Purification method for hemopexin using mixed-mode chromatography

Abstract

Disclosed herein is a method for purifying proteins, particularly hemopexin, comprising passing a solution containing hemopexin and other proteins through a mixed-mode cation exchange chromatography resin under conditions that promote selective binding of hemopexin to the resin, and eluting the bound hemopexin from the resin. The disclosure also extends to compositions containing purified hemopexin and uses thereof.

Description

[Technical Field]

[0001] The present invention relates generally to methods for purifying proteins. More specifically, the present invention relates to methods for purifying hemopexin and uses thereof. [Background technology]

[0002] Hemolysis is characterized by the destruction of red blood cells and is characteristic of red blood cell abnormalities such as enzyme deficiencies, hemoglobinopathies, hereditary spherocytosis, paroxysmal nocturnal hemoglobinuria, and spur cell anemia, as well as anemic disorders associated with exogenous factors such as splenomegaly, autoimmune disorders (e.g., hemolytic disease of the newborn), genetic disorders (e.g., sickle cell disease or G6PD deficiency), microangiopathic hemolysis, gram-positive bacterial infections (e.g., streptococci, enterococci, and staphylococci), parasitic infections (e.g., plasmodium), toxins, and trauma (e.g., burns). Hemolysis is also a common disorder in blood transfusions, especially in patients receiving massive transfusions and extracorporeal cardiopulmonary support.

[0003] The adverse effects observed in patients with hemolysis-related conditions are primarily due to the release of iron and iron-containing compounds, such as hemoglobin (Hb) and heme, from red blood cells. Under physiological conditions, released hemoglobin binds soluble proteins, such as haptoglobin, and is transported to macrophages and hepatocytes. However, when hemolysis accelerates and becomes pathological, the buffering capacity of haptoglobin becomes insufficient. As a result, hemoglobin is rapidly oxidized to ferrihemoglobin, which then releases free heme (containing protoporphyrin IX and iron). Although heme plays an important role in several biological processes (e.g., as part of essential proteins such as hemoglobin and myoglobin), free heme is highly toxic. Free heme is a redox-active source of iron that generates highly toxic reactive oxygen species (ROS) that damage lipid membranes, proteins, and nucleic acids. Heme toxicity is further exacerbated by its ability to intercalate into lipid membranes, where it causes oxidation of membrane components and promotes cell lysis and death.

[0004] The evolutionary pressure of continuous exposure to low levels of extracellular Hb / heme has led to compensatory mechanisms that suppress the harmful effects of free Hb / heme under physiological steady-state conditions and during mild hemolysis. These systems involve the release of a group of plasma proteins that bind Hb or heme, including the Hb scavenger protein, haptoglobin, and the heme scavenger proteins, hemopexin and α1-microglobin. However, while endogenous haptoglobin and hemopexin suppress the harmful effects of free Hb / heme under physiological steady-state conditions, they are largely ineffective in maintaining steady-state Hb / heme levels under pathophysiological conditions, such as those associated with hemolysis.

[0005] Hemopexin preparations have been shown to exhibit serine protease activity (Non-Patent Document 1), anti- and pro-inflammatory activity, inhibition of cell adhesion, and binding of certain divalent metal ions. Furthermore, hemopexin infusion has been shown to attenuate heme-induced endothelial activation, inflammation, and oxidative damage in animal models of hemolytic disorders such as sickle cell disease and β-thalassemia. Although purified hemopexin shows significant therapeutic potential, the amount of hemopexin required to meet anticipated market demand requires a high-capacity purification process. However, previously developed processes for purifying hemopexin from human plasma are limited to small-scale production methods for research purposes only, such as toxicity testing and Phase I clinical manufacturing (see, e.g., Patent Document 1; Non-Patent Document 2; and Non-Patent Document 3). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2014 / 055552 [Non-patent literature]

[0007] [Non-Patent Document 1] Lin et al., 2016, Molecular Medicine, 22:22-31 [Non-patent document 2] Tsutsui and Mueller, 1981, Analytical Biochemistry, 121:244-250 [Non-patent document 3] Muller-Eberhard, 1988, Methods in Enzymology, 163:563-565. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, there remains a need to develop improved methods for the purification of hemopexin. [Means for solving the problem]

[0009] In one aspect of the invention, there is provided a method for purifying hemopexin from a solution containing hemopexin and other proteins, comprising the steps of: (i) providing a solution containing hemopexin and other proteins, wherein the solution contains less than about 300 mM NaCl; (ii) passing the solution of step (i) through a mixed-mode cation exchange chromatography resin under conditions that promote the selective binding of hemopexin to the resin over the binding of other proteins to the resin; (iii) washing the resin after step (ii) to remove unbound proteins; (iv) eluting the resin-bound hemopexin after step (iii); (v) recovering the hemopexin eluted in step (iv); A method is provided which includes:

[0010] In one embodiment, the method comprises: (vi) passing the recovered hemopexin eluate of step (v) through a mixed-mode anion exchange chromatography resin under conditions that allow any impurities in the recovered hemopexin eluate to bind to the resin while allowing hemopexin to pass through the resin as an unbound fraction; (vii) recovering the unbound fraction containing hemopexin; Further includes:

[0011] In another aspect of the present invention, compositions are provided comprising hemopexin recovered by the methods disclosed herein.

[0012] In another aspect of the present invention, there is provided a formulation comprising a composition disclosed herein and a pharmaceutically acceptable carrier.

[0013] In another aspect of the present invention, there is provided a composition or formulation disclosed herein for use as a medicament for treating a condition associated with hemolysis.

[0014] In another aspect of the present invention, there is provided a method of treating a condition associated with hemolysis, comprising administering to a subject in need thereof a composition or formulation disclosed herein.

[0015] In another aspect of the present invention, there is provided the use of a composition or formulation disclosed herein in the manufacture of a medicament for treating a condition associated with hemolysis. [Brief explanation of the drawings]

[0016] [Figure 1-1]

[0023] Figure 1 shows that the mixed-mode anion exchange chromatography resin Capto Adhere can separate hemopexin from other impurity proteins. Photographic representation of non-reducing SDS-PAGE of fractions eluted in a stepwise pH elution from a mixed-mode anion exchange chromatography screen using (A) Capto Adhere (Cytiva); (B) HEA Hypercel (Pall); (C) PPA Hypercel (Pall); and (D) MEP Hypercel (Pall). Lanes 1 and 2 = flow-through, lane 3 = wash, lane 4 = pH 6, lane 5 = pH 5, lane 6 = pH 4, lane 7 = pH 3, lane 8 = blank, and lane 9 = control. [Figure 1-2] Same as above. [Figure 2] Figure 1 shows the amount of protein (%; y-axis) in solution extracted from Fraction IV-4 paste over the pH range of pH 4 to pH 8 (pH; x-axis). Line denoted t = transferrin, line denoted a = albumin, line denoted h = haptoglobin, and line denoted H = hemopexin. [Figure 3] Figure 1 shows the consistency of FIV-4 paste extraction between batches. Graphical representation of hemopexin concentration (g / L; y-axis) and FIV-4 paste batches. Hemopexin concentration measured by reverse-phase HPLC. [Figure 4] Figure 1 shows that hemopexin yield from FIV-4 paste can be increased by using a higher pH extraction buffer. Graphical representation of hemopexin concentration (g / L; y-axis) and FIV-4 paste batch (x-axis) after extraction at pH 6.2 (black bars) and 7.5 (gray bars). [Figure 5-1]

