Integrated process for isolation of plasma proteins

Abstract

Methods that provide an integrated, scalable process for isolating both immunoglobulin G and albumin from plasma at high purity and high yield are described. These methods utilize a combination of salt precipitation and ion exchange chromatography to separately recover immunoglobulin G and albumin at high yield and high purity from flow-through fractions of chromatography steps. This provides a streamlined, scalable process that avoids binding and elution steps and that utilizes relatively small amounts of chromatography media.

Description

Attny Dkt No. 101658.0023PCT INTEGRATED PROCESS FOR ISOLATION OF PLASMA PROTEINS

[0001] This application claims the benefit of United States Patent Application No. 18 / 427623, filed January 30, 2024, and United States Provisional Patent Application Serial No. 63 / 544,576 filed on October 17, 2023. These and all other referenced extrinsic materials are incorporated herein by reference in their entirety. Where a definition or use of a term in a reference that is incorporated by reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein is deemed to be controlling. Field of the Invention

[0002] The field of the invention is isolation of proteins from plasma, in particular immunoglobulin G (IgG) and albumin. Background

[0003] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Albumin is the most abundant circulating protein (providing about half of the total protein content) in blood plasma of an individuals in good health. It is a relatively small (66.5 kD), single chain globular protein (molecular weight: 66.5 kDa) produced and released into circulation by the liver. Due to its high concentration and slight negative charge, albumin provides an oncotic effect that modulates fluid distribution between different body compartments, Albumin also has the property of binding and carrying a wide variety of small, hydrophobic endogenous and exogenous molecules. In addition, albumin acts to maintain capillary permeability and provides an anti-thrombotic effect.

[0005] Albumin has been utilized as a therapeutic protein to treat or prevent a variety of conditions. Notably, albumin has been used as to treat shock, sepsis, trauma, acute respiratory distress syndrome, burns, hypoalbuminaemia, hypoproteinaemia, and acute clinical situationsAttny Dkt No. 101658.0023PCT associated with hypovolaemia. Accordingly, there is a considerable demand for albumin as a therapeutic protein.

[0006] Typically, albumin is separated from blood plasma using ethanol precipitation at low temperatures. This process requires the use of high concentrations of ethanol, which can lead to protein denaturation, and does not provide albumin at high purity. In addition, the need to use low temperatures and the resulting stream of organic solvent-containing waste complicates performing such methods at a large scale. Attempts have been made to address purity issues by incorporating additional chromatography steps, however the size of chromatography columns required for conventional chromatography methods (in which the protein of interest binds to the chromatography media, followed by washing to remove contaminants and elution of the bound protein) can complicate the implementation of such methods at a large scale.

[0007] Immunoglobulin G (IgG) is a protein found in blood plasma, and as a component of the immune system it has been used for the treatment of primary immunodeficiency and a wide variety of other immunological disease conditions. In addition, IgG can be used for passive immune transfer to accelerate the clearance of pathogens (e.g., SARS-CoV2) and prevent or treat infection. For over 70 years, IgG has been produced with the Cohn process, which separates, or fractionates, plasma proteins based on their differential solubility in ethanol with variances in temperature, pH, ethanol concentration, ionic strength, and protein concentration. While the overall IgG yield from the Cohn process is not generally disclosed by commercial plasma fractionators, the modern Cohn process is widely believed to achieve yields of only about 50- 60% of the IgG present in the starting material. In addition, it is understood that this process takes approximately 7 to 10 days to complete.

[0008] United States Patent No. 7,879,331, to Zurlo et al., describes a method for isolation of IgG that utilizes successive fractionation of plasma using citrate salts to yield a precipitate that contains IgG. Following dissolution and buffer exchange, the resulting IgG-rich solution is treated by ion exchange chromatography to generate an eluate that contains the IgG. This eluted fraction is further subjected to anion exchange chromatography. Unfortunately, this process can result in rapid fouling of diafiltration membranes if this technique is utilized for buffer exchange into buffers suitable for the ion exchange steps. This limits the scale at which diafiltration can beAttny Dkt No. 101658.0023PCT applied. In addition, binding and subsequent elution of IgG from the ion exchange media inevitably results in loss of the protein.

[0009] Thus, there is still a need for rapid, robust, efficient, and scalable methods for isolation of plasma proteins such as albumin and IgG at high yield and purity. Summary of The Invention

[0010] Methods of the inventive concept include methods for isolation of albumin from blood products (such as plasma) as well as methods for isolation of immunoglobulin G (IgG) and albumin from a blood product as separate product streams. Such methods utilize at least one initial salt precipitation step and include ion exchange steps. In methods of the inventive concept albumin and IgG are recovered in flow-through fractions from column chromatography steps, advantageously avoiding costly and time-consuming bind and elute steps of prior art methods.

[0011] Methods of the inventive concept include methods of isolating albumin from a blood product (e.g., cryo-poor plasma) by adding a citrate salt to the cryo-poor plasma to a concentration of about 11% to 13% by weight to generate a first supernatant and first precipitate. Following separation of these phases (e.g., by filtration, decanting, centrifugation, or any suitable method) additional citrate salt is added to the first supernatant to generate a second precipitate and a second supernatant. Following separation of these phases (e.g., by filtration, decanting, centrifugation, or any suitable method) caprylate is added to the second supernatant to a concentration of about 40 mM to generate a caprylate treated second supernatant. This caprylate treated second supernatant is heat treated by raising its temperature to about 50°C to about 60° for a time sufficient to generate a third precipitate and a third supernatant (e.g., about one hour). Following separation of these phases (e.g., by filtration, decanting, centrifugation, or any suitable method). The pH of the third supernatant is then adjusted (e.g., by diafiltration using a membrane with a suitable molecular weight cutoff, for example about 30 kD) to from pH 4 to pH 6 to generate a pH adjusted third supernatant, which is applied to an anion exchange medium. Purified albumin is recovered as a flow-through (e.g. the second of two flow-through peaks) from the anion exchange medium. In some embodiments the flow-through obtained from the anion exchange medium us applied to a cation exchange media, followed by collection of a flow- through from the cation exchange media that contains highly purified albumin.Attny Dkt No. 101658.0023PCT

[0012] Methods of the inventive concept include an integrated process for isolating both immunoglobulin G (IgG) and albumin from a blood product (e.g. cryo-poor plasma) as separate product streams, by adding a citrate salt to the blood product to a concentration of about 11% to 13% by weight to generate a first supernatant and first precipitate. Following separation of these phases (e.g., by filtration, decanting, centrifugation, or any suitable method), an additional amount of citrate salt is added to the first supernatant to generate a second precipitate and a second supernatant. Following separation of these phases (e.g., by filtration, decanting, centrifugation, or any suitable method), the second precipitate is dissolved to generate a dissolved second precipitate that includes the IgG. Caprylate is added to the dissolved second precipitate to generate a caprylate treated second precipitate. Solids are removed from the caprylate treated second precipitate by any suitable method (e.g., by filtration, decanting, centrifugation, or any suitable method) and the clarified material applied to a first anion exchange medium to generate a first anion exchange flow-through that contains IgG. This first anion exchange flow-through is applied to a first cation exchange media to generate a first cation exchange flow-through fraction comprising purified IgG.

[0013] In such an integrated process albumin is isolated from the second supernatant generated by salt precipitation by adding caprylate to the second supernatant to a concentration of about 40 mM to generate a caprylate treated second supernatant. This caprylate treated second supernatant is heat treated by raising its temperature to about 50°C to about 60° for a time sufficient to generate a third precipitate and a third supernatant (e.g., about one hour). Following separation of these phases (e.g., by filtration, decanting, centrifugation, or any suitable method), the pH of the third supernatant is then adjusted (e.g., by diafiltration using a membrane with a suitable molecular weight cutoff, for example about 30 kD) to from pH 4 to pH 6 to generate a pH adjusted third supernatant, which is applied to a second anion exchange medium. Purified albumin is recovered as a flow-through (e.g. the second of two flow-through peaks) from the second anion exchange medium. In some embodiments the flow-through obtained from the anion exchange medium is applied to a second cation exchange media, followed by collection of a flow-through from the second cation exchange media that contains highly purified albumin. In such embodiments the highly purified IgG has a purity of 95% or higher and a yield of at least 70%.. In such embodiments the high purified albumin has a purity of 95% or higher and a yield of at least 60%.Attny Dkt No. 101658.0023PCT

[0014] In some embodiments of the inventive concept one or more additional (i.e., non-IgG and non-albumin) therapeutic proteins can be isolated from one or more of the first supernatant, the first precipitate, the second supernatant, and the second precipitate, for example by affinity chromatography.

[0015] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components. Brief Description of The Drawings

[0016] FIG. 1: FIG. 1 shows typical results of SDS-PAGE electrophoresis of products resulting from treatment of a second salt precipitate (S2) with about 40 mM caprylate at different pH values, followed by incubation at 60°C for one hour.

