Method for preparing Cohn cell concentrate from plasma by ultrafiltration

Abstract

The present invention provides methods for fractionating human plasma in some embodiments using a Cohn fractionation procedure. Improvements include the use of physiologically active concentrated plasma as starting material for the fractionation procedure.

Description

Field of the Invention

[0001] The present invention belongs to the field of plasma fractionation for separating therapeutically active plasma proteins from plasma. Background of the Invention

[0003] Clinical utilization of plasma protein-based therapies has steadily increased over the past decade. For example, with the increased awareness of primary immunodeficiency diseases (PIDs), there has been an increase in the effective clinical use of intravenous immunoglobulin (IVIG) in patient populations affected by PIDs. IVIG is increasingly being used for off-label indications and conditions such as chronic inflammatory demyelinating polyneuropathy (CIPD).

[0004] In 2019, the plasma products market is expected to grow at a CAGR of 6.8%, from $20.5B in 2018 to $28.5B in 2023. In 2016, the global annual fractionation capacity was approximately 70.7 million liters.

[0005] The plasma fractionation process is an infrastructure / facility intensive and highly regulated process. To meet the growing demand for plasma-derived therapies, existing infrastructure must be able to meet this demand, or infrastructure must be modified or increased, with the latter two of these options requiring significant capital expenditures, potential disruption to production lines, and potential regulatory review and certification of modified or new facilities. Therefore, options to improve the efficiency of existing infrastructure without significant modifications to existing infrastructure are very attractive.

[0006] Methods for reducing the volume of liquid plasma input during fractionation can achieve the dual benefits of maximizing the productivity of existing fractionation infrastructure and reducing the level of resources required during plasma fractionation. An apparently unexplored method prior to the present invention is a method in which a reduced volume of liquid is processed to produce the same amount of plasma-derived protein product as produced from a larger volume of liquid (e.g., fresh frozen plasma (FFP)).

[0007] Thus, until the present invention described herein, it was not known that proteins in various fractions (e.g., cold ethanol fractions) could be recovered by fractionating a sufficient amount of concentrated plasma Cohen pools to make the expense of concentrating and fractionating concentrated physiologically active plasma worthwhile. In addition, it was not clear whether the concentrated plasma Cohen pools functioned in a manner similar to fresh frozen plasma in Cohen fractionation (or its known modifications). The inventors have discovered that fractionation pathways derived from concentrated plasma Cohen pools are truly feasible. Concentrated Cohen pools are an integral part of economically viable fractionation processes, such as Cohen fractionation or Kistler-Nitschman fractionation, or other methods that begin with concentrated plasma Cohen pools (e.g., Gerlough, Hink, and Mulford methods). See, e.g., 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] In view of the increasing use of therapeutic plasma-derived blood protein compositions (such as immunoglobulin compositions, albumin, protease inhibitors, coagulation factors, coagulation factor inhibitors and proteins of the complement system), it is of vital importance to ensure sufficient, economical, environmentally friendly, effective and safe plasma-derived blood protein compositions and sustainable access to the blood protein compositions.

[0009] It is now found that improving the fractionation process by inputting a concentrated plasma Cohen pool into a standard plasma protein fractionation process can produce process efficiencies that are substantially proportional to the increase in concentration of the plasma input introduced into the process due to the reduction in the volume of the liquid to be fractionated and in some embodiments surprisingly. The improved method results in increased utilization of treatment equipment and increased throughput, which is roughly proportional to the concentration factor in some embodiments and can therefore be significant. In various embodiments, the invention provides a method for improved plasma fractionation with one or more of these improved properties. A plasma protein product prepared using an improved procedure is also provided.

[0010] By the present invention, it has been found that concentrated plasma Cohen pools are effective starting materials for preparing protein therapeutics by fractionating the concentrated Cohen pools. In various embodiments, proteins that are typically present in various Cohen fractions downstream of the concentrated plasma Cohen pool are present in these fractions at a yield and purity similar to the yield and purity of proteins present in the corresponding fractions in a process starting with (non-concentrated) plasma starting materials such as cryo poor plasma, plasma after one or more absorption steps, recovered plasma, plasma from plasmapheresis, frozen plasma, thawed plasma, etc.

[0011] An exemplary method of the present invention comprises: (a) subjecting a concentrated plasma Cohn pool to one or more plasma fractionation processes (e.g., cold ethanol fractionation). In an exemplary embodiment, the present invention provides that prior to (a), (b) preparing a concentrated plasma Cohn 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 Cohen pool. In various embodiments, the improvement also includes concentrating the plasma input before subjecting the concentrated input to the first alcohol fractionation step. An exemplary plasma input is concentrated cryogenically depleted plasma.

[0013] In various embodiments, the reduction in plasma input volume and the concomitant increase in plasma protein concentration results in a plasma fractionation method characterized (to a surprising degree) by a reduction in volume / increase in plasma protein concentration.

[0014] In various embodiments, the present invention provides an improved plasma fractionation process that is performed until a selected step is completed within a determined amount of time. The improvement of the plasma fractionation process includes completing a selected final step in a reduced time relative to an otherwise identical plasma fractionation process (except that the plasma input to the first alcohol fractionation step in the method of the present invention is concentrated relative to the plasma input in the otherwise identical process). An exemplary plasma input is concentrated cryogenically depleted plasma, and thus in this embodiment, the concentrated cryogenically depleted plasma is a concentrated Cohen pool.

[0015] For example, a fractionation process initiated with a Cohen pool that is concentrated by about 10%, about 20%, or about 30% relative to a standard plasma fractionation input results in the use of about 10%, about 20%, or about 30% less reagents than a similar process that starts with an unconcentrated input. Similarly, a fractionation process initiated with a Cohen pool that is concentrated by about 10%, about 20%, or about 30% relative to a standard plasma fractionation input results in a process that takes about 10%, about 20%, or about 30% less time from start to finish than a similar process that starts with an unconcentrated input. This result is unexpected because the quality of the plasma proteins in the concentrated input is substantially unchanged between the concentrated input and the non-concentrated input.

[0016] In an exemplary embodiment, the invention provides an improved method for preparing a protein fraction from plasma, wherein the fraction is enriched in a plasma protein product selected from the group consisting of coagulation factors (e.g., factors V, VII, VIII, IX, X, XI, XII, and XIII), prothrombin complex, von Willebrand factor, factor VIII / von Willebrand factor, fibrin, fibrinogen, thrombin, multivalent and hyperimmune (such as anti-RhO, anti-hepatitis B, anti-rabies, or anti-tetanus) immunoglobulins (IgG), protease inhibitors (such as α1 antitrypsin and C1 inhibitor), anticoagulants (such as antithrombin), C1 esterase inhibitors, protein C, and albumin, and combinations thereof. The improvement comprises concentrating the plasma input, preparing a concentrated Cohen pool, and then introducing the concentrated Cohen pool into the first alcohol fractionation step of the plasma fractionation method. An exemplary plasma input is concentrated poor cryopreserved plasma.

[0017] The improved process of the present invention is applicable to any plasma fractionation process, for example, Cohen, Gerlough, Hink, Mulford, Kistler Nitschmann, hot ethanol fractionation, Hao, etc.

[0018] The present invention also provides a plasma processing system, preferably a cGMP compliant system, particularly useful for fractionating plasma from a concentrated Cohn pool input (eg, concentrated cryo-poor plasma). BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a general flow chart of an exemplary Cohn fractionation procedure.

