Method for preparing corn pool concentrate from plasma by ultrafiltration

Abstract

The present invention provides a method for fractionating human plasma using, in some embodiments, the Cohn fractionation procedure. The improvement involves using bioactive enriched plasma as the starting material for the fractionation procedure.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention is in the field of plasma fractionation for the separation of therapeutically active plasma proteins from plasma. [Background technology]

[0002] 2. Background of the Invention Over the last decade, the clinical utilization of plasma protein-based therapeutics has steadily increased. For example, as awareness of primary immune deficiencies (PIDs) has increased, the effective clinical use of intravenous immunoglobulin (IVIG) has increased across patient populations affected by PIDs. IVIG is increasingly being utilized for off-label indications, as well as conditions such as chronic inflammatory demyelinating polyneuropathy (CIPD).

[0003] In 2019, the plasma products market was expected to grow at a CAGR of 6.8% from $20.5 billion in 2018 to $28.5 billion by 2023. The global annual fractionation capacity was approximately 70.7 million liters in 2016.

[0004] The process of plasma fractionation is infrastructure / facility intensive and highly regulated. To meet the growing demand for plasma-derived therapeutics, either the existing infrastructure must be able to meet this demand or the infrastructure must be modified or augmented, the latter two of these options requiring significant capital investments, possible interruptions to production lines, and possible regulatory review and certification of the modified or new facilities. Options to increase efficiency without substantially modifying the existing infrastructure are therefore very attractive.

[0005] A method for reducing the volume of liquid plasma input material to the fractionation process can achieve the dual benefit of maximizing the productivity of the existing fractionation infrastructure while also reducing the level of resources required during the plasma fractionation process. Clearly unexplored prior to the present invention is the ability to process smaller volumes of liquid to produce the same amount of plasma-derived protein product as would be produced from a larger volume of liquid, e.g., fresh frozen plasma (FFP).

[0006] Thus, prior to the invention described herein, it was not clear that the proteins in the various fractions (e.g., cold ethanol fractionation) could be recovered in sufficient meaningful amounts by fractionation of concentrated plasma Cone pools to make the cost of concentrating and fractionating concentrated bioactive plasma worthwhile. Furthermore, it was unclear whether concentrated plasma Cone pools would perform similarly to fresh frozen plasma in Cone fractionation (or known modifications thereof). The inventors have discovered that fractionation pathways originating from concentrated plasma Cone pools are indeed viable. Concentrated Cone pools are a component of economically viable fractionation processes, such as Cone fractionation or Kistler-Nitschman fractionation, or other methods (e.g., Gerlough, Hink, and Mulford) that start with concentrated plasma Cone pools. See, for example, Kistler et al., Vox. Sang. (1962); 7(4), pp. 414-424; Graham, et al. Subcellular Fractionation, a Practical Approach. Oxford University Press. 1997 (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kistler et al., Vox. Sang. (1962);7(4), pp.414-424, Graham, et al.Subcellular Fractionation, a Practical Approach.Oxford University Press.1997 Summary of the Invention

[0008] BRIEF SUMMARY OF THE INVETION Given the increasingly widespread use of therapeutic plasma-derived blood protein compositions, such as immunoglobulin compositions, albumin, protease inhibitors, blood clotting factors, clotting factor inhibitors, and proteins of the complement system, ensuring adequate, economical, environmentally friendly and sustainable access to effective and safe plasma-derived blood protein compositions is of paramount importance.

[0009] Modifying a standard plasma protein fractionation process by feeding the process with a concentrated plasma cone pool leads to process efficiencies that are a result of a reduction in the volume of liquid to be fractionated, and in some embodiments have been surprisingly found to be essentially proportional to the increase in the concentration of the plasma input material introduced into the process. The improved methods increase processing equipment utilization and increase throughput, which in some embodiments is roughly proportional to the concentration ratio, which in turn can have a large impact. In various embodiments, the present invention provides improved plasma fractionation methods having one or more of these improved properties. Also provided are plasma protein products prepared using the improved procedures.

[0010] In the present invention, it has been found that concentrated plasma Cone pools are an effective starting material for preparing protein therapeutics by fractionation of the concentrated Cone pools. In various embodiments, proteins that are typically found in the various Cone fractions downstream of the concentrated plasma Cone pool are found in those fractions in yields and purities that are comparable to those that they are found in the corresponding fractions in processes that start with (non-concentrated) plasma feedstock, e.g., cryo-poor plasma, plasma after one or more adsorption steps, recovered plasma, plasma after plasma exchange, frozen plasma, thawed plasma, etc.

[0011] An exemplary method of the invention includes (a) subjecting a concentrated plasma Cone pool to one or more plasma fractionation processes (e.g., cold ethanol fractionation). In an exemplary embodiment, the invention provides that prior to (a), (b) preparing the concentrated plasma Cone pool.

[0012] Thus, in various embodiments, the present invention provides an improved process for fractionating plasma. The improvement includes starting the plasma fractionation process with a concentrated plasma cone pool. In various embodiments, the improvement further includes concentrating the plasma input material before subjecting the concentrated input material to an initial alcohol fractionation step. An exemplary plasma input material is concentrated cryo-poor plasma.

[0013] In various embodiments, the reduction in volume of plasma input material and the concomitant increase in plasma protein concentration(s) results in a plasma fractionation process that is characterized by a reduced volume / increased plasma protein concentration(s) to a surprising degree.

[0014] In various embodiments, the present invention provides an improved plasma fractionation process that proceeds to completion of selected steps within a defined time. The plasma fractionation process improvement includes completing selected final steps in a reduced time relative to an otherwise identical plasma fractionation process, where the plasma input to the initial alcohol fractionation step in the method of the present invention is concentrated compared to the plasma input to the otherwise identical process. An exemplary plasma input is concentrated cryo-poor plasma, which in turn is concentrated Cone pool in this embodiment.

[0015] For example, a fractionation process beginning with a Cone pool that is about 10%, about 20%, or about 30% concentrated relative to a standard plasma fractionation input material uses about 10%, about 20%, or about 30% less reagents than a comparable process beginning with unconcentrated input material. Similarly, a fractionation process beginning with a Cone pool that is about 10%, about 20%, or about 30% concentrated relative to a standard plasma fractionation input material consumes about 10%, about 20%, or about 30% less time from start to completion than a comparable process beginning with unconcentrated input material. This result was unexpected because the mass of plasma proteins contained in the concentrated input material is essentially unchanged between the concentrated and unconcentrated input materials.

[0016] In an exemplary embodiment, the invention provides an improved method for preparing a protein fraction from plasma, the fraction being enriched in a plasma protein product selected from clotting factors (e.g., Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, and Factor XIII), prothrombin complex, von Willebrand factor, Factor VIII / von Willebrand factor, fibrin, fibrinogen, thrombin, polyvalent and hyperimmune (e.g., anti-RhO, anti-hepatitis B, anti-rabies, or anti-tetanus) immunoglobulins (IgG), protease inhibitors (e.g., alpha 1-antitrypsin and C1 inhibitor), anticoagulants (e.g., antithrombin), C1 esterase inhibitor, protein C, and albumin, and combinations thereof. The improvement includes concentrating the plasma input material, preparing a concentrated Cohn's pool, and then introducing the concentrated Cohn's pool into the initial alcohol fractionation step of the plasma fractionation process. An exemplary plasma input material is concentrated cryo-poor plasma.