[0017] FIG. 2: FIG. 2 provides a schematic representation of an exemplary albumin isolation method of the inventive concept.

[0018] FIG. 3: FIG. 3 schematically depicts an exemplary IgG isolation phase of an integrated process of the inventive concept, as applied to isolation of immunoglobulin G (IgG) and albumin from a blood product using two anion exchange steps in succession.

[0019] FIG. 4: FIG. 4 shows results of SDS-PAGE performed on supernatants and precipitates generated during a second precipitation step at different citrate concentrations, under reducing conditions. The arrows denote the heavy (HC) and Light chains (LC) of IgG under reducing conditions.

[0020] FIG. 5: FIG. 5 shows results of IgG precipitation studies and mean (N=3) second precipitate IgG recoveries.

[0021] FIG. 6: FIG. 6 shows typical results of bis-tris SDS-PAGE of samples obtained from Q- Sepharose™ column breakthrough studies.Attny Dkt No. 101658.0023PCT

[0022] FIG. 7: FIG. 7 shows results of reduced bis-Tris SDS-PAGE of samples obtained from different ion exchange column arrangements. IgG product derived from various ion exchange chromatography column arrangements.

[0023] FIG. 8: FIG. 8 shows typical A280 measurement obtained during chromatography of a 2 L pilot scale method performed on successive 2 L Q-Sepharose™ columns. Individual fractions are denoted by vertical lines.

[0024] FIG. 9: FIG. 9 shows typical results of non-reduced bis-tris SDS-PAGE of samples obtained during chromatography of a 2 L scale process of the inventive concept. Lane labels correspond to the labeled fractions in Figure 8. The "final pool" consists of fractions 1 to 5.

[0025] FIG. 10: FIG. 10 shows typical results of non-reduced bis-tris SDS-PAGE of four commercially available intravenous IgG products and final product from three 2 L scale batches produced by a method of the inventive concept. 5 µg protein were loaded per lane.

[0026] FIG. 11: FIG. 11 schematically depicts an exemplary process of the inventive concept utilizing anion exchange and minimal cation exchange.

[0027] FIG. 12: FIG. 12 schematically depicts an exemplary process of the inventive concept utilizing anion exchange and restricted-capacity cation exchange.

[0028] FIG. 13: FIG. 13 schematically depicts an exemplary process of the inventive concept as applied to isolation of immunoglobulin G (IgG) from a blood product using anion exchange and restricted-capacity cation exchange.

[0029] FIG. 14: FIG. 14 provides a schematic representation of an exemplary integrated immunoglobulin G (IgG) and albumin isolation process of the inventive concept. Detailed Description

[0030] The inventive subject matter provides apparatus, systems and methods that provide an at- scale process for isolation of albumin and / or both immunoglobulin G (IgG) and albumin from plasma (e.g., cryo-poor plasma) using a combination of salt precipitation and ion exchange chromatography. Remarkably, both IgG and albumin can be recovered at high yield and highAttny Dkt No. 101658.0023PCT purity from flow-through fractions of chromatography steps. The result is a streamlined process utilizing relatively small amounts of chromatography media which avoids protein loss and denaturation that occurs in conventional bind and elute chromatography steps.

[0031] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

[0032] One should appreciate that the disclosed techniques provide many advantageous technical effects including providing a simple and efficient method for isolation albumin and / or both IgG of therapeutic quality in a scalable process and providing an integrated method for rapid, high yield isolation of both IgG and albumin from plasma at manufacturing scale.

[0033] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0034] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.Attny Dkt No. 101658.0023PCT

[0035] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0036] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0037] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0038] Inventors have developed methods for isolation of plasma proteins having therapeutic value from blood products (e.g., serum, plasma, cryo-depleted plasma, cryo-depleted plasma into which cryoprecipitate has been re-dissolved) that utilize two citrate precipitation steps. Each precipitation step produces a supernatant (S) and a precipitate (P), between which proteins present in the starting material are distributed. In such a processes a supernatant from a first precipitation step (S1) can include a subset of proteins present in a blood-based starting material (e.g., plasma, serum, cryo-free plasma, cryo-free plasma into which cryoprecipitate has been re- dissolved, etc.). This S1 supernatant can be treated by the addition of more citrate salt toAttny Dkt No. 101658.0023PCT generate a second supernatant (S2) and second precipitate (P2) between which the proteins remaining in S1 are further segregated. It should be appreciated that protein segregation between supernatant and precipitate phases is a function of both the concentration of the citrate salt and the protein composition and relative concentrations of protein species in the solution being treated. Concentrations of the precipitating citrate salt in a precipitation step or a set of precipitation steps can be selected such that essentially all of the immunoglobulin G (IgG) is found in P2, whereas essentially all of the albumin remains in S2.

[0039] In some embodiments, an albumin containing solution can be treated to provide highly purified albumin at high yield utilizing flow-through (i.e., unbound) fractions obtained from ion exchange chromatography steps. Such albumin-containing solution can be provided by any suitable method. Suitable methods include, but are not limited to, precipitation of a blood product or cell culture product with an organic solvent (e.g., ethanol, acetone, etc.), precipitation of a blood product or cell culture product with a precipitating salt (e.g., sulfate, phosphate, citrate, acetate, etc.), and or a precipitating polymer (e.g., polyethylene glycol, dextran, polyvinylpyrrolidone, etc.).

[0040] In some embodiments the albumin-containing solution can be a process stream produced during a protein isolation process, for example an effluent (e.g., an eluted bound fraction, a flow- through or unbound fraction) of a chromatography step. Suitable chromatography steps include, but are not limited to, anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, affinity chromatography, and dye affinity chromatography.

[0041] In preferred embodiments the albumin-containing material is a supernatant resulting from precipitation of a blood-based material using a citrate salt. For example, such a supernatant can be a second supernatant (S2) generated by an initial precipitation step utilizing 10% w / v to 13% w / v sodium citrate to produce a first supernatant (S1) containing the majority of albumin (and IgG) and a first precipitate (P1). The second supernatant (S2) containing most of the albumin can be produced by adding sodium citrate to S1 to generate the second supernatant (S2) and the second precipitate (P2), between which albumin and IgG are segregated.Attny Dkt No. 101658.0023PCT

[0042] Inventors have found that treatment of S2 with caprylate at elevated temperature induces precipitation of the majority of non-albumin protein contaminants from S2. FIG. 1 shows results of treatment of S2 with about. 40 mM caprylate at different pH values, followed by incubation at 60°C for one hour. As shown the majority of non-albumin contaminants are precipitated. Similar studies performed at 50° provide similar results.

[0043] As shown, treatment with caprylate at pH 6 to 8 is effective in precipitating major contaminants from the S2 material while leaving albumin in solution.

[0044] Surprisingly, Inventors have found that albumin can be recovered at high purity (e.g., 90%, 95%, 97%, or higher) and high yield (e.g., 70%, 80%, 90%, or higher) from supernatants recovered from caprylate treatment as described above using ion exchange chromatography where albumin is recovered in the flow-through from the ion exchange media. This advantageously avoids cumbersome bind, wash, and elute steps and dramatically reduces the volume of ion exchange media required (a sufficient media to bind contaminants is required). These features in turn support at-scale processes where 50L to 5,000 L or more of plasma are processed.

[0045] In an example of such an albumin purification process, frozen plasma is thawed, and cryoprecipitate removed to generate a cryo-poor plasma. A precipitating salt (e.g., a citrate or acetate salt) is added to provide a final concentration of 10% to 13% or about 11% by weight, and the resulting mixture separated into supernatant (S1) and precipitate (P1) fractions. Albumin and IgG is retained in the S2 fraction, to which additional precipitating salt is added to give a concentration of from 22% to 30% by weight, or about 26% by weight. This generates a second precipitate (P2) and a second supernatant (S2). Most or all of the albumin is found in the secondAttny Dkt No. 101658.0023PCT supernatant. This S2 can be processed for further purification of albumin, or it can be frozen for storage and thawed prior to processing for further purification of albumin.

[0046] The pH of S2 is adjusted to about 7 and sodium caprylate is added to give a final concentration of about 10 mM to 100 mM or about 40 mM caprylate. The resulting mixture is then incubated at about 60°C for one hour and the resulting precipitate (P3) removed. The precipitate can be removed from the supernatant (S3) by any suitable method, including centrifugation, settling and decanting, and filtration. For example, following caprylate / heat treatment the resulting mixture can be passed through a depth filter, such as a PDD1 depth filter or a V100P filter. Surprisingly, Inventors have found that use of a PDD1 depth filter is effective in reducing some low molecular weight (i.e., lower MW than albumin) contaminants that are not removed by the V100P depth filter.