[0020] Figure 2 is an exemplary flow diagram for concentrating the starting plasma to form a concentrated Cohn pool that is input into a fractionation process.

[0021] Figure 3An exemplary improved fractionation process of the present invention is shown, wherein the filtration steps are identified and located with red dots. DETAILED DESCRIPTION

[0022] I. Introduction

[0023] Comprising approximately 55% of the total volume of whole blood, plasma is the component of whole blood in which blood cells and other components of whole blood are suspended. Plasma also contains a mixture of over 700 proteins and additional substances that perform functions necessary for good health, including, among others, coagulation, protein storage, and electrolyte balance. When extracted from whole blood, plasma can be used to replace body fluids, antibodies, and coagulation factors. As a result, plasma is widely used in medical treatments.

[0024] As explained above and in the following sections, by initiating fractionation with a concentrated plasma Cohn pool, the present invention imparts numerous efficiency and other advantages to the fractionation process.

[0025] Reference will now be made in detail to the implementation of the exemplary embodiments of the present disclosure as shown in the accompanying drawings. The same reference numerals will be used throughout the accompanying drawings and in the following detailed description to refer to the same or similar parts. Those of ordinary skill in the art will appreciate that the following detailed description is merely illustrative and is not intended to be limiting in any way. Such technicians who benefit from the present disclosure will themselves easily associate other embodiments of the present disclosure.

[0026] For the sake of clarity, not all conventional features of the implementations described herein are shown and described. It will be appreciated that in the development of any such actual implementation, many implementation-specific decisions will be made to achieve the specific goals of the plasma product manufacturer, such as compliance with application-related constraints and business-related constraints, and these specific goals will vary between various implementations and between various plasma product manufacturers. In addition, it will be appreciated that such development work may be complex and time-consuming, but is still a routine task of engineering design for those of ordinary skill in the art who benefit from the present disclosure.

[0027] Without departing from the spirit and scope of the exemplary embodiments, many modifications and variations may be made to the exemplary embodiments set forth in this disclosure, which 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 appended claims and the full scope of equivalents to which such claims are entitled.

[0028] II. Abbreviations and definitions

[0029] Unless defined otherwise, 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 the invention belongs. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, pharmaceutical formulation, and medical imaging are those well known and commonly employed in the art.

[0030] b. Definition

[0031] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "a protein" means one protein or more than one protein.

[0032] "Cohn process" and "Cohn fractionation" are used interchangeably herein and as generally understood, refer to a method of separating human plasma by a series of steps that produce fractions enriched in certain plasma proteins, including ethanol precipitation at different concentrations, changes in pH, changes in temperature, changes in ionic strength. See, e.g., U.S. Pat. No. 2,390,074. Figure 1 An exemplary flow chart of the Cohn process is provided. As used herein, the terms "Cohn process" and "Cohn fractionation" also refer to many variations and improvements of this pioneering process, for example, the Kistler-Nitschmann process (Kistler et al. (1952), Vox Sang, 7, 414-424). Other processes for the methods of the present invention include the method for separating IgG described in U.S. Patent No. 8,940,877.

[0033] "Plasma" is the fluid that remains after blood has been centrifuged (for example) to remove cellular materials such as red blood cells, white blood cells, and platelets. Plasma is typically yellow and transparent to opaque. Blood that has been donated and processed to separate plasma from other specific blood components and has not been frozen is referred to as "never frozen" plasma. Plasma that has been frozen to the temperatures described herein within 8 hours is referred to herein as "fresh frozen plasma" ("FFP"). It contains dissolved components of blood, such as proteins (6% to 8%; e.g., serum albumin, globulin, fibrinogen, etc.), glucose, coagulation factors (clotting proteins), electrolytes (Na, + , Ca 2+ Mg 2+ 、HCO3 - , Cl - etc.), hormones, etc. Whole blood (WB) plasma is plasma separated from whole blood with no additives other than anticoagulants. As the name indicates, citrate phosphate dextrose (CPD) plasma contains citric acid, sodium phosphate, and sugar, usually dextrose, added as an anticoagulant.

[0034] "Recovered plasma" refers to plasma that is separated no later than 5 days after the expiration date of whole blood and stored at 1 to 6 ° C. The characteristics of the plasma proteins in liquid plasma are not fully characterized. The level and activation state of the coagulation proteins in liquid plasma depend on the cells, as well as the storage conditions and duration and vary with the time of contact with the cells. This component serves as a source of plasma proteins. The level and activation state of the coagulation proteins can vary and change over time.

[0035] "Thawed plasma" refers to plasma derived from a source, FFP or FP24, prepared using aseptic techniques (closed system), thawed at about 10°C to about 37°C, and maintained at about 1°C to about 6°C until about 4 days after the initial 24 hour post-thaw period has elapsed. Thawed plasma contains stable coagulation factors (such as factor II and fibrinogen) at concentrations similar to those of FFP, but contains variably reduced amounts of other factors. Exemplary thawed plasma is a component of the first fractionation step, in which plasma (source, recovery, FF, etc.) is removed from a plastic container and thawed in a jacketed container (exchange fluid with a temperature of up to 37°C).

[0036] "Fresh frozen plasma" ("FFP") refers to plasma that is prepared from whole blood or apheresis collection and frozen at about -18°C or colder (e.g., frozen within eight hours of extraction) within a time frame as specified in the instructions for use of the relevant blood collection, processing, and storage system. On average, a unit contains 200 to 250 mL, but apheresis-derived units can contain as much as 400 to 600 mL. FFP contains plasma proteins including all clotting factors. FFP contains higher levels of labile clotting factors V and VIII.

[0037] As used herein, "factor" followed by a Roman numeral refers to a series of plasma proteins related by a complex cascade of enzyme-catalyzed reactions involving the orderly cleavage of large protein molecules to produce peptides, each of which converts an inactive zymogen precursor into an active enzyme, resulting in the formation of a fibrin clot. They include: factor I (fibrinogen), factor II (prothrombin), factor III (tissue thromboplastin), factor IV (calcium), factor V (prothrombinogen), factor VI (no longer considered to have hemostatic activity), factor VII (proconvertin), factor VIII (antihemophilic factor), factor IX (plasma thromboplastin component; Christmas factor), factor X (Stuart factor), factor XI (plasma thromboplastin precursor), factor XII (hageman factor), and factor XIII (fibrin stabilizing factor).

[0038] "Plasma proteins" include, for example, coagulation factors (such as factors V, VII, VIII, IX, X, XI, XII, and XIII), prothrombin complex, von Willebrand factor, factor VIII / von Willebrand factor, fibrin, fibrinogen, thrombin, multivalent and hyperimmune (such as anti-RhO, anti-hepatitis B, anti-rabies, or anti-tetanus) immunoglobulins (IgG), protease inhibitors (such as alpha 1 antitrypsin and C1 inhibitor), anticoagulants (such as antithrombin), C1 esterase inhibitors, protein C, and albumin, and combinations thereof.