[0017] The improved process is applicable to any plasma fractionation process, e.g., Cohn, Gerlrough, Hink, Mulford, Kistler Nitschmann, thermal ethanol fractionation, Hao, etc.

[0018] The present invention also provides, inter alia, a plasma processing system, preferably a cGMP compliant system, used to fractionate plasma from concentrated Conn Pool input material, such as concentrated cryo-poor plasma. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a general flow diagram of an exemplary Cone fractionation procedure. [Diagram 2] FIG. 1 is an exemplary flow diagram for concentrating starting plasma to form concentrated Cone Pool input material to the fractionation process. [Figure 3-1] 1 illustrates an exemplary improved fractionation process of the present invention, with the filtration step identified and located by a red dot. [Figure 3-2] See description of Figure 3-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Detailed Description of the Invention I. Introduction Plasma, which accounts for about 55% of the total volume of whole blood, is the component of whole blood in which blood cells and other constituents of whole blood are suspended. Plasma further contains a mixture of more than 700 proteins and additional substances that perform functions necessary for physical health, including clotting, protein storage, and electrolyte balance, among others. When extracted from whole blood, plasma can be used to replenish bodily fluids, antibodies, and clotting factors. Thus, plasma is widely used in medical treatments.

[0021] As described above and in the following sections, the present invention imparts many efficiencies and other advantages to the fractionation process by starting the fractionation with a concentrated plasma Cone pool.

[0022] Reference will now be made in detail to the practice of exemplary embodiments of the present disclosure, as illustrated in the accompanying drawings. The same reference designations are used throughout the drawings and the following detailed description to refer to the same or similar parts. Those skilled in the art will understand that the following detailed description is illustrative only and is not intended to be in any way limiting. Other embodiments of the present disclosure will be readily suggestible to those skilled in the art having the benefit of this disclosure.

[0023] For clarity, not all of the specific features of the embodiments described herein are shown and described. It will be understood that in the development of any such actual embodiment, many embodiment-specific decisions will be made to achieve the particular goals of the plasma product manufacturer, such as compliance with application-related and business-related constraints, and that these particular goals will vary from embodiment to embodiment and from plasma product manufacturer to plasma product manufacturer. Moreover, it will be understood that such a development effort, while potentially complex and time-consuming, would be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

[0024] Many modifications and variations of the exemplary embodiments described in this disclosure may be made without departing from the spirit and scope of the exemplary embodiments, as will be apparent to those skilled in the art. The specific exemplary embodiments described herein are provided by way of example only, and the disclosure is limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0025] II. Abbreviations and Definitions Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, pharmaceutical formulation, and medical imaging are those commonly used and well known in the art.

[0026] b.Definition As used herein, the articles "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "a protein" means one protein or multiple proteins.

[0027] "Cohn process" and "Cohn fractionation" are used interchangeably herein and refer to a method of separating human plasma through a series of steps including ethanol precipitation at different concentrations, changes in pH, temperature, and ionic strength, as commonly understood, resulting in fractions enriched in certain plasma proteins. See, e.g., U.S. Pat. No. 2,390,074. FIG. 1 provides an exemplary flow diagram of the Cohn process. As used herein, the terms "Cohn process" and "Cohn fractionation" also refer to the many variations and improvements on this pioneering process, such as the Kistler-Nitschmann process (Kistler et al. (1952), Vox Sang, 7, 414-424). Other processes that may be used in the methods of the present invention include the method of isolating IgG described in U.S. Pat. No. 8,940,877.

[0028] "Plasma" is the fluid remaining after blood has been centrifuged to remove (for example) cellular material, e.g., red blood cells, white blood cells, and platelets. Plasma is generally yellow in color and clear to opaque. Blood that has been donated, processed to separate the plasma from certain other blood components, and has not been frozen is referred to as "non-frozen" plasma. Plasma that has been frozen for less than 8 hours at temperatures described herein is referred to herein as "fresh frozen plasma" ("FFP"). Fresh frozen plasma contains the dissolved components of blood, e.g., proteins (6-8%, e.g., serum albumin, globulins, fibrinogen, etc.), glucose, clotting factors (clotting proteins), electrolytes (Na + , Ca 2+ , Mg 2+ , HCO 3 - , Cl - etc.), hormones, etc. Whole blood (WB) plasma is plasma isolated from whole blood that contains no additives other than the anticoagulant(s). Citrate phosphate dextrose (CPD) plasma, as the name suggests, contains citric acid, sodium phosphate, and a sugar, usually dextrose, added as anticoagulants.

[0029] "Recovered plasma" refers to plasma separated within 5 days of the expiration date of whole blood and stored at 1-6°C. The profile of plasma proteins in liquid plasma is difficult to characterize. The levels and activation state of coagulation proteins in liquid plasma depend on and change with the time of contact with cells, as well as the conditions and duration of storage. This component serves as a source of plasma proteins. The levels and activation state of coagulation proteins are variable and change over time.

[0030] "Thawed plasma" refers to plasma derived from a source, FFP or FP24, prepared using aseptic techniques (closed system), thawed at about 10 to about 37°C, and maintained at about 1 to about 6°C for up to about 4 days after an initial 24 hour post-thaw period. Thawed plasma contains stable clotting factors, e.g., factor II and fibrinogen, in concentrations similar to FFP, but with variably reduced amounts of other factors. An exemplary thawed plasma is a component of the first fractionation step, where plasma (source, harvest, FF, etc.) is removed from a plastic container and thawed in a jacketed container (containing exchange fluid at a temperature up to 37°C).

[0031] "Fresh frozen plasma" ("FFP") refers to plasma prepared from whole blood or apheresis collection and frozen at approximately -18°C or below within the time frame specified in the instructions for use of the associated blood collection, processing, and storage system (e.g., frozen within 8 hours of collection). On average, a unit contains 200-250 mL, although apheresis-derived units may contain as much as 400-600 mL. FFP contains plasma proteins including all clotting factors. FFP contains high levels of the labile clotting factors Factors V and VIII.

[0032] As used herein, "factors" followed by a Roman numeral refer to a series of plasma proteins related through a complex cascade of enzyme-catalyzed reactions involving the sequential cleavage of large protein molecules to generate peptides, each of which converts an inactive zymogen precursor into an active enzyme, resulting in the formation of a fibrin clot. These include factor I (fibrinogen), factor II (prothrombin), factor III (tissue thromboplastin), factor IV (calcium), factor V (proaccelerin), factor VI (no longer considered active in hemostasis), factor VII (proconvertin), factor VIII (antihemophilic factor), factor IX (plasma thromboplastin component, Christmas factor), factor X (Stewart factor), factor XI (plasma thromboplastin precursor), factor XII (Hageman factor), and factor XIII (fibrin stabilizing factor).

[0033] "Plasma proteins" include, for example, clotting factors (e.g., Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, and Factor XIII), prothrombin complex, von Willebrand factor, Factor VIII / von Willebrand factor, fibrin, fibrinogen, thrombin, polyvalent and hyperimmune (e.g., anti-RhO, anti-hepatitis B, anti-rabies, or anti-tetanus) immunoglobulin (IgG), protease inhibitors (e.g., alpha 1-antitrypsin and C1 inhibitor), anticoagulants (e.g., antithrombin), C1 esterase inhibitor, protein C, and albumin, and combinations thereof.