[0047] S3 can be subjected to diafiltration in order to remove contaminants and / or provide suitable conditions for ion exchange chromatography. For example, S3 can be diafiltered using a 10 kD cutoff membrane or a 30 kD cutoff membrane. Buffers suitable for ion exchange typically have relatively low ionic strength (e.g., less than about 100 mM) and have a pH appropriate for the protein’s isoelectric point and the nature of the fixed charge on the ion exchange media. For example, to recover albumin in a flow-through fraction the pH can be slightly acidic (e.g., pH 4.5 to pH 6.5, pH 5 to pH 6, or about pH 5.3) when a weak anion exchange chromatography media (e.g., DEAE media) is used. Surprisingly, when S3 was exchanged into 50 mM acetate buffer at pH 5.3 and applied to a DEAE, approximately 97% pure albumin (i.e., purified albumin) was recovered in the flow-through fraction as a second broad peak that followed an initial flow-through peak. This pattern suggests partial retention of albumin on the DEAE media. This degree of purity can be sufficient for pharmaceutical use. Accordingly, the albumin-containing peak obtained as a flow-through fraction from DEAE chromatography can be further processed for use, for example by nanofiltration and adjustment of protein concentration prior to filling.

[0048] Optionally, a second chromatography step can be applied to this purified albumin flow- through peak to provide a highly purified albumin. For example, this albumin-containing flow- through peak can be applied to a small amount of cation exchange media (e.g., a cation exchangeAttny Dkt No. 101658.0023PCT filter) to remove additional contaminants if required. Such a small amount of ion exchange media can be selected such that its capacity for albumin does not significantly impact yield (e.g., more than about 25%, 20%, 156% 10%, 5%, or less) while retaining tightly-bound contaminants. A schematic representation of an albumin isolation method of the inventive concept is provided in FIG. 2.

[0049] Although the example provided above utilizes citrate precipitation in initial purification steps, Inventors contemplate that precipitation using an organic solvent, a non-citrate salt, and / or a polymer can also provide an albumin-containing solution suitable for caprylate treatment and ion exchange chromatography with retention of albumin in unbound, flow-through fractions as described above.

[0050] As noted above, Inventors have also developed methods for the isolation of IgG from plasma using a combination of salt precipitation and ion exchange, with recovery of IgG at high yield and high purity from flow-through fractions from the ion exchange chromatography steps. These methods generate S1, P1, S2, and P2 fractions during salt precipitation steps that correspond to the S1, P1, S2, and P2 fractions described above for the isolation of albumin.

[0051] Embodiments of the inventive concept include integrated methods in which IgG isolation and albumin isolation are combined to provide a single, integrated process for IgG and albumin isolation from plasma without the use of fractions obtained from binding and elution from chromatography media.

[0052] Such integrated methods provide a simple and scalable process that can isolate two or more proteins of interest (e.g., IgG and albumin) from a blood product (e.g.,, serum plasma, cryo-depleted plasma, cryo-depleted plasma into which cryoprecipitate has been re-dissolved) at high yield (e.g., greater than 60% of protein content of the starting material) and high purity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, by weight or greater).

[0053] Such integrated methods can utilize two chromatography steps performed in succession and utilizing the same chromatography media, where flow through material containing unbound proteins from the first chromatography step are directed to the second chromatography step, withAttny Dkt No. 101658.0023PCT the flow through from this second chromatography step containing a therapeutic protein of interest.

[0054] Inventors have found methods of the inventive concept are particularly useful in the isolation of IgG and albumin from blood plasma, although application to other solutions containing IgG and albumin (e.g., cell culture media, cell lysates, dissolved inclusion bodies, other body fluids, etc.) is contemplated. Within the context of this application plasma is considered to include freshly collected serum, freshly collected plasma, reconstituted lyophilized plasma, refrigerated plasma, frozen plasma, recovered plasma, cryo-poor plasma, cryo-poor plasma into which the cryoprecipitate has been re-dissolved, and mixtures of two or more of these. Such plasma can, for example, be obtained as pooled material from a blood or plasma collection center. An example of a method of the inventive concept for isolation of IgG from plasma is shown in FIG. 3.

[0055] For the first two steps of the integrated process the Inventors modified and / or optimized a base fractionation process using a range of salt concentrations for two precipitation steps to determine the optimum concentrations (e.g., about 11% in the first salt precipitation and about 26% in the second salt precipitation) and to maximize IgG and albumin yield while minimizing unwanted proteins. While citrate or acetate salts are preferred, any suitable salt can be used. Such salts can be added rapidly, as a salt solution (e.g., as a calculated volume of a 50% by weight salt solution) and / or in dry form (e.g., as a powder or crystalline solid). As shown the first precipitation step produces an IgG-rich and albumin-rich supernatant (S1), and the second precipitation step produces an IgG-rich precipitate or paste (P2) and an albumin-rich supernatant (S2).

[0056] In an IgG isolation phase of the integrated process this IgG-rich precipitate is dissolved (e.g., in water) prior to ion exchange steps. In such embodiments a buffer exchange step (e.g., dialysis, diafiltration, ultrafiltration followed by dilution, size exclusion chromatography, etc.) can be performed prior to ion exchange steps. Alternatively, the re-dissolved precipitate can be diluted until a desired conductivity (e.g., 2 to 10 mS) or ionic strength is achieved. It should be appreciated that precipitation can occur during buffer exchange steps, and that additional filtration steps (e.g., depth filtration, or depth filtration followed by a clarifying or polishingAttny Dkt No. 101658.0023PCT filtration step) can be implemented to remove such precipitated materials prior to the initial ion exchange step. As shown in FIG. 3, in some embodiments of the inventive concept IgG can be recovered in the flow-through (i.e., unbound) fraction from the second of two successive anion exchange steps.

[0057] In some embodiments a virus inactivation step is applied to the dissolved IgG-rich precipitate prior to an ion exchange step. For example, the pH can be lowered to 5.7 and caprylate added to inactivate enveloped viruses. Such steps do not appear to contribute to any significant IgG losses and caprylate can be effectively removed in subsequent process steps.

[0058] In conventional protein purification processes yield is sacrificed for purity of the final protein product. Surprisingly, in the methods described herein IgG is typically recovered at both high yield (greater than 70%, 75%, 80%, 85%, 90%, or 95% of the IgG present in the starting material) and high purity (greater than 80%, 85%, 90%, 95%, 98%, or 99%). Examples

[0059] Multiple base fractionations of IgG and albumin from cryo-precipitate poor plasma in two precipitation steps utilizing sodium citrate as the precipitant were performed. Two-liter test batches were prepared in pilot facilities. The IgG yield achieved was 90% in the second precipitate (i.e., precipitate resulting from the second precipitation step, P2) and with only 3% in the second supernatant (i.e., supernatant resulting from the second precipitation step, S2), while the second achieved 95% yield in the second precipitate and only 3% in the second supernatant. The uniform presence of ~3% IgG in second supernatant indicates that the base fractionation process is robust and yields approximately 95% of the starting IgG in plasma.

[0060] A range of sodium citrate concentrations were explored to develop an optimal protocol for IgG yield and purity through the base fractionation process. Cryo-poor plasma was subjected to an initial 11% sodium citrate (w / v) precipitation step for 1 hour followed by centrifugation at 4,500 x g. The resulting first supernatant 1 was further fractionated in a second sodium citrate precipitation step for 2 hours at citrate concentrations ranging from 22% to 28% (w / v) sodium citrate followed by centrifugation at 4,500 x g. Pastes from the first precipitation steps (i.e., first precipitates) and second precipitation steps (i.e., second precipitates) wereAttny Dkt No. 101658.0023PCT dissolved in water for injection at a concentration of 10 mL water per gram of wet paste. During fractionation processes both temperature (2-8°C) and pH (7.0±0.1) were maintained. Citrate concentration was adjusted by adding 50% w / v sodium citrate at ambient temperature (20-25°C).

[0061] Analysis of total protein and IgG content was carried out largely by A280measurement and total IgG ELISA (Invitrogen), respectively. While the total IgG ELISA’s measurement precision can be less accurate in complex solutions, such as cryo-poor plasma, it is suitable method for measuring relative IgG concentrations in process steps with increasingly pure IgG.

[0062] Typically, a 98% recovery of IgG in the first supernatant (S1) was found in the initial 11% (w / v) citrate precipitation step (data not shown). As shown in Table 1, IgG yield in the second precipitate from the second precipitation step increases as citrate concentrations increase from 22% to 24% (w / v), then plateaus at citrate concentrations of from 26% and 28% w / v (at about 85% IgG recovery from the first supernatant). These data are further corroborated by the IgG concentrations observed in the corresponding second supernatants, which display the inverse trend. 2ndPrecipitate IgG Recovery S m l w i ht ( ) (% )Table 1

[0063] The increase in second precipitate weight without corresponding increases in IgG yield indicates that precipitation beyond 26% (w / v) citrate accumulates undesired contaminating proteins. This was confirmed by SDS-PAGE (FIG. 4). FIG. 4 shows results from a 4-12% SDS-Attny Dkt No. 101658.0023PCT PAGE gel run under reducing conditions of the fractionation process applied to cryo-poor plasma. Arrows denote the heavy (HC) and light chains (LC) of IgG. The gel was loaded with 20 A280 units of protein per lane. As shown, IgG is evident in the first supernatants (S1) and second precipitates (Pellet 2), with increasing protein contamination observed in the second precipitate at high (e.g., 26%, 28% w / v) citrate concentrations in the second precipitation step.