[0039] As used herein, the term concentrated plasma "Cohn pool" refers to a pool of plasma (e.g., cryo-depleted, coagulation factor-depleted, inhibitor-depleted plasma) that is concentrated and subsequently subjected to a plasma fractionation process in the methods of the present invention. An exemplary concentrated plasma Cohn pool is physiologically active plasma that is concentrated by about 10% to about 30% from its original volume (e.g., the volume when collected from a donor or donor pool, received by a fractionation facility, etc.) and includes proteins that have not been destroyed to the extent of losing substantially all of their physiological activity.

[0040] In exemplary embodiments, the concentration process results in substantially no reduction in the activity of selected plasma proteins (e.g., IgG, A1PI, factors, etc.) that are subsequently separated (or enriched) by fractionation of the concentrated Cohn pool. In various embodiments, the activity of the selected plasma proteins fractionated from the physiologically active concentrated Cohn pool is at least about 99%, at least about 90%, at least about 85%, or at least about 80% of the activity of the selected plasma proteins in the plasma material prior to its concentration. In various embodiments, the selected plasma protein is a multivalent or hyperimmune IgG.

[0041] A "disease" is a health state in an animal in which the animal cannot maintain homeostasis and if the disease is not ameliorated, the animal's health continues to deteriorate. In various embodiments, one or more proteins from the fractionated physiologically active concentrated Cohn pool are used to treat one or more diseases.

[0042] III. Implementation Plan

[0043] A. Composition and Device

[0044] Embodiments of the present disclosure relate to methods of concentrating a physiologically active plasma feedstock to form a concentrated Cohen pool and subsequently fractionating the resulting physiologically active concentrated Cohen pool using an art-recognized fractionation process. An exemplary fractionation process includes an alcohol fractionation step as its first step. Also provided are plasma protein preparations prepared by a fractionation process starting with a concentrated Cohen pool.

[0045] In an exemplary embodiment, the invention provides a physiologically active concentrated plasma. When compared to the starting plasma from which the concentrated plasma was produced, the biological and physiological activity of the concentrated plasma is not substantially reduced. Substantially not reduced means that, for any selected plasma protein, its activity in the concentrated plasma is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% 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 concentrated plasma is not substantially reduced when compared to the starting plasma.

[0046] In an exemplary embodiment, the concentrated plasma of the present invention does not substantially include a greater fraction of plasma protein aggregates than the plasma protein aggregates present in the starting plasma. Substantially no greater fraction of plasma protein aggregates means no more than about 2%, no more than about 5%, no more than about 10%, no more than about 15%, or no more than about 20% more aggregates on a wt% basis. The wt% basis is calculated based on the total protein weight in the concentrated plasma pool and the starting plasma, i.e., no more than about X% of the plasma proteins in the concentrated plasma pool are present as aggregates.

[0047] In an exemplary embodiment, the plasma protein fraction separated according to the inventive method has substantially the same characteristics as the characteristics of the same fraction separated from not yet concentrated plasma (e.g., frozen plasma) using methods recognized in the art. In various embodiments, the characteristics of the plasma protein fraction are different from the characteristics of the same protein fraction separated from not yet concentrated plasma using methods recognized in the art in the same way. In a preferred embodiment, the different characteristics between the two plasma protein fractions correspond to one or more regulatory-related parameters necessary for approval of the sale of therapeutic plasma proteins, and the characteristics are within the range of the one or more parameters, with an amount that is considered to be insignificant relative to the relevant regulatory requirements of the fraction, that is, the plasma fraction incorporated into the downstream of concentrated plasma or the pharmaceutical formulation of the protein separated from the plasma fraction does not require new regulatory considerations or sales approval. In various embodiments, the plasma fraction or protein separated from the downstream of concentrated plasma is substantially the same as the corresponding plasma fraction or protein separated from non-concentrated plasma.

[0048] In an exemplary embodiment, the concentrated plasma pool is the starting input of the improved fractionation process. In various embodiments, the concentrated plasma pool improves the process by contributing to and / or promoting one or more of the following items: (i) reducing the fractionation time, (ii) reducing the fractionation material expenditure, (iii) reducing waste, such as the use of pollutants of VOCs, and (iv) increasing the output using the existing fractionation device infrastructure. In an exemplary embodiment, these results are achieved without substantially reducing the yield of the selected plasma protein fraction. In this case, substantially not reducing the yield means that, when compared with the same fractionation process starting with non-concentrated plasma input, the total yield of plasma proteins is at least about 2%, at least about 5%, at least about 10%, at least about 15% or at least about 20% of the total yield of plasma proteins in the process starting with non-concentrated input.

[0049] In various embodiments, the processing of the physiologically active concentrated plasma is performed by adding one or more components used in plasma fractionation (e.g., alcohol, acid, base). In an exemplary embodiment, the physiologically active concentrated plasma is maintained or passed through components of a fractionation system and incorporated into a process using such a system. In an exemplary embodiment, the fractionation system is a Cohen fractionation system, or a known modification of that system.

[0050] In an exemplary embodiment, the invention provides one or more plasma protein fractions, products of a plasma fractionation process starting with physiologically active concentrated plasma. In an exemplary embodiment, the plasma protein fraction is a Cohn fraction, as that term is understood in the art.

[0051] In various embodiments, the present invention provides one, two, three, four, five or more unique plasma fraction compositions located downstream of the input of physiologically active concentrated plasma. In various embodiments, the composition is a fraction I paste and contains fibrinogen, or a fraction I supernatant. In various embodiments, the composition is a fraction II+III (or fraction I+II+III) paste and contains IgG, or a fraction II+III (or fraction I+II+III) supernatant. In some embodiments, the composition is a fraction IV-1 paste and contains A1PI and / or AT-III, or a fraction IV-1 supernatant. In an exemplary embodiment, 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 contains albumin, or a fraction V supernatant. In an exemplary embodiment, one or more fractions are one or more Cohen fractions.

[0052] In an exemplary embodiment, the present invention provides a preparation of a protease inhibitor prepared by the method of the present invention. In various embodiments, the protease inhibitor is selected from α1 antitrypsin, C1 inhibitor, etc.) and combinations thereof.

[0053] In an exemplary embodiment, the invention provides a preparation of albumin prepared by the method of the invention.

[0054] In an exemplary embodiment, the method provides an aqueous albumin solution containing at least about 5% by volume or at least about 25% by volume albumin and suitable for intravenous injection in a human subject, the solution being stable without precipitation of albumin after exposure to a temperature of about 45° C. for a period of one month. In an exemplary embodiment, the solution is separated by fractionating the physiologically active concentrated human plasma of the invention.

[0055] In an exemplary embodiment, the invention provides a preparation of IgG separated from physiologically active concentrated human plasma. The preparation comprises an IgG of at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the amount present in the same preparation of separating IgG from non-concentrated plasma (e.g., fresh frozen plasma). In various embodiments, the activity of IgG is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the activity of IgG separated from non-concentrated plasma (e.g., fresh frozen plasma). IgG can be multivalent or highly immune IgG.

[0056] In an exemplary embodiment, the present invention provides a plasma protein isolated from fraction IV-1 of fractionated physiologically active concentrated human plasma, the plasma protein being selected from A1PI, AT-III and combinations thereof. In an exemplary embodiment, the plasma protein is isolated at a yield of at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the yield of the protein isolated from non-concentrated plasma (e.g., fresh frozen plasma). In various embodiments, the protein in fraction IV-1 separated from physiologically active concentrated human plasma has at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the activity of the protein isolated from non-concentrated plasma (e.g., fresh frozen plasma).