[0034] As used herein, the term concentrated plasma "cone pool" refers to a plasma pool that, in the methods of the invention, is concentrated and then undergoes a plasma fractionation process (e.g., cryo-, coagulation factor-, inhibitor-depleted plasma). An exemplary concentrated plasma cone pool is bioactive plasma that is about 10% to about 30% concentrated from its original volume (e.g., volume upon collection from a donor or donor pool, upon receipt by a fractionation facility, etc.) and contains proteins that have not been damaged to the extent that they have lost substantially all of their bioactivity.

[0035] In exemplary embodiments, the enrichment process does not substantially reduce the activity of selected plasma proteins, e.g., IgG, A1PI, factors, etc., that are subsequently isolated (or enriched) by fractionation of the enriched Cohn pool. In various embodiments, the activity of the selected plasma protein fractionated from the bioactive enriched Cohn pool is about 99% or more, about 90% or more, about 85% or more, or about 80% or more of the activity of the selected plasma protein contained in the plasma source prior to enrichment. In various embodiments, the selected plasma protein is a multivalent or hyperimmune IgG.

[0036] A "disease" is a condition in the health of an animal in which the animal is unable to maintain homeostasis and, if the disease is not ameliorated, the health of the animal will continue to deteriorate. In various embodiments, one or more proteins from the fractionated bioactive enriched Cohn pool are used to treat one or more diseases.

[0037] III. Embodiment A. Compositions and Devices The present disclosure is directed to a method of concentrating a bioactive plasma feedstock to form a concentrated Cone pool, and then fractionating the resulting bioactive concentrated Cone pool using an art-recognized fractionation process. An exemplary fractionation process includes an alcohol fractionation step as its first step. Also provided is a plasma protein preparation prepared by a fractionation process starting with the concentrated Cone pool.

[0038] In an exemplary embodiment, the present invention provides a bioactive enriched plasma. The biological and physiological activity of the enriched plasma is essentially not decreased compared to the starting plasma from which the enriched plasma is derived. By essentially not decreased, it is meant that for any selected plasma protein, its activity in the enriched plasma is about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more of its activity in the starting plasma. In an exemplary embodiment, this applies to at least two selected plasma proteins, at least five selected proteins, or at least ten selected proteins. In an exemplary embodiment, the total plasma protein activity of the enriched plasma is essentially not decreased compared to the starting plasma.

[0039] In an exemplary embodiment, the concentrated plasma of the present invention is essentially free of more plasma protein aggregate fractions than were present in the starting plasma. Essentially free of more plasma protein aggregate fractions means less than about 2%, less than about 5%, less than about 10%, less than about 15%, or less than about 20% more aggregates on a weight percent basis. The weight percent basis is calculated from the total protein weight in the concentrated plasma pool and the starting plasma. That is, less than about X% of the plasma proteins in the concentrated plasma pool are present as aggregates.

[0040] In exemplary embodiments, the plasma protein fractions isolated according to the methods of the invention have substantially identical properties to those of the same fractions similarly isolated from non-enriched plasma (e.g., frozen plasma) using art-recognized methods. In various embodiments, the properties of the plasma protein fractions are different from those of the same protein fractions similarly isolated from non-enriched plasma using art-recognized methods. In preferred embodiments, the property(ies) that differ between the two plasma protein fractions correspond to one or more regulatory parameters required for marketing approval of a therapeutic plasma protein, and the property varies within such one or more parameters by an amount that is considered insignificant with respect to the regulatory requirements associated with that fraction. That is, pharmaceutical preparations incorporating downstream plasma fractions of the enriched plasma or proteins isolated from the plasma fractions do not require new legal considerations or marketing approvals. In various embodiments, the downstream plasma fractions of the enriched plasma or isolated proteins are essentially identical to the corresponding plasma fractions or isolated proteins from non-enriched plasma.

[0041] In exemplary embodiments, the concentrated plasma pool is the starting input material for an improved fractionation process. In various embodiments, the concentrated plasma pool improves the process by facilitating and / or promoting one or more of the following: (i) shortening fractionation time, (ii) reducing fractionation material investment, (iii) reducing waste, pollutant use, e.g., VOC use, and (iv) increasing throughput with existing fractionation plant infrastructure. In exemplary embodiments, these results are achieved without essentially reducing the yield of the selected plasma protein fraction. In this case, essentially without reducing the yield means that the total plasma protein yield is about 2% or more, about 5% or more, about 10% or more, about 15% or more, or about 20% or more of the total plasma protein yield from a process starting with a non-concentrated plasma input material, as compared to the same fractionation process starting with a non-concentrated plasma input material.

[0042] In various embodiments, the bioactive enriched plasma is treated by adding one or more components used in plasma fractionation, such as alcohol, acid, base. In an exemplary embodiment, the bioactive enriched plasma is maintained in or passes through a component of a fractionation system and is incorporated into the process using such a system. In an exemplary embodiment, the fractionation system is a Cohn fractionation system or a known modification of this system.

[0043] In an exemplary embodiment, the invention provides one or more plasma protein fractions, i.e., the product(s) of a plasma fractionation process beginning with said bioactive enriched plasma. In an exemplary embodiment, said plasma protein fraction is a Cohn fraction, as that term is understood in the art.

[0044] In various embodiments, the invention provides one, two, three, four, five, or more unique plasma fraction composition(s) downstream of the bioactive enriched plasma input material. In various embodiments, the composition is a fraction I paste and comprises fibrinogen, or is a fraction I supernatant. In various embodiments, the composition is a fraction II+III (or fraction I+II+III) paste and comprises IgG, or is a fraction II+III (or fraction I+II+III) supernatant. In some embodiments, the composition is a fraction IV-1 paste and comprises A1PI and / or AT-III, or is a fraction IV-1 supernatant. In exemplary embodiments, the plasma fraction composition is a fraction IV-4 paste and / or a fraction IV-4 supernatant. In various embodiments, the plasma fraction composition is a fraction V paste and comprises albumin, or is a fraction V supernatant. In exemplary embodiments, the fraction(s) are one or more Cohn fractions.

[0045] In an exemplary embodiment, the invention provides a preparation of a protease inhibitor prepared by the method of the invention, in various embodiments, the protease inhibitor is selected from alpha 1-antitrypsin, C1 inhibitor, and the like, and combinations thereof.

[0046] In an exemplary embodiment, the invention provides a preparation of albumin prepared by the methods of the invention.

[0047] In an exemplary embodiment, the method provides an aqueous albumin solution suitable for intravenous injection in a human subject, comprising at least about 5% by volume or at least about 25% by volume albumin, which solution remains stable without precipitation of albumin after exposure to a temperature of about 45° C. for one month. In an exemplary embodiment, the solution is isolated by fractionation of bioactive enriched human plasma of the present invention.

[0048] In an exemplary embodiment, the invention provides a preparation of IgG isolated from bioactive enriched human plasma. The preparation contains IgG in an amount that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more of the amount found in an identical preparation in which IgG is isolated from non-enriched plasma (e.g., fresh frozen plasma). In various embodiments, the activity of the IgG is 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more of the activity of IgG isolated from non-enriched plasma (e.g., fresh frozen plasma). The IgG can be a multivalent or hyperimmune IgG.