[0023] Figure 1. Development and optimization of Capto MMC chromatography as an effective capture step for commercial purification of hemopexin. Series of photographic representations of non-reducing SDS-PAGE analysis of fractions from Capto MMC chromatography of clarified FIV-4 extract paste loaded at (A) pH 5.0, (B) pH 6.0, and (C) pH 7.0 and eluted with a stepwise NaCl gradient. Lane 1 = protein marker, lane 2 = load, lane 3 = drop-through, lane 4 = wash, lane 5 = 50 mM NaCl, lane 6 = 150 mM NaCl, lane 7 = 300 mM NaCl, lane 8 = 500 mM NaCl, lane 9 = 1 M NaCl, and lane 10 = hemopexin control. [Figure 5-2] Same as above. [Figure 6] Figure 1. Optimization of loading and elution conditions for Capto MMC chromatography. Photographic representation of non-reducing SDS-PAGE analysis of fractions from Capto MMC chromatography loaded at pH 6.5, 200 mM NaCl. Lane 1 = protein marker (MW standard), lane 2 = hemopexin standard, lane 3 = extract, lane 4 = drop-through, lane 5 = wash, pH 6.5, 200 mM NaCl, lane 6 = wash, pH 7.0, lane 7 = eluate, pH 7.5, 150 mM NaCl, lane 8 = eluate, pH 7.5, 500 mM NaCl, lane 9 = eluate, pH 7.5, 1 M NaCl. [Figure 7] FIG. 1 shows the concentration of hemopexin (g / L; y-axis) in the unbound fractions from Capto MMC chromatography columns with various loading amounts (g / L resin; x-axis). [Figure 8-1]Figure 1 shows the purity of hemopexin products obtained through a three-step chromatography process (Capto MMC, Capto Adhere, and Eshmuno CPS) after various loading conditions in the Capto MMC step. (A) Photographic representation of non-reducing SDS-PAGE analysis of Eshmuno CPS eluates containing hemopexin products obtained from various Capto MMC loading conditions. (B) Graphical representation of hemopexin purity (%; y-axis) measured by RP-HPLC at various process stages with Capto MMC loaded under various conditions. Circles represent Capto MMC eluates, triangles represent Capto Adhere eluates, and squares represent Eshmuno CPS eluates. [Figure 8-2] Same as above. [Figure 9] Representative chromatogram of Capto MMC mixed-mode cation exchange chromatography showing eluted protein concentration as UV absorbance (AU; y-axis) and time (min; x-axis). Hemopexin-containing peaks are labeled. [Figure 10] Figure 1 shows the development and optimization of conditions for further purification of hemopexin by Capto Adhere chromatography. Recovery (%; y-axis) of various proteins from the unbound fraction of a Capto Adhere mixed-mode anion-exchange chromatography column loaded with Capto MMC eluate under various pH and NaCl conditions (x-axis). Bars indicated with t = transferrin, bar indicated with a = albumin, bar indicated with h = haptoglobin, and bar indicated with H = hemopexin. [Figure 11-1] Figure 1. Development and optimization of conditions for further purification of hemopexin by Capto Adhere chromatography. Recovery (%; y-axis) of (A) hemopexin and (B) transferrin from Capto MMC eluates applied to a Capto Adhere mixed-mode anion-exchange chromatography column loaded under various pH and NaCl conditions (x-axis). [Figure 11-2] Same as above. [Figure 12] Figure 1 shows the purity of hemopexin obtained from Capto Adhere mixed-mode anion exchange chromatography of Capto MMC eluate. Photographic representation of non-reducing SDS-PAGE analysis of the unbound fraction from Capto MMC-purified hemopexin loaded at pH 7.5 and various NaCl concentrations. [Figure 13] Figure 1 shows optimization of the hemopexin load limit for a Capto Adhere mixed-mode anion-exchange chromatography column. Photograph of a non-reducing SDS-PAGE analysis of the unbound fraction from Capto Adhere chromatography. Lane labels indicate the amount of protein loaded per mL of resin. [Figure 14-1] Figure 1 shows the robustness of Capto Adhere loading conditions. (A) Photographic representation of non-reducing SDS-PAGE analysis of unbound fractions from Capto Adhere loaded under various pH and NaCl conditions. Lane 1 = MMC eluate feed; Lane 2 = pH 7.0, 100 mM NaCl; Lane 3 = pH 7.0, 200 mM NaCl; Lane 4 = pH 7.2, 150 mM NaCl; Lane 5 = pH 7.5, 150 mM NaCl; Lane 6 = pH 7.8, 150 mM NaCl; Lane 7 = pH 8.0, 100 mM NaCl; Lane 8 = pH 8.0, 200 mM NaCl; and Lane 9 = pH 7.5, 150 mM NaCl. (B) Graphical representation of transferrin recovery (%, y-axis) from eluates from Capto Adhere loaded under various pH and NaCl conditions (x-axis), as measured by nephelometry or RP-HPLC. [Figure 14-2] Same as above. [Figure 15] Figure 1 shows a representative chromatogram from a Capto Adhere mixed-mode anion-exchange chromatography column showing protein recovery as UV absorbance (AU; y-axis) and time (min; x-axis). Hemopexin is found in the unbound fraction. [Figure 16]Figure 1 shows viral inactivation kinetics during solvent-detergent incubation of Capto MMC eluates. Graphical representation of titer (log10 TCID50 / mL; y-axis) and time (min; x-axis) is shown. [Figure 17]

