globulin-subcutaneous-igsc, March 4, 2022). Symptoms in patients experiencing the hypersensitivity events range from urticaria, pruritis, and orofacial swelling to anaphylactic-like reactions.

[0006] An autoantibody to a human antigen, FcsRip, has been identified that can be used as a marker for screening individual plasma units, donor plasma pools, or plasma-based products such as immune globulins to prevent the occurrence of these hypersensitivity reactions. Further, its presence or the presence of its autoantibodies may be used to prepare plasma-based products such as plasma fractions or plasma proteins with a greatly reduced risk of producing hypersensitivity adverse reactions. FcsRip is part of the FcsRI receptor complex found on human mast cells and basophils. It is involved in cellular activation occurring in IgE-mediated allergy reactions. The association of FcsRip with IgE-mediated allergy reactions presented here is surprising because it has been shown that the FcsRIa segment of the FcsRI receptor complex is where IgE binding occurs, and FcsRip is only involved downstream of receptor binding.

[0007] INCORPORATION BY REFERENCE

[0008] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0009] DETAILED DESCRIPTION OF THE INVENTION

[0010] A. Introduction

[0011] Mast cells, which are found in tissues, and basophiles, which are found in blood, are the cells responsible for allergic reactions. When activated, these cells release tryptase, histamine, prostaglandins, leukotrienes (LTs), and cytokines among other mediators which in turn manifest as allergic-type reactions. These reactions include a range of symptoms including urticaria, pruritis (itchy skin), and orofacial swelling to anaphylaxis.

[0012] A primary activation mechanism for allergic reactions begins with the binding of allergens to IgE residing on FcsRIa receptors on mast cells and basophils. When an allergen binds multiple IgE at once, cross-linking of the FcsRI receptor occurs and a cascade of signaling is initiated. This culminates in degranulation of the cells and consequential release of histamine and other mediator. It is well known that autoantibodies to IgE or to FcsRIa exist in some people and can cause cross-linking and subsequent activation of mast cells and basophils in the absence of a specific allergen / IgE interaction.

[0013] People with autoantibodies to IgE or to FcsRIa often develop chronic urticaria. What has not been reported previously is the existence of autoantibodies to the FcsRip subunit of the FcsRI complex. The FcsRip subunit is encoded by the gene MS4A2 (membrane spanning four domains, A2) and is a transmembrane protein. The FcsRip subunit is known to be involved in mast cell and basophil signaling downstream allergen cross-linking of the FcsRI receptor, but it was not known that it could provide a pathway to activation by itself. Furthermore, activation can be such that the presence of autoantibodies to FcsRip in a single donor can be enough that immune globulin products made from plasma pooled with many other donors can still manifest as hypersensitivity adverse events in patients receiving those products. A test to detect FcsRip in the donors our its autoantibodies could screen out the plasma and thus avoid the hypersensitivity adverse events in the patients. Further, improved preparations of pooled blood plasma, plasma fractions, or plasma proteins (e.g., Intravenous IgG [IGIV] or other IgG preparations) can be manufactured by determining amounts of FcsRip or FcsRip autoantibodies in donated blood plasma.

[0014] B. Definitions

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0016] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the peptide” includes reference to one or more peptides and equivalents thereof, e.g., polypeptides, known to those having skill in the art, and so forth.

[0017] In describing methods of the present invention, the terms “host”, “subject”, “individual” and “patient” are used interchangeably and refer to any mammal in need of such treatment according to the disclosed methods. Such mammals include, e.g., humans, ovines, bovines, equines, porcines, canines, felines, non-human primate, 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 mammalian. In certain instances, the subject is human. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees, and monkeys). As such, subjects of the invention, include but are not limited to mammals, e.g., humans and other primates, such as chimpanzees and other apes and monkey species; and the like, where in certain embodiments the subject are humans. The term subject is also meant to include a person or organism of any age, weight or other physical characteristic, where the subjects may be an adult, a child, an infant or a newborn.

[0018] As used herein, “treatment” refers to any of (i) the prevention of the disease or disorder, or (ii) the reduction or elimination of symptoms of the disease or disorder. Treatment may be effected prophylactically (prior to the onset of disease) or therapeutically (following the onset of the disease). The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. Thus, the term “treatment” as used herein covers any treatment of a condition including a disease including: (a) preventing the condition from occurring in a subject; (b) inhibiting the condition, i.e., arresting its occurrence; or (c) relieving the condition, i.e., causing regression of the condition. Treatment may result in a variety of different physical manifestations, e.g., modulation in gene expression, rejuvenation of tissue or organs, decreasing inflammation, etc. The therapeutic agent may be administered before, during or after the onset of the condition. The subj ect therapy may be administered during the symptomatic stage of the condition, and in some cases after the symptomatic stage of the condition.