[0049] In an exemplary embodiment, the invention provides a plasma protein isolated from fraction IV-1 of fractionated bioactive enriched human plasma selected from A1PI, AT-III, and combinations thereof. In an exemplary embodiment, the plasma protein is isolated in a yield of about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% or more of the yield when the protein is isolated from non-enriched plasma (e.g., fresh frozen plasma). In various embodiments, the protein isolated in fraction IV-1 from the bioactive enriched human plasma has an activity of about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% or more of the activity of the protein isolated from non-enriched plasma (e.g., fresh frozen plasma).

[0050] In some embodiments, the invention provides methods in which albumin isolated from fraction V of the bioactive enriched human plasma is isolated in a yield that is about 80% or greater than the yield when the protein is isolated from a non-enriched plasma input, e.g., fresh frozen plasma. In various embodiments, the albumin isolated from the bioactive enriched human plasma has an activity that is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% or greater than the activity of albumin isolated from non-enriched plasma (e.g., fresh frozen plasma).

[0051] In various embodiments, the invention provides pharmaceutical formulations comprising one of the plasma protein fractions produced by the methods of the invention, or one or more protein components of such fractions further purified from such fractions. Various pharmaceutical formulations also comprise a pharma- ceutical acceptable vehicle in which the plasma fraction, or proteins in / or derived from the fraction (or further purified downstream), are formulated.

[0052] In various embodiments, the invention provides a pharmaceutical formulation of the invention packaged in a device, e.g., a syringe, infusion bag, etc., for administering the pharmaceutical formulation to a subject in need thereof. In various embodiments, the device includes a unit dosage formulation of an active protein for administration to a subject in need thereof. In an exemplary embodiment, the unit dosage is an art-recognized unit dosage for a subject.

[0053] B. Method In various embodiments, the present invention provides novel plasma fractionation methods that begin with the bioactive enriched plasma of the present invention as input material. An exemplary method of the present invention includes, in an exemplary embodiment, providing a bioactive enriched plasma solution prepared by ultrafiltration, and subjecting the bioactive enriched plasma to one or more fractionation processes, including the Cohn fractionation process (FIG. 1), and variations thereof.

[0054] In an exemplary embodiment, the starting plasma used in the method of the present invention is concentrated after pooling (de-cryo, de-coagulation factor, de-inhibitor plasma pool). The concentration of the cone pool can be achieved by several techniques, including but not limited to tangential flow filtration, ultrafiltration, and combinations thereof. For example, the method of concentrating the starting plasma includes (a) batch TFF using UF cassette followed by prefiltration, or (b) single-pass TFF using UF cassette followed by prefiltration, or (c) UF hollow fiber system with or without prefiltration.

[0055] FIG. 3 illustrates an exemplary improved fractionation process of the present invention, with the filtration step identified and located by a red dot.

[0056] The bioactive enriched Cohn pool is concentrated in one embodiment by ultrafiltration, which can be performed in any useful format (i.e., order of addition, temperature, dilution, etc.).

[0057] Proteins can undergo physical degradation by several mechanisms (e.g., clipping, oxidation, unfolding, aggregation, insoluble particulate formation). Many proteins are structurally unstable in solution and are prone to conformational changes due to various stresses encountered during purification, processing, and storage. These stresses include temperature changes, pH changes and exposure to extremes of pH, shear stress, surface adsorption / interfacial stress, etc.

[0058] As will be appreciated by those of skill in the art, these methods of enriching the Cohn pool may be performed alone or in any combination or order. In various embodiments, the bioactive enriched Cohn pool is comprised of at least about 65 g / L of plasma protein.

[0059] In various embodiments, after the cryoprecipitation reaction, the plasma is separated into a cryoprecipitate and a cryosupernatant. The cryosupernatant or the cryosupernatant after adsorption is optionally subjected to a further fractionation step. The separation can be accomplished in any useful manner, such as, but not limited to, centrifugation, filtration, or a combination thereof.

[0060] In embodiments where cooling of the bioactive enriched plasma is desired, any useful cooling means may be utilized. In various embodiments, the vessel or line containing the enriched plasma is jacketed with a cooling device. In exemplary embodiments, the cooling and / or plasma solution is held in a vessel, e.g., a jacketed vessel, and in some embodiments, the plasma solution is cooled during in-line flow (the "radiator method").

[0061] In some embodiments, the physiologically enriched plasma comprises albumin in an amount of about 3.5 to about 5.5 g / dL. In various embodiments, the albumin concentration of the physiologically enriched plasma is about 40% to about 70%, e.g., about 50% to about 60%, of the total plasma protein content of the physiologically enriched plasma.

[0062] In various embodiments, the albumin in the bioactive enriched plasma retains at least about 80%, 85%, 90%, or at least about 95% of its activity on a unit basis of albumin in plasma.

[0063] In some embodiments, the physiologically concentrated plasma contains A1PI in an amount of about 50 to about 300 mg / dL, for example, about 100 to about 200 mg / dL.

[0064] In various embodiments, the A1PI in the bioactive enriched plasma retains at least about 80%, 85%, 90%, or at least about 95% of its activity on a unit basis of A1PI in plasma.

[0065] In various embodiments, the physiologically concentrated plasma contains IgG in an amount of about 500 to about 1600 mg / dL, for example, about 700 to about 1500 mg / dL.

[0066] In various embodiments, the IgG in the bioactive enriched plasma retains at least about 80%, 85%, 90%, or at least about 95% of its activity on a unit basis of IgG in plasma.

[0067] In some embodiments, the bioactive enriched plasma has a mean particle size of about 30 microns or less. In some embodiments, the bioactive enriched plasma has a maximum particle size of about 100 microns or less.

[0068] In some embodiments, the bioactive enriched plasma comprises at least 30% by weight plasma proteins.

[0069] In some embodiments, the bioactive enriched plasma is sterile.

[0070] In an exemplary embodiment, the present invention provides a method for fractionation of bioactive enriched human plasma using a Cohn fractionation procedure, such as that described in U.S. Patent No. 2,390,074, the improvement including the use of bioactive enriched human plasma as the starting material for the fractionation procedure. Figure 1 provides an exemplary process diagram of the Cohn fractionation method.

[0071] Thus, for example, the bioactive enriched plasma is subjected to a protein fractionation method. An exemplary method includes precipitating a selected protein fraction from a solution containing a plurality of protein fractions. The solution is adjusted to have a pH above the isoelectric point of one or more proteins in the fraction from which precipitation is desired. In an exemplary embodiment, the pH of the fractionation solution is lowered to approximately the same as the isoelectric point of the fraction from which precipitation is desired. An exemplary method includes bringing the ionic strength of the solution to 0.1-0.2. Various methods include lowering the temperature of the solution to between about 0° C. and the freezing point of the solution. In some embodiments, an organic precipitant for the plasma protein fractionation is added to the protein solution, the amount of precipitant added being such as to cause precipitation of the desired fraction from the protein solution at the temperature and to separate the precipitate from the solution. In a preferred embodiment, the conditions are adjusted such that substantially only the desired plasma protein fraction precipitates from the solution.

[0072] In various embodiments, a method is provided for fractionating proteins by precipitation from a solution of bioactive enriched human plasma containing a plurality of protein fractions, the method comprising: providing a pH of the solution near the isoelectric point of the desired protein fraction to be precipitated; providing an ionic strength of the solution between 0.01 and 0.2; lowering the temperature of the solution to between about 0° C. and the freezing point of the solution; adding an organic precipitant for proteins to the protein solution, wherein the amount of precipitant added, the pH, the ionic strength, and the temperature are such as to cause precipitation of only the desired fraction from the protein solution; and separating the precipitate from the solution.