[0057] In some embodiments, the present invention provides a method wherein albumin isolated from fraction V of physiologically active concentrated human plasma is isolated at a yield of at least about 80% of the yield of the protein isolated from a non-concentrated plasma input (e.g., fresh frozen plasma). In various embodiments, the albumin isolated from physiologically active concentrated human plasma has at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the activity of the albumin isolated from non-concentrated plasma (e.g., fresh frozen plasma).

[0058] In various embodiments, the invention provides a pharmaceutical formulation comprising one of the plasma protein fractions produced by the method of the invention, or one or more protein components of such fractions further purified from such fractions. Various pharmaceutical formulations also include a pharmaceutically acceptable vehicle in which the plasma fraction or proteins in / from the fraction (or downstream when further purified) are formulated.

[0059] In various embodiments, the invention provides a pharmaceutical formulation of the invention packaged in a device for administering the pharmaceutical formulation to a subject in need of such administration, e.g., a syringe, an infusion bag, etc. In various embodiments, the device contains a unit dose formulation of an active protein administered to a subject in need of such administration. In an exemplary embodiment, the unit dose is an art-recognized unit dose for a subject.

[0060] B. Methods

[0061] In various embodiments, the present invention provides novel plasma fractionation methods starting with the physiologically active concentrated plasma of the present invention as input. Exemplary methods of the present invention include: providing a physiologically active concentrated plasma solution prepared by ultrafiltration in exemplary embodiments; and subjecting the physiologically active concentrated plasma to one or more fractionation processes. Figure 1 ) and variations thereof.

[0062] In an exemplary embodiment, the starting plasma for the inventive method is concentrated after pooling (poor low temperature, poor coagulation factor, poor inhibitor plasma pool). The concentration of Cohen's pool can be achieved by including but not limited to many technologies of tangential flow filtration, ultrafiltration and combinations thereof. For example, the method of concentrating the starting plasma includes: (a) pre-filtration, followed by batch TFF with UF boxes, or (b) pre-filtration, followed by single-pass TFF with UF boxes, or (c) UF hollow fiber system with or without pre-filtration.

[0063] Figure 3 An exemplary improved fractionation process of the present invention is shown, wherein the filtration steps are identified and located with red dots.

[0064] In one embodiment, the physiologically active concentrated Cohn pool is concentrated by ultrafiltration. Ultrafiltration can be performed in any useful format (ie, order of addition, temperature, dilution, etc.).

[0065] Proteins can be physically degraded by many mechanisms (e.g., trimming, oxidation, unfolding, aggregation, insoluble particle formation). Many proteins are structurally unstable in solution and are susceptible to conformational changes due to various stresses encountered during purification, handling, and storage. These stresses include temperature fluctuations, exposure to pH changes and extreme pH, shear stress, surface adsorption / interfacial stresses, etc.

[0066] As will be appreciated by one skilled in the art, any of these Cohn cell concentration modes may be performed alone or in any combination or order.

[0067] In various embodiments, the physiologically active concentrated Cohn pool consists of at least about 65 g / L plasma protein.

[0068] In various embodiments, after cryoprecipitation, 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. Separation can be accomplished in any useful manner such as, but not limited to, centrifugation, filtration, or a combination thereof.

[0069] In those embodiments where it is desired to cool the physiologically active concentrated plasma, any useful cooling means may be utilized. In various embodiments, the container or line containing the concentrated plasma is jacketed with a cooling device. In exemplary embodiments, the cooling and / or plasma solution is maintained in a container (e.g., a jacketed container), and in some embodiments, the plasma solution is cooled during inline flow ("radiator method").

[0070] In some embodiments, the physiologically concentrated plasma contains albumin in an amount of about 3.5 to about 5.5 g / dL. In various embodiments, the albumin concentration of the physiologically active concentrated plasma is about 40% to about 70%, such as about 50% to about 60%, of the total plasma protein content of the physiologically active concentrated plasma.

[0071] In various embodiments, the albumin in the physiologically active concentrated plasma retains at least about 80%, 85%, 90%, or at least about 95% of the activity of the albumin in the plasma on a per unit basis.

[0072] In some embodiments, the physiologically concentrated plasma contains AlPI in an amount of about 50-300 mg / dL, such as about 100 to about 200 mg / dL.

[0073] In various embodiments, the physiologically active concentrated plasma AlPI retains at least about 80%, 85%, 90%, or at least about 95% of the activity of AlPI in plasma on a per unit basis.

[0074] In various embodiments, the physiologically concentrated plasma contains IgG in an amount of about 500 to about 1600 mg / dL, such as about 700 to about 1500 mg / dL.

[0075] In various embodiments, the physiologically active concentrated plasma IgG retains at least about 80%, 85%, 90%, or at least about 95% of the activity of the IgG in the plasma on a per unit basis.

[0076] In some embodiments, the physiologically active concentrated plasma has an average particle size of about 30 microns or less. In some embodiments, the physiologically active concentrated plasma has a maximum particle size of about 100 microns or less.

[0077] In some embodiments, the physiologically active concentrated plasma comprises at least 30% by weight plasma proteins.

[0078] In some embodiments, the physiologically active concentrated plasma is sterile.

[0079] In an exemplary embodiment, the invention provides a method for fractionating physiologically active concentrated human plasma using a Cohen fractionation procedure (e.g., the procedure described in U.S. Pat. No. 2,390,074), wherein the improvement of the invention includes using physiologically active concentrated human plasma as the starting material for the fractionation procedure. Figure 1 An exemplary process diagram of the Cohn fractionation method is provided.

[0080] Thus, for example, physiologically active concentrated plasma is subjected to a method for fractionating proteins. Exemplary methods involve precipitating a selected protein fraction from a solution containing a plurality of protein fractions. The solution is adjusted to have a pH higher than the isoelectric point of one or more proteins in the fraction to be precipitated. In an exemplary embodiment, the pH of the fractionation solution is reduced so that the solution also reaches the approximate isoelectric point of the desired fraction to be precipitated. Exemplary methods include bringing the ionic strength of the solution to between 0.1 and 0.2. Various methods include reducing the temperature of the solution to a temperature between approximately 0°C and the freezing point of the solution. In some embodiments, an organic precipitant for the plasma protein fraction is added to the protein solution, the amount of the precipitant added being such that the desired fraction is precipitated from the protein solution at the temperature, and the precipitate is separated from the solution. In a preferred embodiment, the conditions are adjusted so that substantially only the desired plasma protein fraction is precipitated from the solution.

[0081] In various embodiments, a method for fractionating proteins by precipitation from a solution of physiologically active concentrated human plasma containing multiple protein fractions is provided, the method comprising: bringing the pH of the solution to approximately the isoelectric point of the desired protein fraction to be precipitated; bringing the ionic strength of the solution to between 0.01 and 0.2; lowering the temperature of the solution to a temperature between approximately 0°C and the freezing point of the solution; adding an organic precipitant for proteins to the protein solution, the amount of precipitant added, the pH, the ionic strength and the temperature being such that only the desired fraction is precipitated from the protein solution, and separating the precipitate from the solution.

[0082] In various embodiments, in a method for fractionating proteins from a solution of physiologically active concentrated human plasma, the steps include: mixing an organic precipitant for proteins with the protein solution; adjusting the temperature to between 0 and -15°C, the amount of the 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 the precipitated proteins that are insoluble therein from the resulting liquid system.