[0019] Blood Products Comprising Plasma Components. By a "blood product comprising plasma components," it is meant any product derived from blood that comprises plasma (e.g., whole blood, blood plasma, or fractions thereof). The term "plasma” is used in its conventional sense to refer to the straw-colored / pale-yellow liquid component of blood composed of about 92% water, 7% proteins such as albumin, gamma globulin, anti-hemophilic factor, and other clotting factors, and 1 % mineral salts, sugars, fats, hormones and vitamins. Non-limiting examples of plasma-comprising blood products suitable for use in the subject methods include whole blood treated with anticoagulant (e.g., EDTA, citrate, oxalate, heparin, etc.), blood products produced by filtering whole blood to remove white blood cells ("leukoreduction"), blood products consisting of plasmapheretically-derived or apheretically-derived plasma, fresh-frozen plasma, blood products consisting essentially of purified plasma, and blood products consisting essentially of plasma fractions. In some instances, plasma product that is employed is a non-whole blood plasma product, by which is meant that the product is not whole blood, such that it lacks one or more components found in whole blood, such as erythrocytes, leukocytes, etc., at least to the extent that these components are present in whole blood. In some instances, the plasma product is substantially, if not completely, acellular, where in such instances the cellular content may be 5% by volume or less, such as 1 % or less, including 0.5% or less, where in some instances acellular plasma fractions are those compositions that completely lack cells, i.e., they include no cells.

[0020] Collection of blood products comprising plasma components. Embodiments of the methods described herein include use or preparation of blood products comprising plasma components which can be derived from donors, including human volunteers. The term, “human-derived” can refer to such products. Methods of collection of plasma comprising blood products from donors are well-known in the art. (See, e.g., AABB TECHNICAL MANUAL, (Mark A. Fung, et al., eds., 18th ed. 2014), herein incorporated by reference).

[0021] In one embodiment, donations are obtained by venipuncture. In another embodiment, the venipuncture is only a single venipuncture. In another embodiment, no saline volume replacement is employed. In a preferred embodiment, the process of plasmapheresis is used to obtain the blood products comprising plasma components. Plasmapheresis can comprise the removal of a weight-adjusted volume of plasma with the return of cellular components to the donor. In the preferred embodiment, sodium citrate is used during plasmapheresis in order to prevent cell clotting. The volume of plasma collected from a donor is preferably between 690 to 880 mL after citrate administration, and preferably coordinates with the donor’s weight. In another embodiment, the obtaining of blood products comprising plasma components is obtained by total plasma exchange.

[0022] C. Plasma Fractions

[0023] During the Second World War, there arose a need for a stable plasma expander which could be employed in the battlefield when soldiers lost large amounts of blood. As a result, methods of preparing freeze-dried plasma were developed. However, use of freeze-dried plasma was difficult in combat situations since reconstitution required sterile water. As an alternative, Dr. E.J. Cohn suggested that albumin could be used, and prepared a ready-to-use stable solution that could be introduced immediately for the treatment of shock. (See Johan, Current Approaches to the Preparation of Plasma Fractions in (Biotechnology of Blood) 165 (Jack Goldstein ed., Ist ed. 1991)). Dr. Cohn’s procedure of purifying plasma fractions utilized cold ethanol for its denaturing effect and employs changes in pH and temperature to achieve separation.

[0024] An embodiment of the methods described herein includes the administration of plasma fractions to a subject. Fractionation is the process by which certain protein subsets are separated from plasma. Fractionation technology is known in the art and relies on steps developed by Cohn et al. during the 1940s. (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), herein incorporated by reference). Several steps are involved in this process, each step involving specific ethanol concentrations as well as pH, temperature, and osmolality shifts which result in selective protein precipitation. Precipitates are also separated via centrifugation or precipitation. The original “Cohn fractionation process” involved separation of proteins through precipitates into five fractions, designated fraction I, fraction II+III, fraction IV- 1, fraction IV-4 and fraction V. Albumin was the originally identified endpoint (fraction V) product of this process. In accordance with embodiments of the invention, each fraction, filtrate (or effluent or sometimes referred to as waste streams from a prior separation step) contains or potentially contains therapeutically useful protein fractions. (See 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 U.S. Patent Nos. 3869431, 5110907, 5219995, 7531513, and 8772461 which are herein incorporated by reference). Adjustment of the above experimental parameters can be made in order to obtain specific protein fractions.

[0025] More recently, fractionation has reached further complexity. This recent increase in complexity has occurred through: the introduction of chromatography resulting in isolation of new proteins from existing fractions like cryoprecipitate, cryo-poor plasma, and Cohn fractions; increasing IgG recovery by integrating chromatography and the ethanol fractionation process; and viral reduction / inactivation / removal. (Id.) In order to capture proteins at physiological pH and ionic strength, anion-exchange chromatography can be utilized. This preserves functional activity of proteins and / or protein fractions. Heparin and monoclonal antibodies are also used in affinity chromatography. Additionally, fractionation using gel filtration, fraction by salt, and fractionation by polyethylene glycol are used. (Hosseini M Iran J Biotech, 14(4): 213-20 (2016) herein incorporated by reference). One of ordinary skill in the art would recognize that the parameters and techniques described above may be adjusted to obtain specifically desired plasma protein-containing fractions.