[0073] In various embodiments, in a method for fractionating proteins from a solution of bioactive enriched human plasma, the steps include mixing an organic precipitant for proteins with a solution of proteins, adjusting the temperature to 0° C. to −15° C., the amount of precipitant to about 8% to about 40%, the pH to about 4.4 to about 7, and the ionic strength to about 0.05 to about 0.2, and separating from the resulting liquid system the protein precipitate that is insoluble therein.

[0074] In some embodiments, in a method of fractionating proteins from a solution of bioactive enriched human plasma, the steps include mixing an organic precipitant for proteins with a solution of proteins, adjusting and maintaining the temperature above its freezing point but below 0° C., the amount of precipitant at about 10% to about 40%, the pH at about 4.4 to about 7, and the ionic strength at about 0.05 to about 0.2, and separating from the resulting liquid system the protein precipitate that is insoluble therein.

[0075] In some embodiments, a method is provided for fractionating proteins from bioactive enriched human plasma, the steps of adding to a mixture of proteins both an electrolyte and an organic precipitant for proteins, the electrolyte being added in an amount sufficient to bring the ionic strength to about 0.01-0.2, and the precipitant being added in an amount to cause precipitation of only the desired protein fraction, adjusting and maintaining the pH of the solution at about 4.4 to about 7, and its temperature at about 0° C. to about −15° C., thereby precipitating proteins from the resulting system.

[0076] In an exemplary embodiment, the invention provides a method for purifying and crystallizing albumin from a solution of concentrated human plasma, the method comprising dissolving impure albumin in an alcoholic solution containing about 15 to about 40% alcohol, having a pH of about 5.5 to about 6.0, an ionic strength of about 0.05 to about 0.5, and a temperature of about 0° C. to about −5° C., and maintaining the solution in that temperature range until albumin crystallizes from the mixture.

[0077] In an exemplary embodiment, in a method for fractionating substances (e.g., proteins) having different solubilities from a solution of concentrated human plasma at controlled temperature and hydrogen ion concentration, the resulting precipitate is removed and multiple successive fractions of the substance are precipitated by altering one or more of the factors.

[0078] In various embodiments, the present invention provides a method for preventing protein denaturation by modifying a reagent that normally causes the denaturation, the method comprising adding the reagent to a protein solution of concentrated human plasma by diffusion through a semi-permeable membrane.

[0079] In one embodiment, a method for fractionating proteins from a solution of bioactive enriched human plasma is provided, the method comprising contacting the bioactive enriched human plasma with an organic precipitant. Exemplary embodiments include controlling one or more of the amount of precipitant contacted, the temperature, hydrogen ion concentration, and ionic strength of the resulting mixture, separating the resulting precipitate from the supernatant, and separating successive protein fractions by varying multiple factors that affect their solubility.

[0080] In an exemplary embodiment, the organic precipitating agent is added at a temperature of about or below about 0°C.

[0081] In an exemplary embodiment, the organic precipitating agent is an alcohol. In various embodiments, it is added at a temperature of about 0° C. or less than about 0° C.

[0082] In an exemplary embodiment, a method for fractionating proteins from a solution of bioactive enriched human plasma is provided, the method comprising precipitating one or more different protein fractions from the plasma with an organic precipitant (e.g., an alcohol) and the bioactive enriched human plasma, and by varying the temperature of a mixture of the bioactive enriched human plasma and the organic precipitant, where precipitation of successive protein fractions occurs as the temperature is gradually lowered and the organic precipitant concentration of the mixture is increased, the temperature and the percentage of alcohol being related such that the temperature used for precipitation of any given protein fraction is close to but above the freezing point of the mixture at the percentage of alcohol and plasma contained in the mixture.

[0083] Exemplary organic precipitants include ethanol, acetone, dioxane, and combinations thereof.

[0084] In exemplary embodiments, IgG isolated from the bioactive enriched human plasma is isolated in a yield that is about 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater, or about 95% or greater than the yield when the protein is isolated from fresh frozen plasma. In various embodiments, the activity of the IgG is about 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater, or about 95% or greater than the activity of IgG isolated from fresh frozen plasma.

[0085] In exemplary embodiments, a protein isolated from fraction IV-1 of the fractionated bioactive enriched human plasma selected from A1PI, AT-III, and combinations thereof is isolated in a yield of about 60%, 65%, 70%, 75%, 80%, 85%, 90% or more, or about 95% or more of the yield when the protein is isolated from fresh frozen plasma. In various embodiments, the protein isolated in fraction IV-1 from the bioactive enriched human plasma has an activity of about 60%, 65%, 70%, 75%, 80%, 85%, 90% or more, or about 95% or more of the activity of the protein isolated from fresh frozen plasma.

[0086] In some embodiments, the invention provides methods in which the albumin isolated from fraction V of the bioactive enriched human plasma is isolated in a yield of about 80% or greater than the yield when the protein is isolated from fresh frozen plasma. In various embodiments, the albumin isolated from the bioactive enriched human plasma has an activity of about 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater, or about 95% or greater than the activity of albumin isolated from fresh frozen plasma.

[0087] The method provided herein allows the preparation of A1PI composition with very high level of purity.For example, in one embodiment, at least about 95% of the total protein in the A1PI composition provided herein is A1PI.In other embodiments, at least about 96% of the protein in this composition is A1PI, or at least about 97%, 98%, 99%, 99.5% or more of the total protein of the composition is A1PI.

[0088] Similarly, the method provided herein allows the preparation of A1PI composition that contains extremely low levels of contaminants.For example, in certain embodiments, the A1PI composition is provided that contains less than about 10mg / L of contaminants.In other embodiments, the A1PI composition contains less than about 5mg / L of contaminants, preferably less than about 3mg / L of contaminants, and most preferably less than about 2mg / L of contaminants.

[0089] In various embodiments, the A1PI in the bioactive enriched plasma retains at least about 80%, 85%, 90%, or at least about 95% of its activity on a unit basis of A1PI in plasma.

[0090] In one embodiment, the present invention provides an aqueous IgG composition comprising a protein concentration of about 150 g / L to about 250 g / L. In certain embodiments, the protein concentration of the IgG composition is about 175 g / L to about 225 g / L, or about 200 g / L to about 225 g / L, or any suitable concentration within these ranges, such as 150 g / L, 155 g / L, 160 g / L, 165 g / L, 170 g / L, 175 g / L, 180 g / L, 185 g / L, 190 g / L, 195 g / L, 200 g / L, 205 g / L, 210 g / L, 215 g / L, 220 g / L, 225 g / L, 230 g / L, 235 g / L, 240 g / L, 245 g / L, 250 g / L, or thereabouts or more. In a preferred embodiment, the aqueous IgG composition comprises a protein concentration of at or about 200 g / L. In a particularly preferred embodiment, the aqueous IgG composition comprises a protein concentration of at or about 204 g / L.

[0091] The method provided herein allows the preparation of IgG compositions with very high levels of purity.For example, in one embodiment, at least about 95% of the total protein in the IgG composition provided herein is IgG.In other embodiments, at least about 96% of the protein is IgG, or at least about 97%, 98%, 99%, 99.5% or more of the total protein of the composition is IgG.