[0033] Figure 1. Purification of hemopexin by Eshmuno CPS chromatography. Photographic representation of non-reducing SDS-PAGE analysis of fractions of SD-treated Capto Adhere eluate under various loading conditions. Lane 1 = Mark 12 MW standard; Lane 2 = thawed Capto MMC eluate; Lane 3 = unbound Capto Adhere; Lane 4 = Eshmuno CPS feed; Lane 5 = unbound Eshmuno CPS; Lane 6 = Eshmuno CPS 100 mM NaCl eluate; Lane 7 = Eshmuno CPS 200 mM NaCl eluate; Lane 8 = pH 8.0, 200 mM NaCl eluate; and Lane 9 = pH 7.5, 150 mM NaCl eluate. [Figure 18] Figure 1 shows the binding capacity of Eshmuno CPS resin. Graphical representation of the amount of hemopexin (mg / mL; y-axis) in the unbound fraction from a 5 mL Eshmuno CPS chromatography column with various load amounts (mg / mL resin; x-axis). [Figure 19-1] Figure 1 shows the robustness of loading conditions for Eshmuno CPS chromatography. (A) Graphical representation of the recovery (%; y-axis) of hemopexin and transferrin under various pH and conductivity conditions (x-axis) as calculated by immunoturbidimetry. (B) Photographic representation of non-reducing SDS-PAGE analysis of eluates from an Eshmuno CPS chromatography column under various pH and conductivity conditions. Lane 1 = molecular weight (MW) marker; lane 2 = pH 5.8, 8 mS / cm; lane 3 = pH 5.8, 10 mS / cm; lane 4 = pH 5.8, 12 mS / cm; lane 5 = pH 6.0, 8 mS / cm; lane 6 = pH 6.0, 10 mS / cm; lane 7 = pH 6.0, 12 mS / cm; lane 8 = pH 6.2, 8 mS / cm; and lane 9 = pH 6.2, 10 mS / cm. [Figure 19-2] Same as above. [Figure 20] Figure 1 shows the effect of pH and conductivity on virus filtration. Graphical representation of filter throughput (L / m2; y-axis) and time (min; x-axis) at conductivities of 18 mS / cm (1), 18 mS / cm (2), 37 mS / cm, and 54 mS / cm. [Figure 21-1] Figure 1 shows the effect of prefilter to virus filter area ratio on virus filtration. (A) Graphical representation of filter throughput (L / m2; y-axis) and time (minutes; x-axis) using a Sartopore 2 XLM prefilter at prefilter to virus filter surface area ratios of 0.75:1 and 0.22:1. (B) Graphical representation of filter throughput (L / m2; y-axis) and time (minutes; x-axis) using a Virosart Max prefilter at prefilter to virus filter surface area ratios of 0.25:1 and 1.6:1. [Figure 21-2] Same as above. [Figure 22-1] Figure 1 shows the effectiveness of the Asahi BioEX filtration process for the removal of minute virus of mice (MVM) from purified hemopexin. (A) Graphical representation of filter throughput (L / m2; y-axis) and time (min; x-axis). (B) Viral titer of purified hemopexin spiked with MVM when filtered using an Asahi BioEx filter. [Figure 22-2] Same as above. [Figure 23] 1 shows the resolution of heme-hemopexin complex and hemopexin on a Capto MMC chromatography column. Graphical representation of absorbance (mAu; y-axis) and volume (mL; x-axis) of a 1:1 mixture of hemopexin and heme-hemopexin complex on Capto MMC chromatography. [Figure 24] 1 is a flow diagram of a hemopexin purification process in one embodiment disclosed herein. [Figure 25]FIG. 1 shows a series of photographic representations of non-reducing SDS-PAGE analysis of fractions from hemopexin purification on a small laboratory scale from several batches of Fraction V paste (A), (B) and (C). [Figure 26-1]

[0023] Figure 1 shows hemopexin recovery and purity after a streamlined lab-scale batch of a hemopexin purification process in one embodiment disclosed herein. (A) Graphical representation of step recovery (%; y-axis) across each chromatography step of a streamlined lab-scale batch (x-axis). (B) Photographic representation of a non-reducing SDS-PAGE analysis of process intermediates. [Figure 26-2] Same as above. [Figure 27] Figure 1 shows characterization of hemopexin drug substance. Photographic representation of reducing SDS-PAGE analysis of hemopexin drug substance (DS) produced in two pilot-scale batches (1) and (2). The gel is a merged version of the two original gels. [Figure 28] 1 is a flow diagram of the steps of the pre-viral inactivation process of the hemopexin purification process according to one embodiment disclosed herein, with process intermediates shown in bold. [Figure 29] 1 is a flow diagram of the steps of the post-viral inactivation process of the hemopexin purification process according to one embodiment disclosed herein, with process intermediates shown in bold. [Figure 30] 1 is a flow diagram of pre-viral inactivation process steps and post-viral inactivation process steps of a hemopexin purification process according to one embodiment disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0017] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.

[0018] Reference in this specification to any prior publication (or information derived therefrom) or to any known matter is not, and should not be construed as, either an acknowledgement or admission, or any form of suggestion, that the prior publication (or information derived therefrom) or known matter forms part of the general common knowledge in the art to which this specification pertains.

[0019] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural embodiments unless the context clearly dictates otherwise. Thus, for example, a reference to "a resin" includes a single resin, as well as two or more resins, and a reference to "the composition" includes a single composition, as well as two or more compositions; and similarly.

[0020] Unless indicated to the contrary, throughout this specification, references made to "%" content should be construed to mean % w / w (weight / weight). For example, a solution containing a hemopexin content of at least 80% of total protein is construed to mean a composition containing a hemopexin content of at least 80% w / w of total protein.

[0021] As used herein, "about" as applied to one or more values ​​refers to a value that is approximately the same as the stated reference value. In certain embodiments, the term "about" refers to a range of values ​​that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) from the stated reference value (except where such number exceeds 100% of possible values), unless otherwise stated or apparent from the context. In certain embodiments, the term "about" refers to ±10% of the recited value.

[0022] The present invention is based, at least in part, on the discovery that hemopexin can be purified from human plasma on a commercial scale. Accordingly, one aspect of the invention comprises a method for purifying hemopexin from a solution containing hemopexin and other proteins, the method comprising: (i) providing a solution containing hemopexin and other proteins, wherein the solution contains less than about 300 mM NaCl; (ii) passing the solution of step (i) through a mixed-mode cation exchange chromatography resin under conditions that promote the selective binding of hemopexin to the resin over the binding of other proteins to the resin; (iii) washing the resin after step (ii) to remove unbound proteins; (iv) eluting the resin-bound hemopexin after step (iii); (v) recovering the hemopexin eluted in step (iv); A method is provided that includes:

[0023] Hemopexin (Hx) is described as a 60 kD plasma β-1B glycoprotein containing a single 439 amino acid long peptide chain forming two domains connected by an interdomain linker. It has the highest known affinity (K) for heme of any characterized heme-binding protein. d <1 pM) and binds heme in equimolar ratios between the two domains of Hx in a pocket formed by the interdomain linker.

[0024] Hemopexin is the first line of defense against heme toxicity, at least in part due to its ability to bind heme with high affinity and act as a heme-specific carrier from the bloodstream to the liver. Hemopexin has also been reported to have serine protease activity and several other functions, such as anti- and pro-inflammatory activity, as well as the ability to inhibit cell adhesion and the binding of certain divalent metal ions.