[0026] Blood plasma fractionation can also be ammonium sulfate-based. (See, e.g., Odunuga OO, Biochem Compounds, 1 :3 (2013); Wingfield PT, Curr Protoc Protein Sci, Appx. 3 (2001), herein incorporated by reference). In addition to obtaining specific blood fractions, ammonium sulfate-based fractionation has been employed to reduce abundant proteins from plasma. (Saha S, el al., J. Proteomics Bioinform, 5(8) (2012), herein incorporated by reference).

[0027] In an embodiment of the invention, blood plasma is fractionated in an industrial setting. Frozen plasma is thawed at 1°C to 4°C. Continuous refrigerated centrifugation is applied to the thawed plasma and cryoprecipitate isolated. Recovered cryoprecipitate is frozen at -30°C or lower and stored. The cryoprecipitate-poor (“cryo-poor”) plasma is immediately processed for capture (via, for example, primary chromatography) of labile coagulation factors such as factor IX complex and its components as well as protease inhibitors such as antithrombin and Cl esterase inhibitor. Serial centrifugation and precipitate isolation can be applied in subsequent steps. Such techniques are known to one of ordinary skill in the art and are described, for example, in U.S. patent nos. 4624780, 5219995, 5288853, and U.S. patent application nos. 20140343255 and 20150343025, which disclosures are incorporated by reference in their entirety herein.

[0028] In an embodiment of the invention, the plasma fraction may comprise a plasma fraction containing a substantial concentration of albumin. In another embodiment of the invention, the plasma fraction may comprise a plasma fraction containing a substantial concentration of IgG or intravenous immune globulin (IGIV) (e.g. Gamunex-C®). In another embodiment of the invention the plasma fraction may comprise an IGIV plasma fraction, such as Gamunex-C® which has been substantially depleted of immune globulin (IgG) by methods well-known by one of ordinary skill in the art, such as for example, Protein A-mediated depletion. (See 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 blood plasma fraction may be one in which substantially all the clotting factors are removed in order to retain the efficacy of the fraction with reduced risk of thromboses. For example, the plasma fraction may be a plasma fraction as described in United States Patent No. 62 / 376,529 filed on August 18, 2016; the disclosure of which is incorporated by reference in its entirety herein.

[0029] D. Clotting Factor-Reduced Fractions

[0030] Another embodiment of the invention uses a blood plasma fraction from which substantially all of the clotting factors are removed in order to retain the efficacy of the fraction with reduced risk of thromboses. Conveniently, the blood product can be derived from a young donor or pool of young donors and can be rendered devoid of IgM in order to provide a young blood product that is ABO compatible. Currently, plasma that is transfused is matched for ABO blood type, as the presence of naturally occurring antibodies to the A and B antigens can result in transfusion reactions. IgM appears to be responsible for transfusion reactions when patients are given plasma that is not ABO matched. Removal of IgM from blood products or fractions helps eliminate transfusion reactions in subjects who are administered the blood products and blood plasma fractions of the invention.

[0031] E. Protein-Enriched Plasma Protein Products Treatment

[0032] Additional embodiments of the invention, as with certain fractions such as Plasma Protein Fraction (PPF), Human Albumin Solution (HAS), Effluent I, and Effluent II / III, Effluent IV- 1, Effluent IV-4, and Effluent V are all effectively devoid of clotting factors. Such plasma fractions are hereinafter referred to as “protein-enriched plasma protein products.” For example, an embodiment of the invention may use a protein-enriched plasma protein product comprised of 82% albumin and 18% a, P, and y globulins and other plasma proteins. Another embodiment of the invention may use a protein-enriched plasma protein product comprised of 81% albumin and 19% of a, P, and y globulins and / or other plasma proteins. Another embodiment of the invention may use a protein- enriched plasma protein product comprised of 80% albumin and 20% of a, P, and y globulins and / or other plasma proteins. Additional embodiments of the invention may use protein-enriched plasma protein products comprised of 70-79% albumin and a corresponding 21-30% of a, P, and y globulins and other plasma proteins. Additional embodiments of the invention may use protein-enriched plasma protein products comprised of 60-69% albumin and a corresponding 31-40% of a, P, and y globulins and other plasma proteins. Additional embodiments of the invention may use protein- enriched plasma protein products comprised of 50-59% albumin and a corresponding 41- 50% of a, P, and y globulins and other plasma proteins. Additional embodiments of the invention may use protein-enriched plasma protein products comprised of 40-49% albumin and a corresponding 51-60% of a, P, and y globulins and other plasma proteins. Additional embodiments of the invention may use protein-enriched plasma protein products comprised of 30-39% albumin and a corresponding 61-70% of a, P, and y globulins and other plasma proteins. Additional embodiments of the invention may use protein-enriched plasma protein products comprised of 20-29% albumin and a corresponding 71-80% of a, P, and y globulins and other plasma proteins. Additional embodiments of the invention may use protein-enriched plasma protein products comprised of 10-19% albumin and a corresponding 81-90% of a, P, and y globulins and other plasma proteins. Additional embodiments of the invention may use protein- enriched plasma protein products comprised of 1-9% albumin and a corresponding 91- 99% of a, P, and y globulins and other plasma proteins. A further embodiment of the invention may use protein-enriched plasma protein products comprised of 0% albumin and 100% of a, P, and y globulins and other plasma proteins. Embodiments of the invention described above may also have total gamma globulin concentrations of 1-5%.