[0083] In some embodiments, in a method for fractionating proteins from a solution of physiologically active concentrated human plasma, the steps include: mixing an organic precipitant for proteins with the protein solution; adjusting and maintaining the temperature above its freezing point but not above 0°C, adjusting and maintaining the amount of the precipitant at about 10% to about 40%, adjusting and maintaining the pH at about 4.4 to about 7 and adjusting and maintaining the ionic strength at about 0.05 to about 0.2; and separating the precipitated proteins insoluble therein from the resulting liquid system.

[0084] In some embodiments, a method for fractionating proteins from physiologically active concentrated human plasma is provided, the steps comprising: adding both an electrolyte and an organic precipitant for proteins to a solution containing a protein mixture, the electrolyte being added in an amount sufficient to achieve an ionic strength of about 0.01 to 0.2, and the precipitant being added in an amount such that only the desired protein fraction is precipitated; adjusting and maintaining the pH of the solution at about 4.4 to about 7 and adjusting and maintaining its temperature at about 0 to about -15°C, thereby precipitating the proteins from the resulting system.

[0085] In an exemplary embodiment, the present 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 at 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 within the temperature range until the albumin crystallizes from the mixture.

[0086] In an exemplary embodiment, in a method for fractionating substances (e.g., proteins) of varying solubility from a solution of concentrated human plasma at controlled temperature and hydrogen ion concentration, the precipitate formed thereby is removed and multiple successive fractions of the substances are precipitated by varying one or more factors.

[0087] In various embodiments, the invention provides a method of preventing protein denaturation by altering an agent that would normally cause denaturation, the method comprising adding the agent to a protein solution of concentrated human plasma by diffusion through a semipermeable membrane.

[0088] In one embodiment, a method for fractionating proteins from a solution of physiologically active concentrated human plasma is provided, the method comprising contacting the physiologically active concentrated human plasma with an organic precipitant. Exemplary embodiments include controlling one or more of the amount of contact precipitant, 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 a plurality of said factors that affect their solubility.

[0089] In an exemplary embodiment, the organic precipitation agent is added at a temperature of about 0°C or less than about 0°C.

[0090] In an exemplary embodiment, the organic precipitation agent is an alcohol. In various embodiments, it is added at a temperature of about 0°C or less than about 0°C.

[0091] In an exemplary embodiment, a method for fractionating proteins from a solution of physiologically active concentrated human plasma is provided, the method comprising: precipitating one or more different protein fractions from the plasma by physiologically active concentrated human plasma and an organic precipitant (e.g., alcohol) and by varying the temperature of the mixture of physiologically active concentrated human plasma and the organic precipitant, gradually decreasing the temperature and increasing the concentration of the organic precipitant of the mixture while precipitating successive protein fractions. The temperature is correlated to the percentage of alcohol, such that at a certain percentage of alcohol and plasma present therein, the temperature for precipitation of any given protein fraction is close to but above the freezing point of the mixture.

[0092] Exemplary organic precipitation agents include ethanol, acetone, dioxane, and combinations thereof.

[0093] In an exemplary embodiment, the IgG isolated from physiologically active concentrated human plasma is isolated with a yield of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least about 95% of the yield of the protein isolated from fresh frozen plasma. In various embodiments, the activity of the IgG is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least about 95% of the activity of the IgG isolated from fresh frozen plasma.

[0094] In an exemplary embodiment, a protein selected from A1PI, AT-III, and combinations thereof isolated from fraction IV-1 of fractionated physiologically active concentrated human plasma is isolated at a yield of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least about 95% of the yield of the protein isolated from fresh frozen plasma. In various embodiments, the protein in fraction IV-1 isolated from physiologically active concentrated human plasma has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least about 95% of the activity of the protein isolated from fresh frozen plasma.

[0095] In some embodiments, the present invention provides a method wherein albumin isolated from Fraction V of physiologically active concentrated human plasma is isolated at a yield of at least about 80% of the yield of the protein isolated from fresh frozen plasma. In various embodiments, the albumin isolated from physiologically active concentrated human plasma has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least about 95% of the activity of the albumin isolated from fresh frozen plasma.

[0096] The methods provided herein allow for the preparation of A1PI compositions having very high levels of purity. For example, in one embodiment, at least about 95% of the total protein in the A1PI compositions provided herein is A1PI. In other embodiments, at least about 96% of the protein in the composition is A1PI, or at least about 97%, 98%, 99%, 99.5% or more of the total protein of the composition is A1PI.

[0097] Similarly, the methods provided herein allow for the preparation of AlPI compositions containing extremely low levels of contaminants. For example, in certain embodiments, AlPI compositions containing less than about 10 mg / L of contaminants are provided. In other embodiments, the AlPI compositions will contain less than about 5 mg / L of contaminants, preferably less than about 3 mg / L of contaminants, and most preferably less than about 2 mg / L of contaminants.

[0098] In various embodiments, the physiologically active concentrated plasma AlPI retains at least about 80%, 85%, 90%, or at least about 95% of the activity of AlPI in plasma on a per unit basis.

[0099] In one embodiment, the 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, for example, at or about 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 more. In preferred embodiments, the aqueous IgG composition comprises a protein concentration at or about 200 g / L. In particularly preferred embodiments, the aqueous IgG composition comprises a protein concentration at or about 204 g / L.

[0100] The methods provided herein allow for the preparation of IgG compositions with very high purity levels. For example, in one embodiment, at least about 95% of the total protein in the IgG compositions provided herein will be IgG. In other embodiments, at least about 96% of the protein will be IgG, or at least about 97%, 98%, 99%, 99.5% or more of the total protein of the compositions will be IgG.

[0101] Similarly, the methods provided herein allow the preparation of IgG compositions containing extremely low levels of contaminants. For example, in certain embodiments, an IgG composition containing less than about 100 mg / L IgA is provided. In other embodiments, the IgG composition will contain less than about 50 mg / L IgA, preferably less than about 35 mg / L IgA, most preferably less than about 20 mg / L IgA. In an exemplary embodiment, the IgG preparation contains less than or equal to about 0.14 mg / mL IgA.

[0102] In some embodiments, the present invention provides a preparation of multivalent and / or highly immune immunoglobulin (IgG) prepared by the method of the present invention. In various embodiments, IgG is selected from anti-RhO highly immune immunoglobulin, anti-hepatitis B highly immune immunoglobulin, anti-rabies highly immune immunoglobulin, anti-tetanus IgG highly immune immunoglobulin and any combination of two or more thereof.

[0103] In various embodiments, the physiologically active concentrated plasma IgG retains at least about 80%, 85%, 90%, or at least about 95% of the activity of the IgG in the plasma on a per unit basis.

[0104] Cohn cell concentration process

[0105] Concentration of the Cohn pool can be achieved by a number of techniques including: (a) prefiltration followed by batch TFF with UF cassettes, or (b) prefiltration followed by single-pass TFF with UF cassettes, or (c) UF hollow fiber systems with or without prefiltration.

[0106] Apparatus and methods for ultrafiltration are known in the art. Figure 2 A process flow diagram of an exemplary method / apparatus for concentrating a starting Cohn pool is provided.