[0092] Similarly, the methods provided herein allow for the preparation of IgG compositions that contain extremely low levels of contaminants.For example, in certain embodiments, IgG compositions are provided that contain less than about 100 mg / L IgA.In other embodiments, the IgG compositions contain less than about 50 mg / L IgA, preferably less than about 35 mg / L IgA, and most preferably less than about 20 mg / L IgA.In exemplary embodiments, the IgG preparations contain less than about 0.14 mg / mL IgA.

[0093] In some embodiments, the invention provides a preparation of multivalent and / or hyperimmune immunoglobulin (IgG) prepared by the methods of the invention. In various embodiments, the IgG is selected from anti-RhO hyperimmune immunoglobulin, anti-hepatitis B hyperimmune immunoglobulin, anti-rabies hyperimmune immunoglobulin, anti-tetanus IgG hyperimmune immunoglobulin, and combinations of any two or more thereof.

[0094] In various embodiments, the IgG in the bioactive enriched plasma retains at least about 80%, 85%, 90%, or at least about 95% of its activity on a unit basis of IgG in plasma.

[0095] Corn Pool Concentration Process Concentration of the Corn pool can be achieved by several techniques, including (a) batch TFF using a UF cassette followed by prefiltration, or (b) single-pass TFF using a UF cassette followed by prefiltration, or (c) a UF hollow fiber system with or without prefiltration.

[0096] Apparatus and methods for ultrafiltration are known in the art. Figure 2 provides a process flow diagram of an exemplary method / apparatus used to concentrate the starting Cohn pool.

[0097] Ultrafiltration membranes or ultrafiltration hollow fiber filter membranes with a nominal molecular weight cut-off (NMWCO) of about 300 kDa or less can be used to concentrate the cone pool in either a recirculation or single pass configuration, and with or without prior prefiltration. In another embodiment, ultrafiltration membranes or ultrafiltration hollow fiber filter membranes with a nominal molecular weight cut-off (NMWCO) of about 200 kDa or less can be used to concentrate the cone pool in either a recirculation or single pass configuration, and with or without prior prefiltration. In some embodiments, ultrafiltration membranes or ultrafiltration hollow fiber filter membranes with a nominal molecular weight cut-off (NMWCO) of about 100 kDa or less can be used to concentrate the cone pool in either a recirculation or single pass configuration, and with or without prior prefiltration.

[0098] The following examples are provided to illustrate exemplary embodiments of the invention, but are not intended to define or limit the scope of the invention. EXAMPLES

[0099] Example 1 Four test runs were performed, one each for Precipitate G (PptG) and Fraction V (FrV). The desired Corn Pool plasma volumes and protein concentrations were obtained from different fractions of Corn Pool plasma without significant loss of various plasma-derived proteins.

[0100] For each test run pair, i.e., Run 1 and Control 1, Run 2 and Control 2, etc., the same Corn pool batch was used as the starting material. Prior to the Corn fractionation process, approximately 5-12 liters of cryo-poor plasma was concentrated using ultrafiltration membranes in four test runs (Run 1, Run 2, Run 3, Run 4), whereas no ultrafiltration was performed in each of the four control runs. This plasma concentration step used various pre-filters and various TFF filters with nominal molecular weight cut-off (NMWCO) of 100 kDa or less. As a result, the starting protein concentrations of Corn pool plasma in the test runs were 10%-27% higher than those in the control runs. The composition of this test run is shown in Table 1.

[0101] TIFF2025514479000001.tif66165

[0102] For each protein of interest, a comparative study was performed for each of the enriched test and non-enriched control runs. As shown in Table 1, the total protein content in the upstream process of IgG up to Precipitate G (PptG) in the test runs is generally comparable to the control runs.

[0103] TIFF2025514479000002.tif107165

[0104] TIFF2025514479000003.tif116165

[0105] TIFF2025514479000004.tif104165

[0106] TIFF2025514479000005.tif106165

[0107] TIFF2025514479000006.tif156165

[0108] Proteins typically found in fractions IV and V downstream of the Corn pool concentrate are found in these fractions in yields and purities comparable to those found in fractions IV and V in processes beginning with the unconcentrated Corn pool. Exemplary separation and purification process conditions for plasma-derived product intermediates made from Corn pool concentrate are set forth in Tables 1-5.

[0109] The supernatant of fraction II+III was subjected to further fractionation procedures. The supernatant of fraction II+III was contacted with 25% ethanol to obtain precipitate of fraction IV-1.

[0110] TIFF2025514479000007.tif83165

[0111] TIFF2025514479000008.tif78165

[0112] TIFF2025514479000009.tif110165

[0113] Example 2 Cohn Pool Concentration (CPC) aims to expand production capacity and reduce operational costs while minimizing interruptions to commercial supply. The process of the present invention allows for the concentration of Cohn Pool Plasma via ultrafiltration. Ultrafiltration allows for the selective separation, concentration, and purification of protein components. Pilot-scale runs have been performed in the present invention to obtain the desired Cohn Pool Plasma volume and protein concentrations from different fractions of Cohn Pool Plasma without significant loss of various plasma-derived proteins.

[0114] Prior to the Cohn fractionation process, approximately 100 liters of cryo-poor plasma was concentrated using ultrafiltration membranes in the test runs (Run 1, Run 2, Run 3) and no ultrafiltration was performed in the control run. Hollow fiber filters with a nominal molecular weight cut-off (NMWCO) of 100 kDa or less were used in this plasma concentration step. As a result, the starting protein concentration of the Cohn pooled plasma in the test runs was approximately 15% higher than that in the control run. For each protein of interest, the technical specifications of the isolated and purified protein, e.g., recovery, quality, are tabulated and analyzed.

[0115] 1. Immunoglobulin recovery and quality were not affected by cone pool concentration After the concentration step, the immunoglobulin content of the entire run was calculated. The IgG content obtained in the cone pool plasma prepared for ethanol treatment is concentrated without significant loss. As shown in Table 1, the efficiency of IgG upstream in the test run is comparable to the control run.

[0116] The recovery of purified IgG from Precipitate G (PptG) was also compared between the test runs and the control. An IgG suspension was formed by suspending the fraction II+III precipitate obtained from the Cohn fractionation process in a suspension buffer. This IgG suspension was filtered and the filtrate was treated with a surfactant. Precipitate G was generated after adjusting the pH of the surfactant-treated filtrate to about 7.0 and adding ethanol to a final concentration of about 20% to about 30%. Table 2 shows that the purification efficiency of IgG was not affected by the CPC process. Comparable IgG yields from PptG between the test runs and the non-concentrated control runs were observed in this pilot-scale study. The quality of IgG was assessed to be formulation-level for all runs. All results met all limits as shown in Table 3.

[0117] TIFF2025514479000010.tif186165

[0118] TIFF2025514479000011.tif233165TIFF2025514479000012.tif83165

[0119] TIFF2025514479000013.tif203165TIFF2025514479000014.tif228165TIFF2025514479000015.tif93165

[0120] 2. Process parameters for separation and purification of immunoglobulins from the corn pool Exemplary plasma-derived immunoglobulin protein separation process conditions, including cone pool enrichment, Fraction I precipitation and separation, Fraction II+III precipitation and separation, Fraction II+III extract precipitation and separation, and Precipitate G (PptG) precipitation and separation, are listed in Tables 4-8.