[0033] “Blood plasma proteins” or “plasma protein products” refer to concentrated proteins derived from plasma fractionation or other associated techniques described above. An embodiment of the invention includes preparation of these products with a reduced likelihood of producing an adverse event due to hypersensitivity. Such products include by way of example and not limitation, intravenous IgG or other preparations of plasma-derived IgG, prolastin-C (alpha- 1 antitrypsin), albumin, and antithrombin III. The specific concentrations of proteins in a plasma fraction may be determined using techniques well-known to a person having ordinary skill in the relevant art. By way of example, and not limitation, such techniques include electrophoresis, mass spectrometry, ELISA analysis, and Western blot analysis.

[0034] F. Preparation of Plasma Fractions

[0035] Methods of preparing PPF and other plasma fractions are well-known to those having ordinary skill in the art. An embodiment of the invention allows for blood used in the preparation of human plasma protein fraction to be collected in flasks with citrate or anticoagulant citrate dextrose solution (or other anticoagulant) for inhibition of coagulation, with further separation of Fractions I, II + III, IV, and PPF as per the method disclosed in Hink et al. (See Hink, J.H., Jr., et al., Preparation and Properties of a Heat- Treated Human Plasma Protein Fraction, VOX SANGUINIS 2(174) (1957), herein incorporated by reference.) According to this method, the mixture can be collected to 2 - 8 °C. The plasma can then subsequently be separated by centrifugation at 7°C, removed, and stored at -20°C. The plasma can then be thawed at 37°C and fractionated, preferably within eight hours after removal from -20°C storage.

[0036] Plasma can be separated from Fraction I using 8% ethanol at pH 7.2 and a temperature at -2 to -2.5°C with protein concentration of 5.1 to 5.6 percent. Cold 53.3 percent ethanol (176 mL / L of plasma) with acetate buffer (200 mL 4M sodium acetate, 230 mL glacial acetic acid quantum satis to 1 L with H2O) can be added using jets at a rate, for example, of 450 mL / minute during the lowering the plasma temperature to -2°C. Fraction I can be separated and removed from the effluent (Effluent I) through ultracentrifugation. Fibrinogen can be obtained from Fraction I as per methods well- known to those having ordinary skill in the art.

[0037] Fraction II + III can be separated from Effluent I through adjustment of the effluent to 21 percent ethanol at pH 6.8, temperature at -6°C, with protein concentration of 4.3 percent. Cold 95 percent ethanol (176 mL / L of Effluent I) with 10 M acetic acid used for pH adjustment can be added using jets at a rate, for example, of 500 mL / minute during the lowering of the temperature of Effluent I to -6°C. The resulting precipitate (Fraction II + III) can be removed by centrifugation at -6°C. Gamma globulin can be obtained from Fraction II + III using methods well-known to those having ordinary skill in the art. Fraction IV- 1 can be separated from Effluent II + III (“Effluent II / III”) through adjustment of the effluent to 19 percent ethanol at pH 5.2, temperature at -6°C, and protein concentration of 3 percent. H2O and 10 M acetic acid used for pH adjustment can be added using jets while maintaining Effluent II / III at -6°C for 6 hours. Precipitated Fraction IV- 1 can be settled at -6°C for 6 hours and subsequently separated from the effluent by centrifugation at the same temperature. Stable plasma protein fraction can be recovered from Effluent IV- 1 through adjustment of the ethanol concentration to 30 percent at pH 4.65, temperature -7°C and protein concentration of 2.5 percent. This can be accomplished by adjusting the pH of Effluent IV- 1 with cold acid-alcohol (two parts 2 M acetic acid and one-part 95 percent ethanol). While maintaining a temperature of - 7°C, to every liter of adjusted Effluent IV- 1 170 mL cold ethanol (95%) is added. Proteins that precipitate can be allowed to settle for 36 hours and subsequently removed by centrifugation at -7°C. Fraction IV-4 paste / precipitate can also be attained using the Cohn fractionation process and can be resuspended. Indeed, Fraction IV-4 and its manufacturing process has been described previously (Schopfer LM, et al., PLoS ONE, 14(l):e0209795 (2018) and herein incorporated by reference in its entirety) (Schopfer LM, et al., PLoS ONE, 14(l):e0209795 (2018), and Bertolini J, Goss N, Curlin J eds., PRODUCTION OF PLASMA PROTEINS FOR THERAPEUTIC USE, 16.4: 231 :232 (2013) herein incorporated by reference in their entirety).