[0107] An ultrafiltration membrane or ultrafiltration hollow fiber filter membrane with a nominal molecular weight cutoff (NMWCO) of about 300 kDa or less can be used for the concentration of the Cohen pool, wherein it is performed in a recirculation or single pass configuration, and is performed with or without prior prefiltration. In another embodiment, an ultrafiltration membrane or ultrafiltration hollow fiber filter membrane with a nominal molecular weight cutoff (NMWCO) of about 200 kDa or less can be used for the concentration of the Cohen pool, wherein it is performed in a recirculation or single pass configuration, and is performed with or without prior prefiltration. In some embodiments, an ultrafiltration membrane or ultrafiltration hollow fiber filter membrane with a nominal molecular weight cutoff (NMWCO) of about 100 kDa or less can be used for the concentration of the Cohen pool, wherein it is performed in a recirculation or single pass configuration, and is performed with or without prior prefiltration.

[0108] The following examples are provided to illustrate exemplary embodiments of the invention and are not intended to define or limit the scope thereof.

[0109] Example

[0110] Example 1

[0111] Four study runs were performed until the precipitate G (PptG) and fraction V (Fr V) appeared, respectively. The desired Cohn pool plasma volume and protein concentration were obtained without significant loss of various plasma-derived proteins from the different fractionations of Cohn pool plasma.

[0112] For each pair of research runs, i.e., Run 1 and Control 1, Run 2 and Control 2, etc., the same Cohen pool batch was used as the starting material. Prior to the Cohen fractionation process, approximately 5-12 liters of poor cryogenic plasma were concentrated using ultrafiltration membranes in 4 test runs (Run 1, Run 2, Run 3, Run 4), while no ultrafiltration was performed in the 4 corresponding control runs. For the plasma concentration step, various prefilters and various TFF filters with a nominal molecular weight cutoff (NMWCO) of 100 kDa or less were used. Therefore, the starting protein concentration of the Cohen pool plasma in the test run was 10% to 27% higher than the control run. The research run configuration is shown in the table.

[0113] Table: Overview of research operation configuration

[0114]

[0115] For each protein of interest, comparative testing was performed in a concentrated test and a non-concentrated control run, respectively. In the test runs, all protein contents of the upstream IgG process up to precipitate G (PptG) were generally comparable to the control runs, as shown in Table - Table.

[0116] Table: Protein step yields - Cohen pool

[0118]

[0119] Table: Protein Step Yields - Fraction I Centrate

[0120]

[0121] Table: Protein Step Yields - Fraction II+III Filtrate

[0122]

[0123] Table: Protein step yield - Fraction II+III paste extract CUNO filtrate

[0124]

[0125] Table: Precipitate G protein composition

[0126]

[0127] The proteins that are typically present in fractions IV and V downstream of Cohn pool concentration are present in these fractions at yields and purities that are comparable to those present in fractions IV and V in a process starting with an unconcentrated Cohn pool. The conditions for exemplary separation and purification processes for those plasma-derived product intermediates produced from a Cohn pool concentrate are set forth in Table TABLE.

[0128] The supernatant of fraction II+III was further subjected to a fractionation procedure.The supernatant of fraction II+III was contacted with 25% ethanol to obtain a fraction IV-1 precipitate.

[0129] Table: Protein Step Yield - Fraction IV-1 Filtrate

[0130]

[0131] Table: Protein Step Yield - Fraction IV-4 Filtrate

[0132]

[0133] Table: Fraction V protein composition

[0134] Control 1 Test 1 Control 2 Test 2 Control 3 Test 3 Control 4 Test 4

[0135]

[0136]

[0137] Example 2

[0138] Cohen pool concentration (CPC) is intended to increase capacity and reduce operating costs while minimizing disruption to commercial supply. The process of the present invention makes it feasible to concentrate Cohen pool plasma by ultrafiltration. Ultrafiltration permits selective separation, concentration and purification of protein components. In the present invention, pilot scale runs were performed and the desired Cohen pool plasma volume and protein concentration were obtained without significant loss of various plasma-derived proteins from different fractionation separations of Cohen pool plasma.

[0139] Prior to the Cohn fractionation process, approximately 100 liters of depleted cryogenic plasma were concentrated using ultrafiltration membranes in the test runs (Run 1, Run 2, Run 3), while no ultrafiltration was performed in the control runs. For the plasma concentration step, a hollow fiber filter with a nominal molecular weight cutoff (NMWCO) of 100 kDa or less was used. Thus, the starting protein concentration of the Cohn pool plasma in the test runs was approximately 15% higher than in the control runs. For each protein of interest, the technical specifications (e.g., recovery, quality) of the isolated and purified protein were tabulated and analyzed.

[0140] 1. Immunoglobulin recovery and quality are not affected by Cohen pool concentration

[0141] After the concentration step, the immunoglobulin content was calculated in all runs. The resulting IgG content in Cohen pooled plasma prepared for ethanol treatment was concentrated without significant loss. The upstream IgG efficiency in the test runs was comparable to the control runs, as shown in Table 1.

[0142] The recovery of the purified IgG from precipitate G (PptG) was also compared between the test run and the control. The fraction II+III precipitate produced by the Cohen fractionation process was suspended in a suspension buffer to form an IgG suspension. The IgG suspension was filtered, and the filtrate was treated with a detergent. After the pH of the filtrate treated with the detergent was adjusted to about 7.0 and ethanol was added to reach a final concentration of about 20% to about 30%, a precipitate G was formed. Table 2 shows that the IgG purification efficiency is not affected by the process of CPC. In a pilot scale study, comparable IgG yields from PptG were observed between the test run and the non-concentrated control run. For all runs, IgG quality was assessed at the drug product level. All results met all restrictions, as shown in Table 3.

[0143] Table 1. Immunoglobulin excess in upstream processing

[0144]

[0145] 1 The same starting material was used for 'Run 1 Control' and 'Run 1', ie Cohen plasma after cryo separation was divided and processed in parallel without Cohen pool concentration ('Run 1 Control') and with Cohen pool concentration ('Run 1').

[0146] 2 Factor VIII inhibitor bypass activity

[0147] 3 Antithrombin III

[0148]

[0149] Table 2. Remaining immunoglobulins in test and control runs after purification

[0150]

[0151]

[0152] Table 3. Immunoglobulin drug product testing results

[0153]

[0154]

[0155]

[0156] 2. Process Parameters for Isolation and Purification of Immunoglobulins by Cohn Pool

[0157] Conditions for an exemplary plasma-derived immunoglobulin separation process including Cohn pool concentration, 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 set forth in Tables 4-8.

[0158] In the exemplary CPC process, the target amount of filter aid and filtration area is calculated based on the volume of the concentrated Cohn pool, rather than the volume of the poor cryopreserved plasma (CPP). The Cohn pool is concentrated by ultrafiltration to at least about 14% (w / v), thereby forming a first Cohn pool concentrate. In all runs, the technical schemes and parameters associated with the ultrafiltration process are indicated in Table 4.

[0159] Table 4. Parameters for the Cohn cell concentration step

[0160]

[0161]

[0162] The first Cohen pool concentrate was further subjected to a fractionation procedure. The first Cohen pool concentrate was contacted with 8% ethanol at a pH of about 7.0 to 7.5 to obtain a Fraction I precipitate and a Fraction I supernatant from the fractionated Cohen pool. In all runs, the technical protocols and parameters associated with the Fraction I precipitation and separation are indicated in Table 5.