[0064] Robustness of the precipitation steps was characterized by fractionating aliquots of the same cryo-poor plasma pool with 11-13% (w / v) citrate and then with 22-26% citrate (w / v). IgG yields in the resulting supernatants and precipitates were analyzed via ELISA. The mean IgG levels obtained are shown in FIG. 5. FIG. 5 provides a histogram of mean results obtained with different pairs of first precipitation and second precipitation citrate concentrations (e.g., “11- 22%” indicates 11% w / v citrate in the first precipitation step and 22% w / v citrate in the second precipitation step). As shown, 11% citrate (w / v) in the first precipitation step and 24% to 26% (w / v) citrate in the second precipitation step 11-24% were optimal to achieve maximum IgG yields. Initial precipitation at 12 or 13% (w / v) citrate appeared to have comparably lower IgG yields when the second precipitation step was conducted at varying concentrations.

[0065] To further characterize robustness of initial precipitation steps, four 2L volumes of a pooled cryo-poor plasma were processed, each using an 11% (w / v) citrate concentration for the initial precipitation and a 26% (w / v) citrate concentration for the second precipitation step. The second precipitate from each of these was dissolved in water for injection (10mL / g of wet paste). IgG concentrations were measured by nephelometry. Results are shown in Table 2. Batch A Batch B Batch C Batch DAttny Dkt No. 101658.0023PCT Precipitate Table 2 As shown, the nephelometry data are very consistent and show a 98.1% mean recovery of IgG in the first supernatant obtained from the starting cryo-poor plasma after the initial 11% precipitation step. This data is consistent with the lack of IgG (<1%) in the first precipitate. The final IgG yield in the second precipitate across the four batches in P2 was 90.3% IgG and mean value of 6.2% IgG remaining in the second supernatant. Inventors believe that some IgG- containing precipitate was not collected during centrifugation, and that recovery of IgG in the second precipitate can be about 95% or greater in the second precipitate with more extensive centrifugation.

[0066] It was noted that on dissolving such second precipitates in water for injection at 10 mL / g wet paste, the solution can appear cloudy and / or opalescent. Inventors believe that cloudiness is likely the result of suspended lipids and non-solubilized impurities. In small-scale studies dialysis was used for buffer exchange (i.e., removal of salts from the IgG-containing solution) prior to subsequent ion exchange steps. Such precipitated materials can be removed by centrifugation or filtration (e.g., using a depth filter) prior to application of the dialyzed material to ion exchange media in order to avoid fouling. Similar precipitation was noted during the use of diafiltration which made diafiltration unsuitable for buffer exchange prior to ion exchange chromatography.

[0067] The Inventors found that on scale-up, when depth filtration was used to remove materials precipitating from the re-dissolved second precipitation it was found that additional non-IgG proteins were being introduced into ion exchange chromatography. At a small scale such non- IgG proteins were removed in the dialysis / centrifugation process and did not contribute to the protein burden on the ion exchange column.

[0068] As noted above, in some embodiments of the inventive concept IgG can be recovered in an IgG isolation phase of the integrated process using serial chromatography on two sets of anion exchange media, with retention of IgG in flow-through fractions of both anion exchange steps. In anion exchange media capacity studies, breakthrough on a Q-Sepharose™ column was determined to occur at approximately 14 A280 units per mL of resin. After 2 injections of 35 A280Attny Dkt No. 101658.0023PCT units (14 A280 / mL) onto a 5mL Q-Sepharose™ column high molecular weight contaminants begin to appear in several places above the 62 kDa marker with significant albumin (~60kDa) breakthrough occurring after a third 35 A280 unit injection (21 A280 / mL). Typical results of such studies are shown in an SDS-PAGE gel in FIG. 6.

[0069] FIG. 6 shows a reduced bis-tris SDS-PAGE page gel of samples obtained from Q- Sepharose™ column breakthrough studies. A 5mL Q-Sepharose™ column was successively loaded with 3 injections of 35 A280of P2 material that had been passed through the DL10 depth filters and XLG filters, respectively. Lane 1 contains molecular weight standards. Lane 2 contains filtered second precipitate. Lane 3 contains flow-through fraction following injection 1 (as described above). Lane 4 contains flow-through fraction following injection 2. Lane 5 contains the flow-through fraction following injection 3. The mean ±SD protein load across three batches of depth-filtered dissolved second precipitate was 23,162 ± 2,000 A280 units. This equates to a Q-Sepharose column size of at least about 1 L / 1 L of starting plasma to satisfy the upper standard deviation and a 10% safety margin.

[0070] Typically, isolation of IgG using ion exchange chromatography utilizes anion exchange as negative selection (i.e., the IgG is not bound) coupled to cation exchange as a positive selector to which IgG is bound and subsequently eluted after contaminants are washed from the bound IgG. Such binding, washing, and elution steps in cation exchange chromatography limit scalability of such approaches and also provide additional opportunities for loss of IgG during the process.

[0071] Inventors used information regarding Q-Sepharose™ column capacity as shown in FIG. 6 to develop a different chromatography framework. Dissolved and depth-filtered second precipitate material was initially processed over a Q-Sepharose™ (an anion exchanger) column (14 A280 / mL) to generate a pool of material that was applied to different downstream chromatography schemes. An amount of such pooled material representative of flow-through from a single 5mL Q-Sepharose™ column run was subsequently applied to each of a second 5mL Q-Sepharose™ column, a 5mL DEAE Sepharose™ column, a 5mL CM-Sepharose™ column, or a 5mL DEAE Sepharose™ column coupled to a 5mL CM Sepharose™ column. In the case of the coupled DEAE / CM columns, the DEAE Sepharose™ column was uncoupledAttny Dkt No. 101658.0023PCT from the CM Sepharose™ column prior to elution of the CM Sepharose™ column. FIG. 7 provides a bis-Tris SDS-PAGE page gel of IgG products derived from various chromatography column schemes. Lane 1 contains a molecular weight standard. Lane 2 contains the initial flow through from a single Q-Sepharose™ column. Lane 3 contains eluate from a CM Sepharose™ column. Lane contains material resulting from applying flow-through from a DEAE Sepharose™ column and eluate obtained from applying this flow-through to a CM Sepharose™ column. Lane 5 contains flow-through from a DEAE Sepharose™ column. Lane 6 contains eluate from a DEAE Sepharose™ column. Lane 7 contains flow through from a second Q- Sepharose column. Lane 8 contains eluate from the second Q-Sepharose™ column. Arrows indicate resin combinations that removed a high molecular weight component, thus producing a higher-purity product.

[0072] Materials indicated by arrows in FIG. 7 were analyzed by LCMS to determine the relative IgG purity produced in each column scheme. The products of all column arrangements appear to have similar purities.

[0073] Three complete runs were performed using cryo-poor plasma at a 2L scale and two similarly sized 2L Q-Sepharose™ columns arranged in succession. Chromatography was performed on an AKTA Explorer FPLC™ system. The final conditions for these processes were as follows: 2 x 2L Q-Sepharose™ columns (1x Axichrome™ 100 / 300 and 1 x Axichrome™ 140 / 300) coupled in sequence and equilibrated in 20mM NaOAC pH 5.7. The columns were loaded at 45 mL / min, washed at 50 mL / min and eluted with a 2M NaCl step (implemented at 9500mL). One-liter fractions were collected based on A280reading. Under these conditions, a high-purity IgG with acceptable FXIa levels was obtained, indicating that the process is scalable. The physical characteristics of the three batches (batches D, E, and F) after filtration are shown below in Table 3. Volume Total yAttny Dkt No. 101658.0023PCT Table 3

[0074] In general, all 2 L scale processes performed identically in chromatography with no significant differences between profile or fraction collection. For illustrative purposes batch E was chosen as representative example. Fraction collection for product was started when the A280reached 50 mAU and terminated when the absorbance decreased to 125 mAU. As shown in FIG. 8, the primary flow-through peak (Fractions 1-5) was, surprisingly, not uniform in shape and contained 3-4 distinct features topping off at a maximum A280of 900 mAU / mL. Two smaller features (Fractions 6, biphasic peak and Fraction 8 single peak) were observed after the main peak. The elution peak (Elution fraction) is primarily uniform in shape with a complex tail of material. In the chromatogram shown in FIG. 8 individual fractions are indicated by vertical bars.