[0038] The recovered proteins (stable plasma protein fraction) can be dried (e.g. by freeze drying) to remove alcohol and H2O. The resulting dried powder can be dissolved in sterile distilled water, for example using 15 liters of water / kg of powder, with the solution adjusted to pH 7.0 with 1 M NaOH. A final concentration of 5 per cent protein can be achieved by adding sterile distilled water containing sodium acetyl tryptophanate, sodium caprylate, and NaCl, adjusting to final concentrations of 0.004 M acetyl tryptophanate, 0.004 M caprylate, and 0.112 M sodium. Finally, the solution can be filtered at 10°C to obtain a clear solution and subsequently heat-treated for inactivation of pathogens at 60°C for at least 10 hours.

[0039] The preceding methods of preparing plasma fractions and plasma protein fraction (PPF) are only exemplary and involve merely embodiments of the invention. One having ordinary skill in the art would recognize that these methods can vary. For example, pH, temperature, and ethanol concentration, among other things can be adjusted to produce different variations of plasma fractions and plasma protein fraction in the different embodiments and methods of the invention. In another example, additional embodiments of the invention contemplate the use of nanofiltration for the removal / inactivation of pathogens from plasma fractions and plasma protein fraction.

[0040] G. Methods of Preparation

[0041] An embodiment of the invention includes a method of preparing a blood plasma product with a reduced likelihood or incidence of hypersensitivity reactions. Additional embodiments include, by way of example and not limitation, preparation of blood plasma products such as IgG (intravenous), albumin, antithrombin III, and prolastin-C (alpha- 1 antitrypsin). Further embodiments include blood plasma products such as whole blood or plasma from a single donor or plasma products that are pooled from multiple donors. Additional embodiments include blood plasma products that originate from one donor center or geographic regions.

[0042] Further embodiments of the invention include obtaining blood plasma from a number of donors for the purpose of creating pooled blood plasma from more than one donor. Another embodiment tests said blood plasma from each donor for a predetermined concentration of autoantibodies to FcsRip receptor or for an amount of FcsRip receptor itself to see if a donor is “positive” for risk of causing a hypersensitivity adverse event. The testing of said blood plasma from each donor may also be in the form of detecting autoantibodies to FcsRip receptor or detecting the FcsRip receptor itself, resulting in identification of a positive donor. An embodiment of the invention includes preventing the blood plasma from any positive donors from being pooled with other donors’ blood plasma.

[0043] Additional embodiments of the invention include testing a pool of blood plasma from multiple donors. A further embodiment determines concentrations or detects autoantibodies to FcsRip receptor from a pool of donors wherein a predetermined concentration of autoantibodies in the blood plasma from a single donor with autoantibodies to FcsRip receptor is sufficient to cause a hypersensitivity adverse reaction in the pooled plasma. Another embodiment excludes use of such “contaminated” pooled plasma from use in preparing blood plasma products or proteins. Another embodiment includes subsequently determining whether the degree of dilution of the single donor in other donors’ pooled blood or plasma meets is sufficient for detection of risk of a hypersensitivity adverse event. Another embodiment of the invention is the degree of dilution is 1 : 1000 to 1 : 10,000. An additional embodiment of the invention excludes the entire pool of plasma from use in patients.

[0044] A further embodiment of the invention is pooling the blood plasma of the donors whose blood plasma was not excluded from the pool. Blood plasma products or proteins can then be prepared from these pools.

[0045] Other embodiments of the invention can include the identification and use of antigens other than FcsRip receptor such as FcsRIa receptor to determine whether certain donors or donor pools should be excluded. Preparations of blood plasma products or proteins with reduced risk of causing hypersensitivity adverse reactions can be made.

[0046] A further embodiment of the invention includes testing a donor’s blood plasma using a basophil activation test (BAT) or histamine release assay on (HRA) on basophils or mast cells from the donor. The degree of BAT or HRA compared to other donors is used to determine whether there is an unacceptable risk of a hypersensitivity adverse reaction.

[0047] Techniques for identifying autoantibodies to an antigen have been described previously. Chemiluminescent technologies have been used to identify autoantibodies in diagnosis a range of autoimmune disease (Maier M et al.. Immunopharmacology and Immunotoxicology, 38: 1(14-20) (2015)). Autoantibodies in 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 utilized in detecting autoantibodies in the serum of rheumatoid arthritis patients (Hansson M et al., Arthritis Res & Therapy, 14:R201 (2012)). Autoantibodies for IgE in its association with hypersensitivity reactions have also been described. (Badloe et al., Clin and Translational Allergy, 10(34) (2020)).