[0025] Extraction and Clarification Any suitable material, including hemopexin, can be used to prepare a solution for use in the methods described herein. Suitable materials will be known to those skilled in the art, and illustrative examples include plasma fractions, such as various supernatants and precipitates derived from plasma fractionation processes. In these processes, plasma is typically subjected to various physical purification methods (e.g., precipitation, filtration, and adsorption) sequentially to produce intermediate products enriched in specific proteins. Separation of individual plasma proteins through fractionation processes is achieved by taking advantage of the fact that different plasma proteins have different solubilities, for example, depending on pH, temperature, and ionic strength, as well as different adsorption properties on (for example) different types of solid supports. Suitable industrial-scale plasma fractionation processes will be known to those skilled in the art, and illustrative examples include precipitation with cold ethanol according to protocols such as the Cohn / Oncley fractionation process or the Kistler / Nitschmann fractionation process. Exemplary fractionation processes are described by Schultze and Heremans (Molecular Biology of human proteins., Vol. I: Nature and Metabolism of Extracellular Proteins (Elsevier Publishing Company 1966), pp. 236-317). Illustrative examples of ethanol fractionation processes, including Cohn fractionation and Kistler-Nitschmann fractionation, are described, for example, by Cohn et al. (J Am Chem Soc., 1946; 68: 459-75), Kistler and Nitschmanns (1962, Vox Sang 7: 414-424), Friedli and Morgenthaler (Lancet, 1985; 1(8439): 1215), and Gregori et al. (Biologicals, 2004; 32: 1-10), the contents of all of which are incorporated herein by reference. Illustrative examples of suitable fractions include fractions derived from Cohn or Kistler-Nitschmann fractions, or analogs obtained from cold ethanol fractionation of blood-derived plasma.Fractions obtained from ethanol-free plasma fractionation processes are also contemplated, illustrative examples of which include affinity purification (e.g., affinity chromatography or immunoaffinity) and those described in Burnouf T. (Transfus. Med. Rev. 2007;21(2):101-117, the entire contents of which are incorporated herein by reference). In some embodiments, the sample containing hemopexin is selected from the group consisting of plasma, cryopreserved plasma, IgG-depleted plasma, or a Cohn fraction or Kistler-Nitchmann fraction or similar obtained from cold ethanol fractionation of blood-derived plasma. In embodiments, the hemopexin-containing sample is selected from the group consisting of cryosupernatant, 8% ethanol supernatant I, suspension A, supernatant II+III, supernatant (I)+II+III, supernatant II, fraction III, fraction IV (e.g., fraction IV1 or fraction IV4 supernatant or precipitate), fraction V, supernatant V, supernatant A, precipitate C, and other similar variant fractions and precipitates. Plasma fractions derived from immunoglobulin purification processes are also contemplated herein. Those skilled in the art will understand that solutions containing hemopexin may contain other proteins, such as haptoglobin, transferrin, and heme-hemopexin complexes. In some embodiments, if proteins such as haptoglobin, transferrin, and / or heme-hemopexin complexes are present in the hemopexin-containing solution, it may be desirable to remove them, such as by chromatographic separation, before performing the methods described herein.

[0026] In one embodiment, the solution comprising hemopexin is a human plasma fraction.

[0027] As described elsewhere herein, the methods disclosed herein can be used for commercial / industrial-scale purification of hemopexin. When using a plasma fraction as the starting material, using the methods described herein on a commercial / industrial scale can suitably include using a plasma fraction derived from at least about 500 kg of plasma. Thus, in one embodiment, the plasma fraction is derived from at least about 500 kg of plasma, preferably at least about 5,000 kg, preferably at least about 7,500 kg, preferably at least about 10,000 kg, or preferably at least about 15,000 kg of plasma. In another embodiment, using the methods described herein on a commercial / industrial scale can suitably include using a batch of Fraction IV-4 paste from 21,000 kg of plasma, and optionally pooling multiple batches (two or more, three or more, four or more, etc.) into a single batch of starting material.

[0028] Those skilled in the art will understand that plasma, for purposes of fractionation, is the liquid component of blood remaining after separation of cellular material from collected blood by suitable means known to those skilled in the art, illustrative examples of which include continuous filtration or apheresis.

[0029] In one embodiment, the solution containing hemopexin is derived from a Cohn fraction or an equivalent fraction from another plasma fractionation process. In one embodiment, the solution containing hemopexin is Cohn fraction IV supernatant, Cohn fraction IV precipitate, or an equivalent from another plasma fractionation process. In one embodiment, the solution containing hemopexin is derived from fraction IV4 precipitate.

[0030] If the hemopexin-containing solution is derived from a precipitate (e.g., a fraction IV4 precipitate), the precipitate is suitably stored before purifying the hemopexin according to the methods disclosed herein. Suitable storage conditions will be known to those skilled in the art, and illustrative examples include freezing the hemopexin-containing precipitate at -20°C, -80°C, or using liquid nitrogen before resolubilization. Thus, in some embodiments, the hemopexin-containing precipitate is frozen Cohn fraction IV. In a particularly preferred embodiment, the hemopexin-containing solution is derived from a frozen fraction IV4 precipitate.

[0031] Those skilled in the art will recognize that a cryoprecipitate containing hemopexin should be thawed prior to carrying out the methods disclosed herein. Thawing of such a cryoprecipitate can be carried out at any temperature, preferably between about 2 and about 30°C (e.g., 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, etc.), more preferably at ambient temperature.

[0032] It will be appreciated that when the starting material is provided as a precipitate (e.g., a fraction IV4 precipitate), it is necessary to solubilize or resuspend the precipitate to provide a suitable starting solution containing hemopexin for the methods described herein. In one embodiment, the hemopexin-containing solution is prepared by: (a) resuspending the hemopexin-containing starting material in an extraction buffer to obtain a solution of resuspended or solubilized hemopexin; (b) passing the resuspended hemopexin solution of step (a) through a filter; and (c) recovering the hemopexin-containing solution from step (b). In one embodiment, the hemopexin-containing starting material is Cohn Fraction IV. In one embodiment, the Cohn Fraction IV is Cohn Fraction IV4. In one embodiment, the Cohn Fraction IV is a Cohn Fraction IV4 precipitate.

[0033] The extraction buffer used to resuspend the starting material containing hemopexin may contain any suitable agent or combination of agents that has the ability to solubilize or resuspend the hemopexin present in the starting material while also providing a matrix that is compatible with, for example, clarification and further downstream purification of the hemopexin.

[0034] In one embodiment, the extraction buffer contains about 20 mM to about 500 mM NaCl (e.g., about 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, , 250mM, 260mM, 270mM, 280mM, 290mM, 300mM, 310mM, 320mM, 330mM, 340mM, 350mM, 360mM, 370mM, 380mM, 390mM, 400mM, 410mM, 420mM, 430mM, 440mM, 450mM, 460mM, 470mM, 480mM, 490mM or 500mM NaCl).

[0035] Thus, in one embodiment, the extraction buffer contains about 20 mM to about 500 mM NaCl, preferably about 20 mM, preferably about 30 mM, preferably about 40 mM, preferably about 50 mM, preferably about 60 mM, preferably about 70 mM, preferably about 80 mM, preferably about 90 mM, preferably about 100 mM, preferably about 110 mM, preferably about 120 mM, preferably about 130 mM, preferably about 140 mM, preferably about 150 mM, preferably about 160 mM, preferably about 170 mM, preferably about 180 mM, preferably about 190 mM, preferably about 200 mM, preferably about 210 mM, preferably about 220 mM, preferably about 230 mM, preferably about 240 mM, preferably about 250 mM, 50 mM, preferably about 260 mM, preferably about 270 mM, preferably about 280 mM, preferably about 290 mM, preferably about 300 mM, preferably about 310 mM, preferably about 320 mM, preferably about 330 mM, preferably about 340 mM, preferably about 350 mM, preferably about 360 mM, preferably about 370 mM, preferably about 380 mM, preferably about 390 mM, preferably about 400 mM, preferably about 410 mM, preferably about 420 mM, preferably about 430 mM, preferably about 440 mM, preferably about 450 mM, preferably about 460 mM, preferably about 470 mM, preferably about 480 mM, preferably about 490 mM or preferably about 500 mM NaCl.