[0075] FIG. 9 shows results of non-reduced bis-tris SDS-PAGE on a gel loaded with 10 A280 units per lane of respective fractions collected from batch E Q-Sepharose™ / Q-Sepharose™ chromatography. Lane labels correspond to the labeled fractions in FIG. 8. The “final pool” consists of a pool of fractions 1 to 5, each of which appears to be pure IgG (150 kDa) with a slight extra band (~200kDa) visible in fraction 5. This band is apparently a “bleed through” from the biphasic peak of material contained in fraction 6 and appears to consist of a ~200kDa protein species mixed with an IgG species. The final pool (fractions 1 to 5) appears to be >95% IgG with no obvious contaminants present.

[0076] Regulatory requirements can necessitate that two virus inactivation / removal steps be included in any biological processing of human-derived products manufactured for human use. To that end, methods of the inventive concept can include a virus inactivation step applied to the second precipitate, and / or nano filtration to remove viruses after ion exchange chromatography.

[0077] Two-liter scale methods can be modified to incorporate, for example, treatment with 20mM sodium caprylate for 1 hour at room temperature for viral inactivation prior to depth filtration. A small amount of white precipitate can be formed upon addition of caprylate to the dissolved second precipitate. Table 4 shows the caprylate content of samples from treatment through Q-Sepharose™ chromatography. After Sartoclear™ DL10 depth filtration 63% of theAttny Dkt No. 101658.0023PCT original bolus of caprylate is detectable in the clarified sample. When dilution of P2 post depth filtration sample is accounted for in the post Sartoclear™ XLG sterilization filter sample there is no loss of caprylate in this filtration step. All remaining caprylate is removed during Q- Sepharose™ chromatography with no detectable levels of caprylate observed in any IgG fraction after this process step. SampleCaprylate(mM)Table 4

[0078] In addition to virus inactivation, virus removal (e.g., by nanofiltration) can be applied for virus removal. For example, nanofiltration can be applied as the second of two virus inactivation / removal steps in methods of the inventive concept. Virus removal can be accomplished using any suitable nanofilter / nanofiltration device and / or method. Suitable nanofiltration devices include, but are not limited to, a Sartorius Virosart Max ™0.1µm prefilter followed by a Sartorius Virosart HC™ 0.02µm virus removal filter. In a typical virus removal step a prefilter and virus removal filter are attached in sequence and flushed with water per manufacturer's instructions prior to addition of the product material. For example, a coupled set of 220cm2Virosart Max™ and Virosart HC™ filters (Sartorius) can be used for processing material derived from about 2 L of plasma. Alternatively, two or more coupled sets of such coupled filters can be used to, decrease loss of flux through the filter assembly and decrease processing time.

[0079] Use of flow-through fractions throughout this IgG isolation phase of the integrated process can yield solutions in which the concentration of IgG is non-optimal for clinical application. Accordingly, methods of the inventive concept can include a terminal protein concentration step that takes place after the IgG has been purified and utilizes techniques known in the art. Such concentration steps can, for example, be performed by ultrafiltration, and can incorporate a diafiltration step that permits buffer exchange into a pharmaceutically acceptableAttny Dkt No. 101658.0023PCT buffer composition. For example, in the isolation of IgG the highly purified product material can be concentrated to about 4% (w / v) or higher (e.g., about 5% w / v) IgG, with diafiltration during the concentration step used to replace the extant buffer with 0.2M Glycine (pH 4.2 to 6.5) or any other pharmaceutically acceptable buffer. For example, such a concentration / diafiltration step can be performed in three steps: (1) concentration of a nanofiltered protein solution to a volume approximately 500 mL, (2) diafiltration of the approximately 500 mL volume into a formulation buffer (e.g., 8 or more diavolumes), and (3) concentration of the diafiltered volume to the target concentration of protein (e.g., about 5% w / v IgG). Protein concentration can, for example, be monitored by absorbance at 280 nm.

[0080] The overall IgG yield of the three batches (E, F, G) was determined using a validated nephelometric assay for the starting plasma as well as for the subsequent fractions of the precipitation steps. Once the purification efforts had reached purities of over 90% IgG, A280 measurement and an extinction coefficient of 1.3 were used to quantify IgG. Table 5 shows the mean total IgG for all three batches present at each process step. The mean process yield is 75% with the most significant process losses occurring in the depth filtration (~8%) and Q- Sepharose™ chromatography (~9%) steps. Protein C n ntr ti n M n Yi ldAttny Dkt No. 101658.0023PCT Flow- ThroughTable 5

[0081] The chromatography step performed using two anion exchange columns arranged in series removes >99% of all proteins other than IgG. Surprisingly, it was found that two small trailing peaks (fractions 6 and 8 in FIG. 8) of material contain IgG species and contain no measurable IgA or IgM (by ELISA). IgG species in both of these fractions are disproportionately (~30%) IgG4. Inventors believe that the atypical shape of the Q-Sepharose™ chromatography flow-through fraction is due to differential, transient retention of the four IgG subclasses found in the sample inputted to the columns. IgG subclass ELISA data indicates that IgG1 continuously appears throughout the flow-through fraction, the second shoulder (fraction 3 on Figure 10) appears to be enriched in types 2 and 3 while IgG4 predominantly elutes towards the end of the main UV ‘peak’ (fraction 5). This unexpected result could be a function of limited IgG interaction the anion exchange resin at the buffer conductivity used (about 7mS), thereby essentially retarding progress of some IgG subclasses through the column without binding. Inventors believe that buffer conditions and anion exchange media selection and / or capacity can be optimized to provide IgG subclass separation in a flow-through fraction from anion exchange chromatography, in an integrated process starting with a blood product.

[0082] In prior art process for protein isolation there is generally an inverse relationship between yield and purity of the protein product. Surprisingly, Inventors have found that methods of the inventive concept can provide both very high yields (in excess of 70% of IgG content of starting material) and high purity. Comprehensive analytical testing was performed to examine the safety and co-purifying non-IgG protein content for several IgG product purifications using a method of the inventive concept employing two Q-Sepharose™ columns arranged in series, with IgG collected in flow-through fractions.

[0083] As shown in FIG. 10, SDS-PAGE indicates a product purity of >98% IgG, matching or exceeding that of current commercial IgG products for human use. Specifically, when examinedAttny Dkt No. 101658.0023PCT in comparison to commercially available intravenous IgG products (Gammagard™, Gamunex™, Privagen™, and Bivigam™), the final product IgG has fewer HMW contaminants.

[0084] Analytical testing results of IgG products from a method of the inventive concept employing two Q-Sepharose™ are shown in Table 6. For this purpose, IgG products from 2 L scale batches produced using such a method of the inventive concept were formulated in 0.2M Glycine pH 4.2, with a target of about 5% total protein. This is consistent with current practice in regard to therapeutic IgG products for human use. IgG concentrations were determined by absorbance at 280nm by spectrophotometry. Additional protein content determinations were performed on the final product formulations. The mean concentrations across methods (N=5) of the three respective final formulations were 41.2, 52.9 and 50.9 mg / mL, respectively. IgG Subclass distribution was measured by IgG subclass ELISA and determined to be 63% IgG1, 28% IgG2, 7% IgG3 and 2% IgG4 across the three final formulations. The respective cryo-poor plasma starting material values were 59% IgG1, 29% IgG2, 7% IgG3, and 4% IgG4. In regard to other immunoglobulin species, only IgA was detected by ELISA (3.3 µg / mL) and was confirmed by LCMS. IgA content, however, is below 4 µg / mL. IgM was not detected by ELISA, and LCMS only detected trace amounts of IgM, IgE and IgD peptides Test Method Batch E Batch F Batch G Acceptance Criteria G %Attny Dkt No. 101658.0023PCT Test Method Batch E Batch F Batch G Acceptance Criteria IgM ND ND ND <1 µg / mLTable 6

[0085] Product from an IgG isolation phase method of an integrated IgG and albumin isolation method of the inventive concept and employing two anion exchange columns arranged in series was characterized for content related to safety and efficacy. Accordingly, Anti A, B and D antibodies, anti-complement activity (ACA), protein Kinase A (PKA) activity, Fc function, activated coagulation factor XI (FXIa) activity by thrombin generation assay or TGA), and non- activated partial thromboplastin time (NAPTT) activity were characterized for several 2 L scale final product formulations. Results were as follows: • Anti A,B,D Antibodies: All three product batches tested passed typical acceptance criteria for products for human use. • Fc Function / Polymerization: Fc function in all three product batches was >100% at the two concentrations of IgG tested, which meets or exceeds typical acceptance criteriaAttny Dkt No. 101658.0023PCT for IgG product for human use. Furthermore, SE-HPLC data indicates that >96% of the IgG is monomeric with <4% dimer, <0.2% higher order polymers, and almost undetectable levels of Ig fragments. This meets both FDA and EU specifications for IgG products for human use. • PKA and ACA Activity: PKA and ACA activity are tests for the presence of contaminants that can lead to activation / inactivation of innate immunity through their respective proteolytic cascades. PKA activity was low (Mean =1.3 IU / mL) and well below the 35 IU / mL acceptance criteria typical for IgG products for human use. However, ACA activity was above the 1.0 CH50 U / mg.