[0048] H. Reagents, Devices, and Kits

[0049] Also provided are reagents, devices, and kits thereof for practicing one or more of the above-described methods. The subject reagents, devices, and kits thereof may vary greatly. Reagents and devices of interest include those mentioned above with respect to the methods of preparing plasma products,

[0050] Kits may also comprise blood collection bags, tubing, needles, centrifugation tubes, and the like. In yet other embodiments, kits as described herein include two or more containers of blood plasma product such as plasma protein fraction, such as three or more, four or more, five or more, including six or more containers of blood plasma product. In some instances, the number of distinct containers of blood plasma product in the kit may be 9 or more, 12 or more, 15 or more, 18 or more, 21 or more, 24 or more 30 or more, including 36 or more, e.g., 48 or more. Each container may have associated therewith identifying information which includes various data about the blood plasma product contained therein, which identifying information may include one or more of the age of the donor of the blood plasma product, processing details regarding the blood plasma product, e.g., whether the plasma product was processed to remove proteins above an average molecule weight (such as described above), blood type details, etc. In some instances, each container in the kit includes identifying information about the blood plasma contained therein, and the identifying information includes information about the donor center of the blood plasma product, e.g., the identifying information provides confirming data of the donor center origin of the blood plasma product in order to aid in testing and / or identifying potential donors or lots of plasma for risk of adverse events of hypersensitivity due to, for example, the presence of FcsRip receptors or its autoantibodies. The identifying information can be present on any convenient component of the container, such as a label, an RFID chip, etc. The identifying information may be human readable, computer readable, etc., as desired. The containers may have any convenient configuration. While the volume of the containers may vary, in some instances the volumes range from 10 ml to 5000 mL, such as 25 mL to 2500 mL, e.g., 50 ml to 1000 mL, including 100 mL to 500 mL. The containers may be rigid or flexible, and may be fabricated from any convenient material, e.g., polymeric materials, including medical grade plastic materials. In some instances, the containers have a bag or pouch configuration. In addition to the containers, such kits may further include administration devices, e.g., as described above. The components of such kits may be provided in any suitable packaging, e.g., a box or analogous structure, configured to hold the containers and other kit components. In addition to the above components, the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, portable flash drive, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.

[0051] I. Experimental Examples

[0052] 1. Example 1 - Manufacturing and Quality Investigation

[0053] An elevated rate of hypersensitivity adverse events was observed in some lots of an intravenous IgG blood plasma protein product causing hives, itching, and rash (on the groin, chest, legs and other locations). Additionally, some adverse events were breathing difficulty, swollen lips, bronchospasm, and laryngeal edema. These symptoms were consistent with mast cell response and were resolved rapidly upon treatment with antihistamines. Lots of the product are typically withdrawn from the market when such hypersensitivity reactions are observed, often from multiple manufacturers. In this case, a significant number were withdrawn.

[0054] Several potential causes of the increase in hypersensitivity reactions were investigated. There were no significant manufacturing deviations for any the withdrawn lots and an extensive review of all raw materials, manufacturing steps, in-process and final container testing revealed no discrepancies. Quality control testing met specifications and no significant differences between withdrawn and comparator lots were found.

[0055] Investigational testing identified no significant difference between withdrawn lots and comparator lots. Testing included the tests identified in TABLE 1. TABLE 1

[0056] 2. Example 2 - Donor Investigations

[0057] Lots exhibiting an elevation in hypersensitivity adverse events that were withdrawn from the market were investigated for a nexus to a common donor center. It was determined that these lots originated from a single donor center. A thorough review of processes, practices, commodities, and quality systems were performed for this center. No identifiable issue was found that related to or contributed to the hypersensitivity adverse events. Subsequently, a common donor analysis for this center was performed. The analysis evolved as additional lots were identified and withdrawn, and it was found that 18 donors could be identified in common to withdrawn lots having a higher rate of hypersensitivity reactions. Those donors were deferred from further plasma donation and their plasma analyzed. Subsequently, no reports of hypersensitivity from this common donor center were reported for any lots from this common donor center once the plasma from the 18 donors was no longer included in any lots.

[0058] The 18 common donors’ donation history and medical information was analyzed, but nothing was deemed remarkable. Each had extensive donation history with no remarkable medications taken. The medical histories themselves were unremarkable. Some had common allergies, but nothing remarkable. Their electrophoresis serum protein profiles were all normal. There were no detectable anti-gal (IgE or IgG) when submitted for diagnostic testing. And several donors had elevated IgE, indicating chronic allergies.

[0059] One of the 18 common donors (“Donor 18”) produced a positive response in the basophil activation test (BAT) and the histamine release assay (HRA). Plasma from the other 17 donors produced negative results by both BAT and HRA tests.