[0036] In one embodiment, the extraction buffer contains about 400 mM NaCl.

[0037] In one embodiment, the extraction buffer comprises about 20 mM to about 60 mM of a buffering agent. Suitable buffering agents will be familiar to those of skill in the art, and illustrative examples include sodium phosphate. In one embodiment, the extraction buffer comprises about 20 mM to about 60 mM (e.g., about 20 mM, 30 mM, 40 mM, 50 mM, 60 mM) sodium phosphate. In one embodiment, the extraction buffer comprises about 30 mM to about 50 mM sodium phosphate. In one embodiment, the extraction buffer comprises about 20 mM sodium phosphate. In one embodiment, the extraction buffer comprises about 30 mM sodium phosphate. In one embodiment, the extraction buffer comprises about 40 mM sodium phosphate. In one embodiment, the extraction buffer comprises about 50 mM sodium phosphate. In one embodiment, the extraction buffer comprises about 60 mM sodium phosphate.

[0038] In one embodiment, the extraction buffer comprises about 40 mM sodium phosphate (Na2HPO4 / NaH2PO4) and about 400 mM NaCl.

[0039] In one embodiment, the extraction buffer has a pH of about 6 to about 8 (e.g., about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0).

[0040] Thus, in one embodiment, the extraction buffer has a pH of about 6 to about 8, preferably about 6, preferably about 6.1, preferably about 6.2, preferably about 6.3, preferably about 6.4, preferably about 6.5, preferably about 6.6, preferably about 6.7, preferably about 6.8, preferably about 6.9, preferably about 7.0, preferably about 7.1, preferably about 7.2, preferably about 7.3, preferably about 7.4, preferably about 7.5, preferably about 7.6, preferably about 7.7, preferably about 7.8, preferably about 7.9, or preferably about 8.0.

[0041] In one embodiment, the extraction buffer has a pH of about 6.2 to about 7.5.

[0042] In another embodiment, the extraction buffer has a pH of about 7.5.

[0043] The conductivity of the extraction buffer may suitably be in the range of about 30 mS / cm to about 45 mS / cm (e.g., about 30 mS / cm, about 31 mS / cm, about 32 mS / cm, about 33 mS / cm, about 34 mS / cm, about 35 mS / cm, about 36 mS / cm, about 37 mS / cm, about 38 mS / cm, about 39 mS / cm, about 40 mS / cm, about 41 mS / cm, about 42 mS / cm, about 43 mS / cm, about 44 mS / cm, or about 45 mS / cm). Thus, in one embodiment, the conductivity of the extraction buffer is about 30 mS / cm to about 45 mS / cm. In one embodiment, the conductivity of the extraction buffer is about 31 mS / cm to about 44 mS / cm. In one embodiment, the conductivity of the extraction buffer is about 32 mS / cm to about 43 mS / cm. In one embodiment, the conductivity of the extraction buffer is about 33 mS / cm to about 42 mS / cm. In one embodiment, the conductivity of the extraction buffer is about 35 mS / cm to about 41 mS / cm. In one embodiment, the conductivity of the extraction buffer is about 35 mS / cm to about 40 mS / cm. In one embodiment, the conductivity of the extraction buffer is about 35 mS / cm to about 39 mS / cm. In one embodiment, the extraction buffer has a conductivity of about 42 mS / cm. Suitable methods for determining (measuring) the conductivity of a solution, including those described herein, will be familiar to those of skill in the art, and an illustrative example thereof includes using a Thermo Fisher Orion Star A212 conductivity meter. The conductivity of the extraction buffer is determined at any suitable temperature, preferably ambient temperature, for example, about 18°C ​​to about 25°C, preferably about 18°C, or preferably about 19°C, or preferably about 20°C, or preferably about 21°C, or preferably about 22°C, or preferably about 23°C, or preferably about 24°C, or preferably about 25°C. In one embodiment, the conductivity is measured at ambient temperature. In one embodiment, the conductivity is measured at a temperature of about 18°C ​​to about 25°C.

[0044] Resuspension of hemopexin in the extraction buffer is preferably achieved by mixing the hemopexin-containing material with the extraction buffer for a period of time and under conditions suitable to achieve resuspension (i.e., dissolution) of the hemopexin. In one embodiment, the hemopexin-containing material and the extraction buffer are mixed for a period of about 10 minutes to about 240 minutes (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 240 minutes). In another embodiment, the hemopexin-containing material and extraction buffer are mixed for a period of 120 minutes or more, for example, from about 120 minutes to about 240 minutes (e.g., 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 240 minutes).

[0045] In one embodiment, the hemopexin-containing material and extraction buffer are mixed for at least about 20 minutes. In a preferred embodiment, the hemopexin-containing material and extraction buffer are mixed (i.e., stirred) for at least about 60 minutes.

[0046] Mixing the hemopexin-containing material with the extraction buffer can be accomplished using any method known to those skilled in the art, illustrative examples of which include stirring, vortexing, shaking, rotating, rocking, and any other suitable form of agitation. In one embodiment, the hemopexin-containing material and extraction buffer are mixed by vortexing. According to this embodiment, the hemopexin-containing material and extraction buffer are vortexed to about 5% to about 20% of the liquid depth (e.g., about 5%, 10%, 15%, or 20% of the liquid depth). In a preferred embodiment, the hemopexin-containing material and extraction buffer are vortexed to about 10% of the liquid depth.

[0047] In one embodiment disclosed herein, the solution containing hemopexin is about 1:2 to about 1:20, preferably about 1:2, preferably about 1:2.5, preferably about 1:3, preferably about 1:3.5, preferably about 1:4, preferably about 1:4.5, preferably about 1:5, preferably about 1:5.5, preferably about 1:6, preferably about 1:6.5, preferably about 1:7, preferably about 1:7.5, preferably about 1:8, preferably about 1:8.5, preferably about 1:9, preferably about 1:9.5, preferably about 1:10, preferably about 1:10.5, preferably about 1:11, preferably about 1:11.5, preferably about 1:12 , preferably about 1:12.5, preferably about 1:13, preferably about 1:13.5, preferably about 1:14, preferably about 1:14.5, preferably about 1:15, preferably about 1:15.5, preferably about 1:16, preferably about 1:16.5, preferably about 1:17, preferably about 1:17.5, preferably about 1:18, preferably about 1:18.5, preferably about 1:19, preferably about 1:19.5, preferably about 1:20 or preferably about 1:20.5.