[0086] Procoagulant Activity: Clotting time measures inherent procoagulant activity (regardless of source), whereas the TGA assay measures FXIa-like activity. At the lowest dilution, batch E (141 sec) barely failed to meet the >150 sec acceptance criteria, while batches F and G passed. FXIa-like activity in all three batches was somewhat high (>2.0 mU / mL) indicating activation of the contact pathway of coagulation at some point during the purification process. In order to eliminate FXIa (i.e., activated factor XI) and other trace contaminations use of a cation exchange column applied to effluent from the serially arranged Q-Sepharose™ columns can be used, by binding IgG found in the Q-Sepharose™ eluate and subsequent elution of IgG from the cation exchange media.

[0087] Alternatively, such integrated methods can utilize a chromatography step utilizing a chromatography media that does not interact with a protein of interest (e.g., IgG), with direction of the flow through from such a chromatography step to a second chromatography step utilizing a different chromatography media that has an affinity for both a protein that is to be isolated (i.e., the protein of interest) and one or more contaminants that are present in solution with a protein of interest. This second chromatography media can be selected to have a higher affinity (i.e., interact more strongly) with one or more the contaminants than a protein of interest.

[0088] The capacity of such a chromatography media used can be calculated on the basis of the contaminant content of the solution containing a protein of interest, and can be selected such that breakthrough of one or more contaminants does not occur to a significant extent (e.g., greater than 0.01%, 0.1%, 0.25%, 0.5%, 1%, 2%, 5%, or 10%) of the contaminant content of theAttny Dkt No. 101658.0023PCT solution being purified during purification. Since only contaminants are bound to this second chromatography media the ion exchange capacity can be quite low. This advantageously supports the use of small volumes of chromatography media and / or the use of ion exchange filters, which reduces the complexity of large scale operations.

[0089] For example, in an IgG isolation portion of such an integrated process an anion exchange step can be performed in which IgG is not bound to the anion exchange media, with the flow through from the anion exchange step being directed to a cation exchange step having a small capacity for cation exchange (e.g., through the use of a small volume of cation exchange media). Due to the limited capacity of the cation exchange media provided, binding of contaminating proteins can displace IgG (which would normally bind to the cation exchange media) and permit recovery of IgG at high purity and yield in the flow through from the cation exchange step. It should be appreciated that such chromatography media capacity is a function of the nature of the chromatography media, the amount of media used, solution composition, solution pH, flow rate, etc., and is readily determined and / or optimized experimentally. Generally the capacity of chromatography media required is relatively small relative to prior art methods, and can advantageously be provided by a functionalized (e.g., charged) filter.

[0090] In practice Inventors have found that, despite using chromatography media having the ability to capture a protein of interest under the buffer conditions used, application of methods of the inventive concept permit recovery of in excess of 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of a total protein of interest applied to chromatography media in the flow-through fraction. Without wishing to be bound by theory, Inventors believe that interaction of contaminants with the chromatography media effectively displaces the protein of interest. This permits highly efficient recovery of a protein of interest from contaminants having similar charge or other characteristics that would otherwise make them difficult to separate from one another. Purity of the recovered protein of interest can be from 80%, 85%, 90%, 95%, 98%, 99%, or higher (by weight).

[0091] An example of chromatography steps of an integrated method is shown schematically in FIG. 1. As shown, a solution containing a protein of interest and one or more contaminants (e.g., a supernatant from a precipitation step, a re-dissolved precipitate from a precipitation step, aAttny Dkt No. 101658.0023PCT flow-through from a chromatography step) is applied to a low capacity chromatography media. Buffer conditions (e.g., ionic strength, pH, temperature, etc.) can be adjusted prior to this application. Buffer conditions can be adjusted by any suitable means, for example addition or removal of one or more salt(s), addition of acid or base, etc. Suitable media include, but are not limited to, ion exchange media (e.g., DEAE, Q, S, or CM media), hydrophobic interaction media (e.g., propyl, butyl, or phenyl media), affinity media, or mixed-mode media. As noted above, the functionality of the chromatography media is selected to interact with both the protein of interest and one or more contaminants, however the capacity of the media is selected to match or slightly exceed (e.g., by 1%, 5%, 10%, 25%, 50%, 100%, 150%, or 200%) the capacity at which breakthrough of contaminant occurs. The protein of interest can then be subsequently collected at high yield and purity (as described above) in a flow-through fraction.

[0092] It should be appreciated that such an approach can greatly simplify processing of protein- containing solutions at large volumes (e.g., greater than 2 L, 10 L, 50 L, 250 L, 1000 L, 2500 L, 5000 L, 8000 L), as collection of the protein of interest does not require a separate set of elution steps and the process utilizes a minimal amount of chromatography media.

[0093] Some embodiments chromatography phases of such an integrated process can utilize two different chromatography media. One of these is selected to not interact with a protein of interest, and to bind a portion of the contaminating species (e.g., contaminating proteins) present. The second chromatography media differs from the first chromatography media, and is selected and used under conditions in which it can bind with the protein of interest. Inventors have surprisingly found that such second chromatography media can be employed in negative selection mode (i.e., with a protein of interest being recovered in the flow-through from the media) provided that the second chromatography media is provided in amounts that provide relatively little protein binding capacity to provide simple, efficient, and highly scalable integrated protein isolation methods. In some embodiments such a second chromatography media can be selected to bind contaminants while having little to no binding capacity for the protein of interest while differing from the first chromatography media.Attny Dkt No. 101658.0023PCT

[0094] In such methods the capacity of the second chromatography media (which is a function of both the chemistry of the chromatography media, the chromatography buffer, and amount used) is selected on the basis of its ability to bind contaminants remaining in the flow-through obtained from the first chromatography media. This capacity can, for example, be determined using break-through studies in which contaminants are applied to the second chromatography media until they appear in the flow-through (under defined conditions of buffer composition and flow rate). An amount of the second chromatography media sufficient to bind contaminant content at the desired scale can then be utilized in a combined process using both the first and second chromatography media. In some embodiments a small excess of capacity in this second chromatography media (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% excess) can be used.

[0095] Notably, the capacity of the second chromatography media is significantly less than that used in a conventional isolation method in which the protein of interest is bound and subsequently eluted (e.g., less than 10%, 5%, 2.5%, 1%, 0.5%, 0.25%, 0.1%). In some embodiments this permits use of functionalized (e.g., charged) filters having appropriate pendant groups in place of traditional bead or particle-based chromatographic media. This advantageously permits combining particle removal and chromatography steps.

[0096] Inventors have found that, despite having the ability to bind with the target protein, integrated methods of the inventive concept typically recover a protein of interest at high yield (in excess of 60%, 70%, 75%, 80%, 85%, 90%, or 95%) in the flow-through from the second chromatography media. Without wishing to be bound by theory, the Inventors believe that utilization of a second chromatography step that utilizes a judiciously minimized amount of media capacity results in displacement of the protein of interest from the media by contaminants present in the first flow-through. This removes the need for an elution step, and results in increased yield. In addition, collection of the protein of interest in successive flow-through fractions from successive chromatography steps greatly simplifies the isolation process and transition from bench scale (up to about 2 L of starting material) to industrial scale (8,000 L or more of starting material).Attny Dkt No. 101658.0023PCT

[0097] Further, such integrated methods can produce more isolated proteins having reduced denatured and / or aggregated protein content since isolated proteins are not bound and subsequently eluted from chromatography media using harsh conditions. This advantageously both enhances yield and reduces the chance of denaturation, while also simplifying the isolation process and greatly reducing processing time. As such it is distinct and different from (and much more cost effective) than current protein isolation processes that involve binding to and elution from chromatographic media, with improved protein stability, increased in vivo half-life, more rapid infusion rates, improved patient tolerance, and reduced immunogenicity of the protein therapeutic.

[0098] In methods of the inventive concept first and second chromatography media can be selected to have complementary binding characteristics. For example, the first chromatography media and the second chromatography media can be ion exchange media that have opposing charges under the separation conditions used (e.g., anion exchange followed by cation exchange, cation exchange followed by anion exchange). Although examples provided below cited the use of ion exchange media, Inventors contemplate that any chromatographic media having complementary binding characteristics can be paired in methods of the inventive concept. Suitable chromatography media include, but are not limited to, ion exchange media (e.g., DEAE, Q, CM, and / or S chromatography media), hydrophobic interaction chromatography media, affinity chromatography media, and mixed-mode chromatography media. For example, in some embodiments first and second chromatography media can be ion exchange media with opposing charges under separation conditions. Alternatively, in some embodiments ion exchange chromatography media or mixed-mode chromatography media can be paired with hydrophobic interaction chromatography media (with appropriate adjustments to ionic strength between chromatographic steps). In still other embodiments, an affinity chromatography media can be used as a first chromatographic media and ion exchange, hydrophobic interaction, or mixed- mode chromatographic media as the second chromatography media.