[0060] 3. Example 3 - Characterization And In Vitro Methods Applied to Investigation of Donor Batches

[0061] (a) 2D-DIGE and LC-MS / MS

[0062] 2-dimensional difference gel electrophoresis was tested on a grouping of three normal and three hypersensitivity batches of plasma, which detected approximately 600 proteins per spots. Advanced imaging software and statistical analysis were used to analyze identified spots. No unique spots or intensities were detected in hypersensitivity batches compared to normal batches.

[0063] Proteins not binding to protein G (non-IgG proteins) were purified and tryptic digested were tested by liquid chromatography combined with tandem mass spectrometry (LC-MS / MS) was used to analyze a grouping of three normal and three hypersensitivity batches of plasma. The proteins were derived from a group of five normal and five hypersensitivity batches of plasma. Tandem MS enabled identification of peptides via database matching (human and non-human). Approximately 30 impurity (non-IgG) proteins were detected in samples, but no significant differences were observed in these impurities between the withdrawn and comparator batches.

[0064] (b) Characterization of Product Batches for Aggregates

[0065] Advanced characterization techniques for aggregates and subvisible particle analysis were performed on normal and hypersensitivity batches. This was due to historical issues of aggregate-related adverse events for previous generations of products. Using these techniques, a wide range of molecular / particle sizing could be examined. Size exclusion chromatography with multiangle light scattering detection and Dawn HELEOS II detector coupled with an OptiLab rEX refractometer for batch mode testing with no chromatography showed no difference between the batches. Nanoparticle Tracking Analysis (NTA) using the Stokes-Einstein equation to calculate hydrodynamic diameters and use of Malvern NanoSight LM10 instrument likewise revealed no differences between the batches. Resonant Mass Measurement (RMM) using a Malvern Archimedes instrument showed no differences between the batch types. And Micro Flow imaging (MFI) performing image analysis for particles in approximate range of 1 pm to mm in diameter reported no differences between the batches.

[0066] 4. Characterization of Autoantibodies Implicated in Chronic Urticaria

[0067] Autoimmune Anti-FcsRIa and Anti-IgE have both been implicated as causing chronic urticaria, which has symptoms consistent with many of the hypersensitivity reactions observed in patients receiving the withdrawn intravenous IgG batches. Anti- FcsRIa and Anti-IgE can be tested at diagnostic laboratories as well as by the use of commercially available enzyme-linked immunosorbent assay (ELISA) and were tested via both methods. There was some Anti-FcsRIa signal observed in product lots, but no differences between hypersensitivity and normal product lots, and no detectable anti-IgE in normal or hypersensitivity batches.

[0068] 5. Functional Methods for Basophil Activation

[0069] (a) Reactivity of Donor Plasma Pools and IgG Final Product

[0070] Two in vitro assays were performed in order to test for basophil activation within plasma pools and IgG final product batches — the histamine release assay (HRA) and the basophil activation test (BAT). The HRA was performed as follows. Fresh human whole blood was a source of basophils. Incubation of samples from donor plasma pools or IgG final product batches was incubated in LDN release buffer with the fresh whole blood samples. Acetylation was used to protect released histamine, and histamine detected by ELISA (Immunotech). Anti-IgE and N-Formylmethionyl-leucyl-phenylalanine (fMLP) were used as positive controls for histamine release.

[0071] The BAT used fresh human whole blood as the source of basophils and incubated these with samples from donor plasma pools or IgG final product batches. Cell surface expression of CD63 or CD203c on basophils by flow cytometry using Buhlmann Lab Flow CAST Basophil Activation Test was measured. Anti-FcsRIa and N- Formylmethionyl-leucyl-phenylalanine (fMLP) were used as positive controls.

[0072] The positive controls for both assays showed activation through independent activation pathways. Anti-FcsRIa and anti-IgE were associated with the FcsRI pathway. fMLP was associated with a G-protein coupled receptor pathway. Ultimately, neither assay detected a signal in either plasma pools or IgG final product batches.

[0073] (b) Reactivity of 18 Specific Donors

[0074] BAT and HRA were performed on the 18 common donors (supra) who were identified from withdrawn lots as having a higher rate of hypersensitivity reactions. These donors’ plasma was mixed with the basophils from five different healthy donors.

[0075] Figure 1 shows that the results from Donor 18 were well above buffer controls and the other 17 samples for all five reagent basophil sources. Due to this consistent response by BAT / HRA, Donor 18’s plasma was further characterized.

[0076] (c) Donor 18 Plasma Dilution Series with BAT

[0077] A series of plasma dilutions in buffer was made from Donor 18 at dilutions from 1 :0 (neat) up to 1 :2000. These dilutions were incubated with fresh reagent blood (the basophil source) and tested for basophil activation (BAT) using CD63 and CD203C.

[0078] Figure 2 shows that the BAT activation signal was reduced to background levels after a dilution factor of 1 : 100. Relevant dilutions that are able to detect a response from a single donor in pools or final product would therefore be 1 : 1000 to 1 : 10,000.