[0048] In one embodiment, the hemopexin-containing solution is prepared by a process comprising resuspending a hemopexin-containing material in an extraction buffer at a ratio of hemopexin-containing material to extraction buffer of about 1:2.5. In one embodiment, the hemopexin-containing starting material is Cohn Fraction IV. In one embodiment, the Cohn Fraction IV is Cohn Fraction IV4. In one embodiment, the Cohn Fraction IV is Cohn Fraction IV4 precipitate.

[0049] In one embodiment, the resuspended hemopexin solution is passed through a depth filter.

[0050] In one embodiment, the depth filter is a cellulose depth filter (e.g., 3M™ 90SP Zeta Plus™, Pall™ EK1P™, ErtelAlsop™ 953P™, Cytiva™ Stax™ depth filter, 3M™ 70CA Zeta Plus™ lenticular depth filter).

[0051] Optimization of filtration throughput is achieved, for example, by adjusting any one or more of the filter area, frame depth, and flow-through pressure. Those skilled in the art will understand that any adjustment to the filtration parameters can alter the clarity, throughput, and viscosity of the clarified solution containing hemopexin.

[0052] In one embodiment, the filtration throughput is about 50 to about 200 L / m 2 In one embodiment, the filtration throughput is about 100 L / m 2 That's why.

[0053] In one embodiment, the filter area is about 0.5 to about 2 m per 9 kg of Cohn Fraction IV-4 sediment. 2 In one embodiment, the filter area is about 1 m per 9 kg of Cohn Fraction IV-4 sediment. 2 is.

[0054] In another embodiment, the filter area is about 1 m per 9 kg of Cohn Fraction IV-4 sediment. 2 (i.e., approximately 0.0312 m per liter of Cohn Fraction IV extract) 2 ) or less. Those skilled in the art will appreciate that the filter area is approximately 1 m per 9 kg of Cohn Fraction IV-4 sediment. 2 It will be appreciated that if the flow rate is reduced below this, corresponding adjustments to other filtration parameters, for example increasing the frame depth / volume due to the amount of solids present, will also be necessary to maintain throughput.

[0055] In one embodiment, the frame depth is about 1 cm to about 10 cm, preferably about 2 cm to about 6 cm, and more preferably about 3 cm to about 5 cm. In one embodiment, the frame depth is 4 cm.

[0056] Further improvement in filtration throughput is achieved by pre-coating (i.e., pre-flushing) the depth filter with a filter aid (e.g., Celpure C1000) under conditions and in an amount sufficient to uniformly coat the filter. The depth filter is optionally washed prior to pre-coating using a suitable buffer. In one embodiment, the depth filter comprises a filter aid. In some embodiments, the depth filter does not comprise a filter aid. In one embodiment, the buffer is an extraction buffer (e.g., 40 mM sodium phosphate, 400 mM NaCl, pH 7.2) described elsewhere herein.

[0057] In one embodiment, the amount of filter aid is about 2 mm, e.g., about 0.625 kg / m 2 The flow rate is sufficient to provide a pre-coat of the filter. In one embodiment, the flush volume for pre-coating with filter aid is about 1 press volume. The flow rate of the filter aid is set to provide a uniform coating of the filter and prevent the filter aid from pooling at the bottom of the frame. In one embodiment, the flow rate is about 6.25 L / m 2 In one embodiment, the filter aid is applied to the filter under pressure (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, or 1 bar).

[0058] In one embodiment, step (b) comprises passing the resuspended hemopexin solution of step (a) through a filter at a flow rate and pressure sufficient to prevent clogging of the filter and maintain throughput and clarity. In one embodiment, the flow rate is 6.25 L / m 2 / min. In one embodiment, the filtration pressure is less than about 2 bar. In another embodiment, the filtration pressure is less than about 1.5 bar. In yet another embodiment, the filtration pressure is less than about 1 bar.

[0059] After filtration, the depth filter can be washed, i.e., post-washed, to optimize hemopexin recovery. Suitable post-wash solutions and conditions will be known to those skilled in the art. In one embodiment, the post-wash solution is an equilibration buffer (e.g., 40 mM sodium phosphate, 225 mM NaCl, pH 6.4) as described elsewhere herein. In one embodiment, the flush volume of post-wash solution sufficient to maximize hemopexin recovery from the filter is 2.5 times the press volume. In one embodiment, the flush volume is about 3.0 times the press volume.

[0060] In one embodiment, the pH of the solution containing hemopexin is adjusted to a value of about 6.2 to about 6.6 (eg, 6.2, 6.3, 6.4, 6.5, or 6.6).

[0061] Thus, in one embodiment, the pH of the solution containing hemopexin is adjusted to a value of about 6.2 to about 6.6, preferably about 6.2, preferably about 6.3, preferably about 6.4, preferably about 6.5, or preferably about 6.6.

[0062] In one embodiment, the pH of the solution containing hemopexin is adjusted to about 6.4 (ie, 6.4±0.1).

[0063] The pH of the hemopexin-containing solution is adjusted with any suitable acidic solution known to those skilled in the art, an illustrative example of which is hydrochloric acid (HCl).

[0064] The conductivity of the hemopexin-containing solution is preferably adjusted to a value of about 24 mS / cm to about 30 mS / cm (e.g., 24 mS / cm, 25 mS / cm, 26 mS / cm, 27 mS / cm, 28 mS / cm, 29 mS / cm, or 30 mS / cm). The conductivity of the hemopexin-containing solution is also preferably adjusted to a value of about 20 mS / cm to about 30 mS / cm (e.g., about 20 mS / cm, about 21 mS / cm, about 22 mS / cm, about 23 mS / cm, about 24 mS / cm, about 25 mS / cm, about 26 mS / cm, about 27 mS / cm, about 28 mS / cm, about 29 mS / cm, or about 30 mS / cm).

[0065] Thus, in one embodiment, the conductivity of the hemopexin-containing solution is adjusted to a value of about 24 mS / cm to about 30 mS / cm, preferably about 26 mS / cm to about 28 mS / cm, preferably about 24 mS / cm, preferably about 25 mS / cm, preferably about 26 mS / cm, preferably about 27 mS / cm, preferably about 28 mS / cm, preferably about 29 mS / cm, or preferably about 30 mS / cm. In one embodiment, the conductivity of the hemopexin-containing solution is about 26 mS / cm to about 28 mS / cm, preferably about 26 mS / cm, preferably about 27 mS / cm, or preferably about 28 mS / cm. In one embodiment, the conductivity of the hemopexin-containing solution is about 27 mS / cm. In another embodiment, the conductivity of the hemopexin-containing solution is adjusted to a value of about 20 mS / cm to about 30 mS / cm, preferably about 20 mS / cm to about 29 mS / cm, preferably about 20 mS / cm to about 28 mS / cm, preferably about 20 mS / cm to about 27 mS / cm, preferably about 20 mS / cm to about 26 mS / cm, preferably about 20 mS / cm to about 25 mS / cm, preferably about 20 mS / cm, preferably about 26 mS / cm to about 28 mS / cm, preferably about 21 mS / cm, preferably about 22 mS / cm, preferably about 23 mS / cm.