[0099] Preferred embodiments of the inventive concept can utilize both anion and cation exchange chromatography (which are relatively inexpensive and available in a wide variety of formats), where buffer conditions and column binding capacity are selected or optimized to provide the target protein (e.g., IgG) in the flow-through fraction of each chromatographic step.Attny Dkt No. 101658.0023PCT Towards this end an initial ion exchange step (e.g., anion exchange, cation exchange) can be performed using a high capacity ion exchange media that does not appreciably bind the protein of interest.

[0100] For example, in an integrated method that incorporates isolation of IgG a large / high capacity anion exchange step can be performed on a re-dissolved second precipitate (P2), providing a flow-through fraction containing IgG (along with some contaminants) and retaining a bound fraction that includes a portion of the contaminating proteins present in the starting material. This flow-through fraction can then be applied (in some embodiments following an adjustment of salt content and / or pH to adjust ionic strength / conductivity and or pH) to a small or low capacity cation exchange media.

[0101] The size of this small or low capacity cation exchange media is selected so that it is near or slightly (e.g., 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%) greater than the amount or capacity for breakthrough of a contaminating protein found in the flow-through of the anion exchange media (taking buffer conditions into account). Without wishing to be bound by theory, the Inventors believe that this permits contaminating proteins to displace IgG that may bind to the cation exchange media. Careful selection of the amount / capacity of the cation exchange media and / or buffer conditions provides efficient removal of contaminating protein while also providing high yields of IgG.

[0102] An example of such an initial step in an integrated process of the inventive concept is shown below in FIG. 2. It should be appreciated that in this context a starting material can be serum, plasma, cryo-poor plasma, cryo-poor plasma into which the cryoprecipitate has been re- dissolved, or a fraction (e.g., a supernatant or a dissolved precipitate) resulting from a precipitation step applied to such materials. Although blood proteins are of interest, a suitable starting material can be any solution containing a protein of interest (e.g., cell cultures, supernatants or lysates of cells from mammalian, bacterial, fungal, insect, or plant-based tissues or tissue cultures, solvated inclusion bodies, animal egg contents, milk, urine, or other body fluids, etc.). As shown, a starting material is applied to a high capacity first chromatography media selected to bind a portion of the contaminants and to not bind the protein of interest. The flow-through of this first chromatography step is directed (in some instances, after an adjustmentAttny Dkt No. 101658.0023PCT in buffer composition and / or pH) to a second, different chromatographic media that is provided at low capacity. This second chromatography media can potentially bind the protein of interest as well as contaminants remaining in the first flow-through, but the capacity of the media is selected such that available binding sites are occupied by contaminants. Under these conditions the target protein is recovered in the flow-through from the second chromatography step at high yield (e.g., with less than 30%, 25%, 20%, 15%, 10%, 5%, 2.5%, or 1% of target protein present in the first flow-through lost).

[0103] Methods of the inventive concept are particularly suitable for isolation of therapeutic proteins, for example from blood and blood products. Examples of blood products include serum, plasma, cryoprecipitate, cryo-poor plasma, and cryo-poor plasma into which cryoprecipitate has been re-dissolved. Similarly, methods of the inventive concept are suitable for isolation of therapeutic proteins from products of conventional processes for protein isolation from blood products, including precipitation (e.g., with an organic solvent, an inorganic salt, an organic acid salt, and / or a hydrophilic polymer), chromatography, ultrafiltration, and / or diafiltration.

[0104] Methods of the inventive concept can also be applied to non-blood sources of therapeutic proteins. These include cell cultures, supernatants or lysates of cells from mammalian, bacterial, fungal, insect, or plant-based tissues or tissue cultures, solvated inclusion bodies, animal egg contents, milk, urine, or other body fluids, etc. products of cell-free protein synthesis, and products of protein conjugation processes.

[0105] Therapeutic proteins towards which methods of the inventive concept can be applied include, but are not limited to two or more of albumin, alpha-one antitrypsin, immunoglobulins (e.g., IgG, IgM, IgA, IgY), clotting factors (e.g., Factor VIII, von Willebrand Factor), and host cell proteins (HSP).

[0106] Inventors have found methods of the inventive concept are particularly useful in the isolation of IgG and albumin from blood plasma, although application to other solutions containing IgG and albumin (e.g., cell culture media, cell lysates, other body fluids, etc.) is contemplated. Within the context of this application plasma is considered to include freshly collected plasma, refrigerated plasma, frozen plasma, cryo-poor plasma, and cryo-poor plasmaAttny Dkt No. 101658.0023PCT into which the cryoprecipitate has been re-dissolved. Such plasma can, for example, be obtained as pooled material from commercial collection centers.

[0107] An example of an initial portion of an integrated method of the inventive concept for isolation of IgG and albumin (specifically, the portion in which IgG is isolated) from plasma is shown in FIG. 3. The Inventors used two salt precipitation steps (i.e., about 11% in the first salt precipitation and about 26% in the second salt precipitation) to generate a protein solution from cryo-poor plasma (CPP). This protein solution severed as starting material for the subsequent chromatography step and contained maximum IgG yield and minimized concentrations of unwanted proteins. As shown, the first precipitation step produces a first supernatant (S1) that includes IgG (and albumin), and the second precipitation step produces an IgG-rich precipitate or paste (P2). The albumin-rich supernatant from this step (S2) is separated and processed to isolate albumin as described below.

[0108] The IgG-rich P2 precipitate is dissolved (e.g., in water) prior to ion exchange steps. As noted below, in preferred embodiments a buffer exchange step (e.g., dialysis, diafiltration, ultrafiltration followed by dilution, size exclusion chromatography, etc.) is not performed prior to ion exchange steps. Table 1

[0109] As shown in FIG. 3, the isolation process of a target protein from re-dissolved P2 precipitate can utilize a first ion exchange step that can be performed using an anion exchange media (e.g., DEAE or Q chromatography media). In some embodiments a strong anion exchange media can be used that maintains a positive charge over a wide range of pH conditions (e.g., pH 1 to 14, pH 2 to 13, pH 3 to 12, pH 4 to 11). Chromatography media (e.g., anion exchange media as cited in this example) can be provided in any suitable form and / or on any suitable support (e.g., agarose, cross linked agarose, cellulose, polyacrylamide, polystyrene, glass, or combinations thereof) and in any suitable configuration (e.g., porous beads, non-porous beads, fibers, wools, filters, etc.).Attny Dkt No. 101658.0023PCT

[0110] As shown in FIG. 3, IgG is recovered in this initial portion of the integrated process in the flow-through (i.e., unbound) fraction from the anion exchange media. This first flow- through fraction can subsequently be applied to a small or low capacity cation exchange media (e.g., media that includes carboxylate or sulfonate groups) in which size / capacity has been selected such that contaminants (e.g., Factor XI, activated Factor XI, Factor XII, activated Factor XII) are retained while IgG (which typically also binds to cation exchange media) passes through in the flow-through fraction (i.e., the second flow-through fraction).

[0111] In such embodiments the capacity or size of the cation exchange media is selected to be at or slightly exceeding (e.g., by 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 75%, or 100%) its capacity for contaminants present in the first flow-through fraction. Without wishing to be bound by theory, Inventors believe that when a cation exchange media is so selected bound protein (e.g., IgG) is displaced by the contaminated protein and released into the second flow- through fraction, thereby increasing yield. In preferred embodiments loss of IgG in the cation exchange step is less than about 30%, 25%, 15%, 10%, 7.5%, 5%, 4%, 3%, 2%, or 1% of IgG content of the starting material.

[0112] Such second chromatography media (e.g., a cation exchange media in this example) can be provided on any suitable support (e.g., agarose, cross linked agarose, cellulose, polyacrylamide, polystyrene, glass, or combinations thereof) and in any suitable configuration (e.g., porous beads, non-porous beads, fibers, wools, filters, etc.). Inventors have found that the capacity of the cation exchange media utilized can be quite small, and in preferred embodiments can be provided by a filter with pendant cation exchange groups. This advantageously combines purification and clarification steps.

[0113] In some embodiments of the inventive concept the buffer composition and / or pH of the first flow-through fraction can be modified prior to application to the second chromatography media (e.g., the cation exchange media of this example) in order to optimize capacity and selectivity for contaminants. For example, buffer conditions can be selected or adjusted such that contaminants have a higher affinity for the second chromatography media than that of the target protein. In preferred embodiments of protein (IgG) isolation methods of the inventive concept salts can be added to increase the ionic strength or conductivity of the first flow-throughAttny Dkt No. 101658.0023PCT fraction to fall within a desired range. Alternatively, the first flow-through can be diluted or subjected to buffer exchange (e.g., dialysis, diafiltration, etc.) prior to application to the second chromatography media.