[0079] (d) BAT Time Course Study for Donor 18 Plasma

[0080] Plasma samples were retained from 22 plasma donations from Donor 18 spanning approximately 3.5 years of donation history. Figure 3 shows that in Donor 18’s first year of donations, there was low reactivity in BAT. However, there was a dramatic increase in activation near the time when elevation of product hypersensitivity adverse events began. And there was some variability in activation signal after the dramatic increase.

[0081] (e) Relationship of BAT Response of Donor 18 and Hypersensitivity Events

[0082] Figure 4 shows hypersensitivity adverse events as a function of BAT activation. The hypersensitivity adverse events were normalized to relative quality of Conor 18 plasma in the final product. A response fit with a four-parameter logistic fit indicates a latent period followed by a rapid increase in BAT. This is consistent with a thresholdrelationship; once the threshold is reached, hypersensitivity occurs.

[0083] (f) BAT Reactivity of IgG from Donor 18

[0084] Figure 5 reports the BAT reactivity of IgG from Donor 18. Donor 18 and control plasma were fractionated using Capture Select IgG affinity resin. The fractions were tested by SDS PAGE to confirm composition of fractions and the fractions were subsequently tested by BAT using both CD63 and CD203c markers. SDS-PAGE confirmed that IgG and IgG depleted fractions had been obtained, and basophil activation remained predominately with the IgG fraction of Donor 18 and was greatly reduced in the IgG-depleted fraction. This suggests that the observed activation of basophils is cause by IgG.

[0085] (g) Donor 18 Reactivity with Purified Basophils

[0086] Basophils were fractionated from whole blood by affinity depletion with commercial reagent (EasySep Human Basophil Isolation Kit). Donor 18 plasma was tested by BAT using the purified basophils. Figure 6 shows that basophil activation by Donor 18 plasma is similar whether in whole blood or using purified basophils. This suggest that there is direct activation of basophils by Donor 18 plasma and not activation through an intermediary reactions or cascade reaction.

[0087] (h) Donor 18 Plasma and FcsRI Signal Inhibition

[0088] Donor 18 plasma was tested by applying a selective phophoinositide-3 -kinase delta (PI3K6) inhibitor, Umbrilisb (Ukoniqu™). Umbrilisb blocks activation signaling downstream of FcsRI. Three concentrations of Umbrilisb were used.

[0089] Figure 7 shows that basophil activation as measured by CD203c is reduced in a dose-depending manner with Umbrilisb. Activation by anti-FcsRI positive control is also locked by Umbrilisb, whereas activation by fMLP, which activates through a different pathway is not blocked.

[0090] (i) Activation of Reagent Basophils by Donor 18 Plasma

[0091] Figure 8 shows basophil activation by Donor 18 plasma tracks with anti- FcsRI / anti-IgG controls. Positive controls anti-FcsRI and anti-IgE activate through the FcsRI pathway. fMLP is a peptide that activates through a G-protein coupled receptor pathway. For each assay, both positive controls were tested. All of the reagent basophils that exhibited activation with positive controls through the FcsRI pathway also showed activation by Donor 18 plasma. However, two reagent basophils only showed activation through the fMLP G-protein coupled receptor pathway and Donor 18 did not cause activation of these two reagent basophils. These results are consistent with Donor 18 plasma causing activation of basophils through the FcsRI pathway and not the fMLP pathway.

[0092] (j) Testing of Plasma from 18 Common Donors by Human

[0093] Antigen Microarray Plasma from 18 different donors and three control plasmas were tested by

[0094] HuProt™ microarray. This microarray includes approximately 21,000 human antigens. Binding to a number of mast cell receptor proteins was determined. These proteins included FcsRI, FcsR2, MRGPRX2, Complement C3a and C5a receptors. Donor 18 exhibited binding above baseline to FcsRip (also referred to as MS4A2) while no other donors exhibited responses above baseline to these receptors as reported in Figure 9. Figure 10 shows the degree of signal of FcsRip versus the other 17 donors and three control plasmas.

Claims

CLAIMS1. A method of preparing a blood plasma product with a reduced risk of adverse hypersensitivity reactions, the method comprising: obtaining blood plasma from a number of donors; testing said blood plasma from each donor for a predetermined concentration of autoantibodies to an antigen; excluding an individual donor’s blood plasma from the pool of the blood plasma if the donor’s blood plasma contains at least the predetermined concentration of autoantibodies; and pooling the blood plasma of the donors whose blood plasma was not excluded.

2. The method of claim 1 wherein the antigen is FcsRip.

3. A method of preparing a blood plasma product with a reduced risk of adverse hypersensitivity reactions, the method comprising: obtaining blood plasma from a number of donors; detecting the presence of autoantibodies to an antigen in said blood plasma from each donor; excluding an individual donor’s blood plasma from the pool of the blood plasma if said autoantibodies are detected in the donor’s blood plasma; and pooling the blood plasma of the donors whose blood plasma was not excluded.

4. The method of Claim 3 wherein the antigen is FcsRip.

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