[0066] In another embodiment, the conductivity of the hemopexin-containing solution is about 20 mS / cm to about 28 mS / cm. In another embodiment, the conductivity of the hemopexin-containing solution is about 20 mS / cm to about 26 mS / cm. In one embodiment, the conductivity of the hemopexin-containing solution is about 21 mS / cm to about 26 mS / cm. In one embodiment, the conductivity of the hemopexin-containing solution is about 22 mS / cm to about 26 mS / cm. In one embodiment, the conductivity of the hemopexin-containing solution is about 23 mS / cm to about 26 mS / cm. In one embodiment, the conductivity of the hemopexin-containing solution is about 22 mS / cm to about 25 mS / cm. In one embodiment, the conductivity of the hemopexin-containing solution is about 23 mS / cm. In one embodiment, the conductivity of the hemopexin-containing solution is about 24 mS / cm. In one embodiment, the conductivity of the hemopexin-containing solution is about 25 mS / cm. The conductivity of the solution is determined at any suitable temperature, preferably ambient temperature, for example, from about 18°C ​​to about 25°C, preferably about 18°C, or preferably about 19°C, or preferably about 20°C, or preferably about 21°C, or preferably about 22°C, or preferably about 23°C, or preferably about 24°C, or preferably about 25°C. In one embodiment, the conductivity is measured at ambient temperature. In one embodiment, the conductivity is measured at a temperature of from about 18°C ​​to about 25°C.

[0067] The hemopexin-containing solution can be stored for future use.

[0068] In one embodiment, a solution containing hemopexin is prepared by further filtering Cohn fraction IV extract. For example, fraction IV-4 (FIV-4) paste can be used as starting material and resuspended in a solution containing 40 mM sodium phosphate, 400 mM NaCl, pH 7.5+ / -0.1 at a ratio of 2.5 kg of buffer per 1 kg of paste. The resuspended FIV-4 paste can then be filtered, for example, at 1 ml 2The extraction paste solution is filtered through 3M 90SP Zeta+ filter media in a filter press using a filter area of ​​32 L and a frame thickness of 4 cm. The filter is optionally pre-coated with a filter aid such as Celpure C1000.

[0069] In one embodiment, a solution containing hemopexin is passed through a fine filter having a pore size of about 0.5 μm or less to obtain a clarified solution containing hemopexin.

[0070] In one embodiment, the area of ​​the fine filter is about 10 cm 2 / L ~ approx. 50cm 2 / L (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 cm 2 / L). In another embodiment, the filter area of ​​the fine filter is about 33 cm 2 / L depth filter.

[0071] In one embodiment, the filtration pressure is less than about 5 bar (e.g., 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 bar). In another embodiment, the filtration pressure is less than 1.5 bar.

[0072] The clarified solution containing hemopexin can be stored for future use, hi one embodiment, the clarified solution containing hemopexin can be stored below 23°C for up to about 24 hours.

[0073] In another embodiment, the clarified solution containing hemopexin can be stored at about 2°C to about 8°C for up to 48 hours.

[0074] Chromatographic purification of hemopexin Protein purification by chromatography can be carried out using axial flow columns such as those available from Cytiva, Sartorius, and Bio-Rad, or using radial flow columns such as those available from Proxcys, Inc. Chromatography can also be carried out using expanded bed techniques known to those skilled in the art.

[0075] Most chromatography processes use a solid support, also referred to interchangeably herein as a resin or matrix. Suitable solid supports will be well known to those skilled in the art, and their selection will depend on the type of product being purified. Examples of suitable solid supports include inorganic supports, such as glass and silica gel, organic supports, synthetic supports, or naturally occurring supports, such as agarose, cellulose, dextran, polyamides, polyacrylamides, vinyl copolymers of bifunctional acrylates, and various hydroxylated monomers. Commercially available supports are sold under the names Eshmuno™, Nuvia™, Sephadex™, Sepharose™, Hypercel™, Capto™, Fractogel™, MacroPrep™, Unosphere™, GigaCap™, Trisacryl™, Ultrogel™, Dynospheres™, Macrosorb™, and XAD™ resins.

[0076] As described elsewhere herein, the present inventors have unexpectedly discovered that mixed-mode cation exchange chromatography resins can be used for the selective retention of hemopexin.

[0077] Mixed-mode chromatography Mixed-mode chromatography (MMC) is a chromatographic technique used to separate proteins based on two or more types of interactions (e.g., hydrophobic, hydrophilic, ionic) between the stationary phase and the proteins being separated. For a given mixed-mode chromatography column, the predominant separation interactions depend on the solution properties and mobile phase conditions.

[0078] Those skilled in the art will understand that any mixed-mode cation exchange chromatography resin can be used to purify hemopexin from a solution, as long as it has the ability to bind to the chromatography resin while allowing some impurities in the solution to pass through the resin. Suitable resins will be known to those skilled in the art. Examples of suitable mixed-mode cation exchange chromatography resins include those represented by formula (I) or (II): [ka] The ligand comprises a ligand having the structure:

[0079] In one embodiment, the mixed-mode cation exchange chromatography resin comprises a ligand having the structure of Formula I (eg, Capto MMC™).

[0080] In one embodiment, the mixed-mode cation exchange chromatography resin comprises a ligand having the structure of Formula II (eg, Nuvia cPrime™).

[0081] Chromatographic steps are generally carried out under non-denaturing conditions at convenient temperatures in the range of about 5° C. to +30° C., more usually at about ambient temperature. Chromatographic steps can be carried out batchwise or continuously, as is convenient.

[0082] Optimization of the chromatographic efficiency of a mixed-mode cation exchange chromatography resin is achieved by adjusting variables such as pressure, temperature, column length, column bed height, height equivalent to a theoretical plate (HETP), and linear flow rate. One skilled in the art will understand that any adjustments to such variables may alter the selective binding of hemopexin and other proteins to the chromatography column.

[0083] The chromatography column bed height will vary according to the specified product load, as well as the pressure and operating range of the column. In one embodiment, the column bed height is about 15 cm (i.e., 15±2 cm). For large-scale processes, the column bed height can be from about 10 cm to about 25 cm.

[0084] Those skilled in the art will appreciate that the column linear flow rate should provide a convenient flow rate without generating significant back pressure. In one embodiment, the column linear flow rate is about 120 cm / hr.