[0114] In some embodiments the second flow-through fraction can be subjected to additional processing steps. Such additional processing steps can include nanofiltration for virus removal, such as filtration using a 0.02 µm pore membrane. Inventors have found that this effectively retains any remaining virus particles while minimizing yield losses.

[0115] Typical IgG yield results for isolation of IgG in an initial step of an integrated process of the inventive concept are shown in Table 1. Tot pleVolu al IgG IgG Total IgG IgG SamConcentrationme Total (A280 / mL)(mL) (A280) [A280] [Neph] [Neph] YieldAttny Dkt No. 101658.0023PCT Total Additive Loss from Sample Removal 0.96 Total yield based on A280 absorbance / initial nephelometry values corrected for sample removal. Extin ti n C ffi i nt f r I G: 13Table 1 Test results obtained from the material shown in Table 1 (i.e., 44.94 mg / mL IgG) are shown in Table 2. Test Result Pass / Fail EP Standard PKA 1.48 IU / mL Pass <35 IU / mL ct 1 yTable 2 In addition to remarkably low levels of Factor XI contamination, the Inventors have found that methods of the inventive concept provide surprisingly low levels of IgA contamination.

[0116] Initial steps of an integrated process for isolating IgG and albumin from plasma were found to provide robust and commercially scalable processes that consistently produce about a 72-78% yield of IgG from starting plasma in just 48 hours. The resulting product is >99% pure IgG product with 100% functionality.

[0117] In such integrated methods the second supernatant (S2) can be separated from the second precipitate (P2, from which IgG can be recovered as described above) and can be furtherAttny Dkt No. 101658.0023PCT processed for albumin isolation to provide an integrated process for isolation of both IgG and albumin and high yield and purity. A schematic representation of such an integrated process is shown in FIG. 14.

[0118] In an example of such an integrated process for isolation of IgG and albumin from a blood product, two successive salt precipitations can be performed as described above. This generates a second precipitate (P2, which can be processed for isolation of IgG as described above) and a second supernatant (S2). Most or all of the albumin is found in the second supernatant. This S2 can be processed for further purification of albumin, or it can be frozen for storage and thawed prior to processing for further purification of albumin.

[0119] The pH of S2 is adjusted to about 7 and sodium caprylate is added to give a final concentration of about 10 mM to 100 mM or about 40 mM caprylate. The resulting mixture is then incubated at about 60°C for one hour and the resulting precipitate (P3) removed. The precipitate can be removed from the supernatant (S3) by any suitable method, including centrifugation, settling and decanting, and filtration. For example, following caprylate / heat treatment the resulting mixture can be passed through a depth filter, such as a PDD1 depth filter or a V100P filter. Surprisingly, Inventors have found that use of a PDD1 depth filter is effective in reducing some low molecular weight (i.e., lower MW than albumin) contaminants that are not removed by the V100P depth filter.

[0120] S3 can be subjected to diafiltration in order to remove contaminants and / or provide suitable conditions for ion exchange chromatography. For example, S3 can be diafiltered using a 10 kD cutoff membrane or a 30 kD cutoff membrane. Buffers suitable for ion exchange typically have relatively low ionic strength (e.g., less than about 100 mM) and have a pH appropriate for the protein’s isoelectric point and the nature of the fixed charge on the ion exchange media. For example, to recover albumin in a flow-through fraction the pH can be slightly acidic (e.g., pH 4.5 to pH 6.5, pH 5 to pH 6, or about pH 5.3) when a weak anion exchange chromatography media (e.g., DEAE media) is used. Surprisingly, when S3 was exchanged into 50 mM acetate buffer at pH 5.3 and applied to a DEAE, approximately 97% pure albumin (i.e., purified albumin) was recovered in the flow-through fraction as a second broad peak that followed an initial flow-through peak. This pattern suggests partial retention ofAttny Dkt No. 101658.0023PCT albumin on the DEAE media. This degree of purity can be sufficient for pharmaceutical use. Accordingly, the albumin-containing peak obtained as a flow-through fraction from DEAE chromatography can be further processed for use, for example by nanofiltration and adjustment of protein concentration prior to filling. Optionally, a second chromatography step can be applied to this purified albumin flow-through peak to provide a highly purified albumin. For example, this albumin-containing flow-through peak can be applied to a small amount of cation exchange media (e.g., a cation exchange filter) to remove additional contaminants if required. Such a small amount of ion exchange media can be selected such that its capacity for albumin does not significantly impact yield (e.g., more than about 25%, 20%, 156% 10%, 5%, or less) while retaining tightly-bound contaminants.

[0121] Embodiments of the inventive concept include those in which additional proteins are recovered from S1, P1, S2, P2, and / or intermediate products of IgG and / or albumin isolation paths of the integrated process (e.g., by affinity chromatography). Examples of such additional proteins include alpha 1 antitrypsin.

[0122] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refer to at least one of something selected from the group consisting of A, B, C …. and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

Claims

CLAIMS What is claimed is:

1. A method of isolating albumin from cryo-poor plasma, comprising adding a citrate salt to cryo-poor plasma to a concentration of about 11% to 13% by weight to generate a first supernatant and first precipitate; isolating the first precipitate from the first supernatant; adding the citrate salt to the first supernatant to generate a second precipitate and a second supernatant; separating the second precipitate from the second supernatant; adding caprylate to the second supernatant to a concentration of about 40 mM to generate a caprylate treated second supernatant; increasing the temperature of the caprylate treated second supernatant to about 50°C to about 60° for a time sufficient to generate a third precipitate and a third supernatant; separating the third supernatant from the third precipitate; adjusting the pH of the third supernatant to from pH 4 to pH 6 to generate a pH adjusted third supernatant; applying the pH adjusted third supernatant to an anion exchange medium; and collecting an anion flow-through fraction comprising the albumin.

2. The method of claim 1, wherein the time sufficient to generate the third supernatant and the third precipitate is about one hour.

3. The method of claim 1 or 2, wherein adjusting the pH is performed by diafiltration using a diafiltration membrane with a 30 kD molecular weight cutoff.

4. The method of one of claims 1 to 3, wherein separating the third supernatant from the third precipitate comprises application to a depth filter.

5. The method of one of claims 1 to 4, wherein the anion flow-through fraction is a second of two flow-through peaks observed from the anion exchange media.

6. The method of one of claims 1 to 5, further comprising applying the anion flow-through fraction to a cation exchange media and collecting a cation exchange flow-through fraction comprising albumin.

7. An integrated process for isolating both immunoglobulin G (IgG) and albumin from cryo- poor plasma, comprising adding a citrate salt to cryo-poor plasma to a concentration of about 11% to 13% by weight to generate a first supernatant and first precipitate; isolating the first precipitate from the first supernatant; adding the citrate salt to the first supernatant to generate a second precipitate and a second supernatant; separating the second precipitate from the second supernatant; dissolving the second precipitate to generate a dissolved second precipitate; adding caprylate to the dissolved second precipitate to generate a caprylate treated second precipitate; removing solids from the caprylate treated second precipitate; applying the caprylate treated second precipitate to a first anion exchange medium to generate a first anion exchange flow-through fraction comprising IgG; applying the first anion exchange flow-through fraction to a second ion exchange step, wherein the second ion exchange step comprises one of a first cation exchange media and a second anion exchange media to generate a first cation exchange flow-through fraction comprising purified IgG; adding caprylate to the second supernatant to a concentration of about 40 mM to generate a caprylate treated second supernatant; increasing the temperature of the caprylate treated second supernatant to about 50°C to about 60° for a time sufficient to generate a third precipitate and a third supernatant; separating the third supernatant from the third precipitate; adjusting the pH of the third supernatant to from pH 4 to pH 6 to generate a pH adjusted third supernatant; applying the pH adjusted third supernatant to a second anion exchange medium; and collecting a second anion flow-through fraction comprising albumin.

8. The method of claim 7, wherein the time sufficient to generate the third supernatant and the third precipitate is about one hour.

9. The method of claim 7 or 8, wherein adjusting the pH is performed by diafiltration using a diafiltration membrane with a 30 kD molecular weight cutoff.

10. The method of one of claims 7 to 9, wherein separating the third supernatant from the third precipitate comprises a depth filter.

11. The method of one of claims 7 to 10, wherein the second anion flow-through fraction is a second of two flow-through peaks observed from the anion exchange media.

12. The method of one of claims 7 to 11, further comprising applying the second anion flow- through fraction to a second cation exchange media and collecting a second cation exchange flow-through fraction comprising albumin.

13. The method of one of claims 7 to 12, wherein the purified IgG has a purity of 95% or higher and a yield of at least 70%.

14. The method of one of claims 1 to 13, wherein albumin has a purity of 95% or higher and a yield of at least 60%.

15. The method of one of claims 1 to 14, comprising isolation of an additional therapeutic protein from at least one of the group consisting of the first supernatant, the first precipitate, the second supernatant, and the second precipitate.

16. The method of claim 15, wherein isolation comprises an affinity chromatography step.

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