[0163] Table 5. Parameters for precipitation and separation of fraction I using 8% ethanol

[0164]

[0165] The Fraction I supernatant was further contacted with about 25% ethanol at a pH of about 6.7 to about 7.3 to form a Fraction II+III precipitate. In all runs, the technical parameters associated with separating proteins from Fraction II+III of the fractionated Cohen pool are indicated in Table 6.

[0166] Table 6. Parameters for precipitation and separation of fraction II+III using 25% ethanol

[0167]

[0168] The fraction II+III precipitate is further suspended in a suspension buffer to form an IgG suspension. After finely ground silicon dioxide (SiO 2) is mixed with the IgG suspension for at least about 30 minutes, the IgG suspension is filtered and a filtrate and a filter cake are produced. In all runs, the technical parameters associated with the separation of proteins from the fraction II+III extracts are indicated in Table 7.

[0169] Table 7. Parameters for precipitation and separation of fraction II+III extracts

[0170]

[0171]

[0172] The filtrate produced by the Fraction II+III precipitate was contacted with a detergent to form a treated filtrate. After 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%, a Ppt G precipitate was formed. In all runs, the technical parameters associated with the precipitation and separation of Ppt G from the Fraction II+III precipitate are indicated in Table 8.

[0173] Table 8. Parameters for Ppt G precipitation and separation

[0174]

[0175] 3. Plasma-derived products isolated from fractions IV and V of the fractionated Cohn pool are not affected by Cohn pool concentration

[0176] The proteins that are typically present in fractions IV and V downstream of Cohn pool concentration are present in these fractions at yields and purities that are comparable to the yields and purities that would be present in fractions IV and V in a process starting with an unconcentrated Cohn pool. The conditions for exemplary separation and purification processes for plasma-derived products including albumin, alpha-1 proteinase inhibitor, and others from a Cohn pool concentrate are set forth in Tables 9-14.

[0177] 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 fraction IV-1 precipitate. The technical protocol and parameters for isolating proteins in fraction IV-1 are indicated in Table 9.

[0178] Table 9. Parameters for precipitation and separation of fraction IV-1 using 25% ethanol

[0179]

[0180]

[0181] The supernatant of fraction IV-1 was then contacted with 40% ethanol to obtain a fraction IV-4 precipitate. The technical parameters for isolating the proteins in fraction IV-4 are indicated in Table 10.

[0182] Table 10. Parameters for precipitation and separation of fraction IV-4 using 40% ethanol

[0183]

[0184]

[0185] The supernatant of fraction IV-4 was then contacted with 40% ethanol to obtain a fraction V precipitate. The fraction V of the present invention mainly includes albumin. In all runs, the technical scheme and parameters for separating the proteins in fraction V of the fractionated Cohen pool are indicated in Table 11. Comparable total protein content in fraction IV-4 was observed between run 1 and the non-concentrated control run, as shown in Table 12.

[0186] Table 11. Parameters for precipitation and separation of fraction V using 40% ethanol

[0187]

[0188]

[0189] Table 12. Remaining amount of protein isolated from fraction IV-4

[0190]

[0191] The quality of the protein isolated from Fraction V was assessed by various biochemical and immunological assays as shown in Table 13. The quality-indicating parameters of the isolated protein in Fraction V of Run 1 were compared to the results of the corresponding non-concentrated controls.

[0192] Table 13. Extended characterization testing of Fraction V

[0193]

[0194]

[0195] The fraction IV-1 intermediate isolated from concentrated Cohn pool plasma was further processed until it became the AlPI drug product. All relevant processing parameters are summarized in Table 14.

[0196] Table 14. Parameters for the isolation and purification of A1PI from fraction IV-1

[0197]

[0198]

[0199] Human albumin and A1PI isolated from concentrated Cohn pool plasma met all predefined limits. All results of additional tests performed at drug product and intermediate levels were within predefined ranges as shown in Tables 15-17.

[0200] All results met the predefined acceptance criteria at the intermediate and drug product levels. Based on the results shown, it can be concluded that Cohn pool concentration does not negatively impact the product quality of A1PI or albumin.

[0201] Table 15. Albumin Drug Product Testing Results for Albumin Run 1 and Run 1 Control

[0202]

[0203]

[0204] Table 16. Albumin Drug Product Testing Results for Albumin Run 2 and Run 3

[0205]

[0206]

[0207] Table 17. A1PI drug product testing results

[0208]

[0209]

[0210] Example 3

[0211] In the present invention, commercial scale runs are performed until the precipitate G (PptG), fraction V (FrV) and fraction IV-1 (Fr IV-1) intermediates appear. The desired Cohn pool plasma volume and protein concentration are obtained without significant loss of various plasma-derived proteins from the different fractionation separations of Cohn pool plasma.

[0212] PptG and Fr IV-1 were further purified at pilot scale as immunoglobulin drug product and A1PI drug product, respectively. FrV was purified at commercial scale as albumin drug product.

[0213] Before the Cohen fractionation process, approximately 4700 liters of poor cryopreservation plasma were concentrated using ultrafiltration membranes. For the plasma concentration step, prefiltration (10 μm + 0.5 μm) was used, followed by batch TFF using a UF cassette with a nominal molecular weight cutoff (NMWCO) of 100 kDa. Therefore, the starting protein concentration of the Cohen pool plasma in the test run was approximately 15% higher than in the control run. For each protein of interest, the technical specifications (e.g., recovery, quality) of the separated and purified proteins were tabulated and analyzed.

[0214] 1. Immunoglobulin process performance and product quality are not affected by Cohen pool concentration

[0215] After the concentration step, the immunoglobulin content was calculated.The resulting IgG content in the Cohn pool plasma prepared for ethanol treatment was concentrated without significant losses.

[0216] The yield and purity of the proteins typically present in the precipitate G and immunoglobulin drug product downstream of Cohn cell concentration were found to be comparable to those present in the precipitate G and immunoglobulin drug product in a process starting with a non-concentrating Cohn cell.

[0217] All operational parameters of upstream and downstream processing such as cycle time and step yields were found to be comparable to historical commercial manufacturing data.IgG quality was assessed at drug product level and all results met the predefined limits as shown in Table 18.

[0218] Table 18. Immunoglobulin Drug Product Test Results

[0219]

[0220] 3. Process Parameters for Immune Globulin Isolation and Purification in Cohn Pools at Commercial Scale

[0221] The Cohn pool was concentrated by approximately 15% (w / v) by ultrafiltration to form a first Cohn pool concentrate. The technical scheme and parameters associated with the ultrafiltration process are indicated in Table 19.

[0222] Table 19. Parameters for the Cohn cell concentration step

[0223]

[0224] In an exemplary CPC process, the target amount of filter aid and filtration area is calculated based on the volume of the concentrated Cohn pool, rather than the volume of cryo-poor plasma (CPP).

[0225] The first Cohen pool concentrate was further subjected to a fractionation procedure.The first Cohen pool concentrate was contacted with 8% ethanol at a pH of about 7.0 to 7.5 to obtain a Fraction I precipitate and a Fraction I supernatant from the fractionated Cohen pool.

[0226] The Fraction I supernatant is further contacted with about 25% ethanol at a pH of about 6.7 to about 7.3 to form a Fraction II+III precipitate.