[0121] In the exemplary CPC process, the target amount of filter aid and filtration area was calculated based on the volume of concentrated corn pool instead of the volume of cryopreserved plasma (CPP). The corn pool was concentrated by ultrafiltration to at least about 14% (w / v) to form the initial corn pool concentrate. The technical protocol and parameters related to the process of ultrafiltration in all runs are shown in Table 4.

[0122] TIFF2025514479000016.tif163165

[0123] The first corn pool concentrate was further subjected to a fractionation procedure. The first corn pool concentrate was contacted with 8% ethanol at pH approximately 7.0-7.5 to obtain a fraction I precipitate and a fraction I supernatant from the fractionated corn pool. The technical protocols and parameters related to the precipitation and separation of fraction I in all runs are shown in Table 5.

[0124] TIFF2025514479000017.tif122165

[0125] The supernatant of fraction I was further contacted with about 25% ethanol at pH about 6.7 to about 7.3 to form a precipitate of fraction II+III. The technical parameters associated with the isolation of proteins from fraction II+III of the fractionated cone pool in the entire run are shown in Table 6.

[0126] TIFF2025514479000018.tif194165TIFF2025514479000019.tif41165

[0127] The precipitate of fraction II+III was further suspended in suspension buffer to form an IgG suspension. 2 ) was mixed with the IgG suspension for at least about 30 minutes, and then the IgG suspension was filtered to obtain a filtrate and a filter cake. The technical parameters associated with the isolation of proteins from the Fraction II+III extracts for all runs are shown in Table 7.

[0128] TIFF2025514479000020.tif213165TIFF2025514479000021.tif35165

[0129] The filtrate obtained from the fraction II+III precipitate was contacted with a surfactant to form a treated filtrate. The pH of the treated filtrate was adjusted to about 7.0 and ethanol was added to a final concentration of about 20% to about 30%, after which precipitation of Precipitate G occurred. The technical parameters associated with the precipitation and separation of Ppt G from the fraction II+III precipitate for all runs are shown in Table 8.

[0130] TIFF2025514479000022.tif128165

[0131] 3. Plasma-derived products isolated from fractions IV and V of the Corn Pool were not affected by Corn Pool enrichment Proteins normally found in fractions IV and V downstream of the Corn pool concentrate are found in these fractions in yields and purities comparable to those found in fractions IV and V in processes beginning with the unconcentrated Corn pool. Exemplary separation and purification process conditions for plasma-derived products, including albumin, alpha-1 proteinase inhibitor, from the Corn pool concentrate are listed in Tables 9-14.

[0132] The supernatant of fraction II+III was further subjected to fractionation procedure. The supernatant of fraction II+III was contacted with 25% ethanol to obtain precipitate of fraction IV-1. The technical protocol and parameters for isolating the proteins in fraction IV-1 are shown in Table 9.

[0133] TIFF2025514479000023.tif230165

[0134] The supernatant of fraction IV-1 was then contacted with 40% ethanol to obtain the precipitate of fraction IV-4. The technical parameters for isolating the proteins in fraction IV-4 are shown in Table 10.

[0135] TIFF2025514479000024.tif196165TIFF2025514479000025.tif48165

[0136] The supernatant of fraction IV-4 was then contacted with 40% ethanol to obtain a precipitate of fraction V. Fraction V of the present invention mainly contains albumin. The technical protocol and parameters for isolating proteins in fraction V of the fractionated cone pool in all runs are shown in Table 11. As shown in Table 12, a comparable total protein content was observed in fraction IV-4 between run 1 and the non-enriched control run.

[0137] TIFF2025514479000026.tif203165

[0138] TIFF2025514479000027.tif101165

[0139] The quality of the protein isolated from Fraction V was assessed by multiple biochemical and immunological assays, as shown in Table 13. Parameters indicative of the quality of the protein isolated from Fraction V of Run 1 are aligned with the results of the respective non-enriched controls.

[0140] TIFF2025514479000028.tif98165

[0141] The fraction IV-1 intermediate isolated from concentrated Cohn pool plasma was further processed to the A1PI formulation. All relevant processing parameters are summarized in Table 14.

[0142] TIFF2025514479000029.tif191165

[0143] Human albumin and A1PI isolated from concentrated Cohn pool plasma meet all prescribed limits. All results of further tests performed at formulation and intermediate levels are within the prescribed ranges, as shown in Tables 15-17.

[0144] All results met the predefined acceptance criteria at the intermediate and formulation levels. Based on the results presented, it can be concluded that the cone pool enrichment does not adversely affect the quality of either the A1PI or albumin products.

[0145] TIFF2025514479000030.tif156165TIFF2025514479000031.tif102165

[0146] TIFF2025514479000032.tif182165

[0147] TIFF2025514479000033.tif215165

[0148] Example 3 In the present invention, commercial scale runs were performed up to the intermediates of Precipitate G (PptG), Fraction V (FrV) and Fraction IV-1 (Fr IV-1). The desired Corn Pool plasma volumes and protein concentrations were obtained from the different fractions of Corn Pool plasma without significant loss of various plasma derived proteins.

[0149] PptG and Fr IV-1 were further purified at pilot scale to immunoglobulin and A1PI preparations, respectively. FrV was purified at commercial scale to albumin preparations.

[0150] Approximately 4700 liters of cryo-poor plasma was concentrated using ultrafiltration membranes prior to the Cone fractionation process. This plasma concentration step used prefiltration (10 μm + 0.5 μm) followed by batch TFF with UF cassettes with a nominal molecular weight cut-off (NMWCO) of 100 kDa. As a result, the starting protein concentration of the Cone pool plasma in the test run was approximately 15% higher than that of the control run. For each protein of interest, the technical specifications of the isolated and purified protein, e.g., recovery, quality, are tabulated and analyzed.

[0151] 1. Immunoglobulin process performance and product quality were not affected by corn pool concentration Immunoglobulin content was calculated after the concentration step. The IgG content obtained in the cone pool plasma prepared for ethanol treatment was concentrated without significant loss.

[0152] Proteins typically found in Precipitate G and immunoglobulin preparations downstream of Cone pool concentration are found in yields and purities comparable to those found in Precipitate G and immunoglobulin preparations in processes that begin with the non-concentrated Cone pool.

[0153] Operating parameters for upstream and downstream processing, e.g., cycle times and step yields, were found to be comparable to historical commercial production data. IgG quality was assessed at the formulation level and all results met predefined limits as shown in Table 18.

[0154] TIFF2025514479000034.tif185165

[0155] 3. Process parameters for isolation and purification of immunoglobulins from corn pools in a commercial-scale run The corn pool was concentrated by ultrafiltration to about 15% (w / v) to form the initial corn pool concentrate. The technical protocol and parameters associated with the process of ultrafiltration are shown in Table 19.

[0156] TIFF2025514479000035.tif155165

[0157] In the exemplary CPC process, the target amount of filter aid and filtration area was calculated based on the volume of concentrated cone pool instead of the volume of cryo-poor plasma (CPP).

[0158] The first corn pool concentrate was further subjected to a fractionation procedure: the first corn pool concentrate was contacted with 8% ethanol at a pH of about 7.0-7.5 to obtain a fraction I precipitate and a fraction I supernatant from the fractionated corn pool.

[0159] The supernatant of Fraction I was further contacted with about 25% ethanol at a pH of about 6.7 to about 7.3 to form a precipitate of Fraction II+III.

[0160] The precipitate of fraction II+III was further suspended in suspension buffer to form an IgG suspension. 2 ) was mixed with the IgG suspension for at least about 30 minutes, and then the IgG suspension was filtered to obtain a filtrate and a filter cake.