[0227] The fraction II+III precipitate is further suspended in a suspension buffer to form an IgG suspension. After mixing finely divided silica (SiO2) with the IgG suspension for at least about 30 minutes, the IgG suspension is filtered and a filtrate and a filter cake are produced.

[0228] The filtrate produced from the Fraction II+III precipitate is contacted with a detergent to form a treated filtrate.After the pH of the treated filtrate is adjusted to about 7.0 and ethanol is added to a final concentration of about 20% to about 30%, a Precipitate G precipitate is formed.

[0229] 3. Plasma-derived products isolated from fractions IV and V of the fractionated Cohn pool are not affected by Cohn pool concentration

[0230] The proteins that are normally present in fractions IV and V downstream of Cohn pool concentration are present in these fractions at a yield and purity that is comparable to that present in fractions IV and V in a process starting with a non-concentrating Cohn pool.

[0231] The supernatant of fraction II+III was further subjected to a fractionation procedure.The supernatant of fraction II+III was contacted with 25% ethanol to obtain a fraction IV-1 precipitate.

[0232] The supernatant of fraction IV-1 was then contacted with 40% ethanol to obtain a fraction IV-4 precipitate.

[0233] The supernatant of fraction IV-4 was then contacted with 40% ethanol to obtain a fraction V precipitate.

[0234] The quality of the protein isolated from Fraction V was evaluated by a variety of biochemical and immunological assays, as shown in Table 20. The quality-indicating parameters of the isolated protein in Fraction V from the commercial scale run were comparable to historical commercial manufacturing data starting with a non-concentrated Cohn pool.

[0235] Table 20. Extended characterization testing of fraction V

[0236] Test Parameters unit Commercial scale operation PKA IU / mL <4 purity ％ 97.2

[0237] Human albumin and A1PI isolated from concentrated Cohn pool plasma were tested at the final drug product level. All results at the drug product level were within the predefined ranges as shown in Tables 21-22.

[0238] All results met the predefined acceptance criteria at drug product level. Based on the results shown, it can be concluded that Cohn pool concentration does not negatively impact product quality of either A1PI or albumin.

[0239] Table 21. Commercial scale run 20% albumin drug product testing results

[0240]

[0241] Table 22. A1PI drug product testing results

[0242]

[0243] In additional laboratory scale filtration experiments, it has been demonstrated that when concentrating Cohn pools, permeate fluxes are comparable for filters up to 300 kDa molecular weight cut-off.

Claims

1. A method for preparing a Cohn pool concentrate from plasma, the method comprising: (a) providing a Cohn pool from plasma; as well as (b) concentrating the Cohn pool to a total protein concentration of at least about 65 g / L, thereby forming a Cohn pool concentrate.

2. The method of claim 1, wherein the Cohn pool is concentrated to a protein concentration of about 50 to about 65 g / L.

3. The method of any preceding claim, wherein the Cohn pool is a member selected from cryo-depleted plasma, factor-depleted plasma, and inhibitor-depleted plasma.

4. A method as claimed in any preceding claim, wherein step (b) is performed using an ultrafiltration membrane having a nominal molecular weight cut-off (NMWCO) of 300 kDa or less, the ultrafiltration membrane being operated in a recirculating or single pass configuration, with or without prior prefiltration.

5. A method as claimed in any preceding claim, wherein step (b) is performed using a hollow fiber filter membrane having a nominal molecular weight cut-off (NMWCO) of 300 kDa or less, the hollow fiber filter membrane being operated in a recirculating or single-pass configuration, with or without prior prefiltration.

6. The method of any preceding claim, wherein there is a total protein concentration in the Cohn pool centrate of at least about 65 g / L.

7. The method of any preceding claim, wherein the Cohn pool concentrate is further subjected to a purification process for preparing a composition selected from the group consisting of an immunoglobulin G (IgG) composition, an albumin composition, an A1-PI composition, and combinations thereof.

8. A method as claimed in any preceding claim, wherein the Cohn pool is concentrated with substantially no loss of protein mass in the resulting Cohn pool concentrate attributable to a member selected from the group consisting of total protein, IgG, albumin, AAT and fibrinogen.

9. The method of any preceding claim, further comprising subjecting the Cohn pool concentrate to a plasma fractionation procedure.

10. The method of claim 9, wherein the fractionation procedure is one of Cohn fractionation or a modification thereof.

11. The method of claim 10, wherein the fractionation procedure comprises: (i) contacting the Cohn pool concentrate with about 6% to about 10% ethanol at a pH of about 7.0 to 7.5 to obtain a Fraction I precipitate and a Fraction I supernatant; and (ii) contacting the Fraction I supernatant or concentrated Cohn pool with about 18% to about 27% alcohol at a pH of about 6.7 to about 7.3 to form a member selected from Fraction II+III precipitate or alternatively Fraction I+II+III precipitate.

12. The method of claim 11, further comprising: (iii) suspending a member selected from the Fraction II+III precipitate, the Fraction II+III (II+III or alternatively I+II+III) precipitate in a suspension buffer, thereby forming an IgG suspension; (iv) mixing finely divided silicon dioxide (SiO2) with the IgG suspension for at least about 30 minutes; (v) filtering the IgG suspension to form a filtrate and a filter cake.

13. The method of claim 12, further comprising: (vi) contacting the filtrate with a cleaning agent to form a treated filtrate; (vii) adjusting the pH of the treated filtrate of step (vi) to about 7.0 and adding ethanol to a final concentration of about 20% to about 30%, thereby forming a precipitate G precipitate; (viii) dissolving the precipitate G in an aqueous solution comprising a member selected from a solvent, a detergent, and a combination thereof, thereby forming a precipitate G solution; (ix) passing the solution through a cation exchange material to allow proteins contained therein to adsorb to the cation exchange material, and subsequently eluting the adsorbed proteins in an eluent; (x) passing the eluent through an anion exchange material to produce a flow-through effluent; (xi) passing the effluent through a nanofilter to produce a nanofiltrate; (xii) concentrating the nanofiltrate by ultrafiltration to produce a first ultrafiltrate; (xiii) diafiltering the first ultrafiltrate against a diafiltration buffer to produce a diafiltrate; as well as (xi) concentrating the diafiltrate by ultrafiltration, thereby producing a second ultrafiltrate having a protein concentration between about 8% (w / v) and about 22% (w / v), thereby forming an IgG enriched fraction.

14. The method of claim 13, further comprising: Prior to (vi), washing the filter cake with at least one filter press dead volume of a wash buffer having a pH of about 4.9 to about 5.3 to form a wash solution; combining the filtrate with the wash solution to form a solution; and treating the solution with a cleaning agent in step (vi).

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

16. A pharmaceutical formulation comprising albumin isolated from the Cohn pool concentrate of claim 1 and a pharmaceutically acceptable vehicle.

17. A pharmaceutical formulation comprising AAT isolated from the Cohn pool concentrate of claim 1 and a pharmaceutically acceptable vehicle.

18. A pharmaceutical formulation comprising IgG isolated from the Cohn pool concentrate of claim 1 and a pharmaceutically acceptable vehicle.

19. A pharmaceutical formulation comprising fibrinogen isolated from the Cohn pool concentrate of claim 1 and a pharmaceutically acceptable vehicle.

20. A pharmaceutical formulation comprising TP isolated from the Cohn pool concentrate of claim 1 and a pharmaceutically acceptable vehicle.

Images