[0161] The filtrate obtained from the precipitate of Fraction II+III was contacted with a surfactant to form a treated filtrate. After adjusting the pH of the treated filtrate to about 7.0 and adding ethanol to a final concentration of about 20% to about 30%, precipitation of Precipitate G occurred.

[0162] 3. Plasma-derived products isolated from fractions IV and V of the Corn Pool were not affected by Corn Pool enrichment Proteins normally found in fractions IV and V downstream of the Corn pool enrichment are found in these fractions in yields and purities comparable to those found in fractions IV and V in processes beginning with the unenriched Corn pool.

[0163] The supernatant of fraction II+III was subjected to further fractionation procedures. The supernatant of fraction II+III was contacted with 25% ethanol to obtain precipitate of fraction IV-1.

[0164] The supernatant of fraction IV-1 was then contacted with 40% ethanol to obtain a precipitate of fraction IV-4.

[0165] The supernatant of fraction IV-4 was then contacted with 40% ethanol to obtain a precipitate of fraction V.

[0166] The quality of the protein isolated from Fraction V was assessed by multiple biochemical and immunological assays, as shown in Table 20. Parameters indicative of the quality of the protein isolated from Fraction V from commercial scale runs are comparable to previous commercial production data starting from non-enriched corn pools.

[0167] TIFF2025514479000036.tif41165

[0168] Human albumin and A1PI isolated from concentrated Corn Pool plasma were tested at the final formulation level. All results at the formulation level were within expected ranges as shown in Tables 21-22.

[0169] All results met the predefined acceptance criteria at the formulation level. Based on the results presented, it can be concluded that the cone pool enrichment does not adversely affect the quality of either the A1PI or albumin products.

[0170] TIFF2025514479000037.tif169165

[0171] TIFF2025514479000038.tif65165

[0172] Further laboratory scale filtration experiments demonstrated that permeate flux was comparable for filters with molecular weight cutoffs up to 300 kDa when concentrating the Corn pool.

Claims

1. A method for preparing a corn pool concentrate from plasma, comprising: ultrafiltration of the corn pool total protein in the corn pool in a tangential flow filtration mode using an ultrafiltration membrane with a nominal molecular weight cutoff (NMWCO) of 100 kDa or less, thereby concentrating the corn pool from a first concentration to a second concentration 10% to 27% higher than the first concentration.

2. A method for preparing a Cornpool concentrate from plasma, (a) providing a cone pool from plasma, and (b) Concentrating the corn pool to a total protein concentration of at least about 65 g / L, thereby forming a corn pool concentrate. The method, including the method described above.

3. The method according to claim 1 or 2, wherein the cone pool is a member selected from decryoplasma, defactored plasma, and deinhibitored plasma.

4. The method according to claim 2, wherein step (b) is performed with or without preceding prefiltration, using a run of an ultrafiltration membrane having a nominal molecular weight cutoff (NMWCO) of 300 kDa or less, in either a recirculation or single-pass configuration.

5. The method according to claim 2, wherein step (b) is performed with or without preceding prefiltration, either in a recirculation or single-pass configuration, using a run of hollow fiber filter membranes having a nominal molecular weight cutoff (NMWCO) of 300 kDa or less.

6. The method according to claim 1 or 2, wherein the corn pool concentrate has a total protein concentration of at least about 65 g / L.

7. The method according to claim 1 or 2, wherein the cornpool concentrate is further subjected to a purification process for preparing a composition selected from an immunoglobulin G (IgG) composition, an albumin composition, an A1-PI composition, and combinations thereof.

8. The method according to claim 1 or 2, wherein the corn pool is concentrated without any essential loss of protein mass due to a member selected from total protein, IgG, albumin, AAT, and fibrinogen in the resulting corn pool concentrate.

9. The method according to claim 1 or 2, further comprising sending the corn pool concentrate to a plasma fractionation procedure.

10. The method according to claim 9, wherein the fractionation procedure is a cone fractionation method or one of its variations.

11. The aforementioned fractionation procedure is (i) In order to obtain the precipitate of fraction I and the supernatant of fraction I, the corn pool concentrate is brought into contact with ethanol at a pH of approximately 6% to approximately 10% at a pH of approximately 7.0 to 7.5, and (ii) To form a precipitate of fraction II + III or, alternatively, a member selected from the precipitates of fraction I + II + III, contact the supernatant or concentrated corn pool of fraction I with alcohol at a pH of approximately 18% to approximately 27% at approximately 6.7 to approximately 7.

3. The method according to claim 10, including the method described in claim 10.

12. (iii) Suspending a member selected from the precipitate of fraction II + III and the precipitate of fraction II + III (II + III or alternatively I + II + III) in a suspension buffer to form a suspension of IgG. (iv) Finely powdered silicon dioxide (SiO ２ Mix the above IgG suspension with the above IgG suspension for at least about 30 minutes. (v) Filtering the IgG suspension to form a filtrate and a filter cake. The method according to claim 11, further comprising:

13. (vi) Contacting the filtrate with a surfactant to form a treated filtrate. (vii) Adjust the pH of the filtrate from step (vi) to approximately 7.0, and add ethanol to a final concentration of approximately 20% to approximately 30%, thereby forming the precipitate G. (viiii) Dissolve the precipitate of precipitate G in an aqueous solution containing a member selected from a solvent, a surfactant, and a combination thereof, to form a solution of precipitate G. (ix) Pass the solution through a cation exchange material to adsorb the proteins contained therein onto the cation exchange material, and then elute the adsorbed proteins into an eluate. (x) Passing the eluate through an anion exchange material to generate a flow-through effluent, (xi) Pass the effluent through a nanofilter to generate a nanofiltrate. (xi) Concentrating the nanofiltrate by ultrafiltration to produce a first ultrafiltrate, (xiii) Dialysis of the first ultrafiltrate against a dialysis buffer to produce a dialysis filtrate, and (xi) The dialysate is concentrated by ultrafiltration to produce a second ultrafiltrate having a protein concentration of about 8% (w / v) to about 22% (w / v), thereby forming a fraction concentrated with IgG. The method according to claim 12, further comprising:

14. (vi) Before that, wash the filter cake with a washing buffer having a pH of about 4.9 to about 5.3 in at least one filter-pressed dead volume, thereby forming a washing solution; The filtrate is combined with the washing solution to form a solution, and the solution is treated with a surfactant in step (vi). The method according to claim 13, further comprising:

15. A plasma protein preparation wherein the protein is a member selected from IgG, A1PI, and albumin prepared by the method described in claim 12.

16. A pharmaceutical formulation comprising albumin isolated from the cornpool concentrate according to claim 1 or 2, and a pharmaceutically acceptable medium.

17. A pharmaceutical formulation comprising AAT isolated from the corn pool concentrate according to claim 1 or 2, and a pharmaceutically acceptable medium.

18. A pharmaceutical formulation comprising IgG isolated from the corn pool concentrate according to claim 1 or 2, and a pharmaceutically acceptable medium.

19. A pharmaceutical formulation comprising fibrinogen isolated from the cornpool concentrate according to claim 1 or 2, and a pharmaceutically acceptable medium.

20. A pharmaceutical formulation comprising TP isolated from the corn pool concentrate according to claim 1 or 2, and a pharmaceutically acceptable medium.

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