[0085] Prior to loading the hemopexin-containing solution, an equilibration buffer can be applied to the mixed-mode chromatography column to ensure that the pH and conductivity are comparable to those of the hemopexin-containing solution (e.g., a clarified Cohn Fraction IV extract). Suitable equilibration buffers will be known to those of skill in the art, and illustrative examples include the wash buffers described elsewhere herein (e.g., 40 mM sodium phosphate, 225 mM NaCl, pH 6.4). In one embodiment, the volume of equilibration buffer required for pre-equilibration is one column volume (CV) or more. In one embodiment, the pH of the mixed-mode chromatography column after equilibration is about pH 6.3 to about 6.5 (e.g., pH 6.3, 6.4, or 6.5). The conductivity of the equilibration buffer may suitably be about 20 mS / cm to about 30 mS / cm (e.g., about 20 mS / cm, about 21 mS / cm, about 22 mS / cm, about 23 mS / cm, about 24 mS / cm, about 25 mS / cm, about 26 mS / cm, about 27 mS / cm, about 28 mS / cm, about 29 mS / cm, or about 30 mS / cm). Therefore, in one embodiment, the conductivity of the equilibration buffer is about 20 mS / cm to about 30 mS / cm, preferably about 21 mS / cm to about 29 mS / cm, preferably about 22 mS / cm to about 28 mS / cm, preferably about 23 mS / cm to about 28 mS / cm, preferably about 23 mS / cm to about 27 mS / cm, preferably about 23 mS / cm to about 26 mS / cm, or more preferably about 23 mS / cm to about 25 mS / cm. In one embodiment, the conductivity of the equilibration buffer is about 20 mS / cm to about 30 mS / cm. In one embodiment, the conductivity of the equilibration buffer is about 20 mS / cm to about 30 mS / cm. In one embodiment, the conductivity of the equilibration buffer is about 20 mS / cm to about 30 mS / cm. In one embodiment, the conductivity of the equilibration buffer is about 23 mS / cm to about 28 mS / cm. In one embodiment, the conductivity of the equilibration buffer is from about 23 mS / cm to about 25 mS / cm.The conductivity of the equilibration buffer is determined at any suitable temperature, preferably ambient temperature, for example, about 18°C ​​to about 25°C, preferably about 18°C, or preferably about 19°C, or preferably about 20°C, or preferably about 21°C, or preferably about 22°C, or preferably about 23°C, or preferably about 24°C, or preferably about 25°C. In one embodiment, the conductivity is measured at ambient temperature. In one embodiment, the conductivity is measured at a temperature of about 18°C ​​to about 25°C.

[0086] The present inventors have unexpectedly found that solutions containing hemopexin and other proteins having a sodium chloride (NaCl) concentration of less than about 300 nM (e.g., 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM) are optimal for loading onto a mixed-mode cation exchange chromatography resin to promote selective binding of hemopexin to the resin.

[0087] Thus, in one embodiment, the solution containing hemopexin contains less than about 300 mM NaCl, preferably about 10 mM, preferably about 20 mM, preferably about 30 mM, preferably about 40 mM, preferably about 50 mM, preferably about 60 mM, preferably about 70 mM, preferably about 80 mM, preferably about 90 mM, preferably about 100 mM, preferably about 210 mM, preferably about 220 mM, preferably about 230 mM, preferably about 240 mM, preferably about 250 mM, preferably about 260 mM, preferably about 270 mM, preferably about 280 mM, or preferably about 290 mM NaCl.

[0088] In one embodiment, the hemopexin-containing solution contains about 160 mM to about 250 mM NaCl. In another embodiment, the hemopexin-containing solution contains about 200 mM to about 250 mM NaCl. In a preferred embodiment, the hemopexin-containing solution contains about 250 mM NaCl. In another embodiment, the hemopexin-containing solution contains about 220 mM to about 230 mM NaCl. In a preferred embodiment, the hemopexin-containing solution contains about 225 mM NaCl.

[0089] The inventors have also shown that solutions containing hemopexin and other proteins with a pH maintained below about 8 (e.g., 7, 6, 5, 4, and values ​​therebetween) are optimal for loading onto mixed-mode cation exchange chromatography resins to promote selective binding of hemopexin to the resin.

[0090] Thus, in one embodiment, the solution comprising hemopexin has a pH of less than about 7, preferably about 6.5, preferably about 6, preferably about 5.5, preferably about 5, preferably about 4.5, or preferably about 4.

[0091] In one embodiment, the solution containing hemopexin has a pH of about 6.2 to about 6.6, hi another embodiment, the solution in step (i) has a pH of about 6.4.

[0092] In one embodiment, the solution comprising hemopexin is (a) a pH of about 6.2 to about 6.6; (b) about 20 mM to about 60 mM phosphate buffer; (c) about 160 mM to about 250 mM NaCl Includes.

[0093] In another embodiment, the solution comprising hemopexin is (a) a pH of about 6.4; (b) about 40 mM phosphate buffer; (c) approximately 225 mM NaCl and Includes.

[0094] In one embodiment, the amount of hemopexin passed through the resin in step (ii) is about 1 mg to about 40 mg per mL of resin (e.g., about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, or about 40 mg per mL of resin).

[0095] Thus, in one embodiment, the amount of hemopexin passed through the resin in step (ii) is preferably about 1 mg, preferably about 2 mg, preferably about 3 mg, preferably about 4 mg, preferably about 5 mg, preferably about 6 mg, preferably about 7 mg, preferably about 8 mg, preferably about 9 mg, preferably about 10 mg, preferably about 11 mg, preferably about 12 mg, preferably about 13 mg, preferably about 14 mg, preferably about 15 mg, preferably about 16 mg, preferably about 17 mg, preferably about 18 mg, preferably about 19 mg, per mL of resin. is about 19 mg, preferably about 20 mg, preferably about 21 mg, preferably about 22 mg, preferably about 23 mg, preferably about 24 mg, preferably about 25 mg, preferably about 26 mg, preferably about 27 mg, preferably about 28 mg, preferably about 29 mg, preferably about 30 mg, preferably about 31 mg, preferably about 32 mg, preferably about 33 mg, preferably about 34 mg, preferably about 35 mg, preferably about 36 mg, preferably about 37 mg, preferably about 38 mg, preferably about 39 mg, or preferably about 40 mg. In one embodiment, the amount of hemopexin passed through the resin in step (ii) is about 10 mg / mL to about 20 mg / mL resin. In one embodiment, the amount of hemopexin loaded onto the resin in step (ii) is about 1 mg to about 40 mg per mL of resin (e.g., about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, or about 40 mg per mL of resin). In one embodiment, the amount of hemopexin loaded onto the resin in step (ii) is from about 10 mg / mL to about 20 mg / mL resin.

[0096] Once hemopexin has bound to the mixed-mode cation exchange chromatography resin, the resin can be washed to remove residual impurities under conditions that retain the hemopexin bound to the resin. Suitable washing solutions and conditions will be known to those skilled in the art. In one embodiment, the washing solution comprises 40 mM sodium phosphate, 225 mM NaCl, pH 6.4. In one embodiment, the volume of the washing solution applied to the mixed-mode cation exchange chromatography resin is approximately 3 CV. The flow-through wash fraction can also be collected and stored for future use, if desired. The bound hemopexin can be eluted from the mixed-mode cation exchange chromatography resin by means known to those skilled in the art.

[0097] Suitable buffers for eluting hemopexin from the resin will also be known to those skilled in the art, illustrative examples of which include phosphate, hi one embodiment, the elution buffer comprises 40 mM sodium phosphate at a pH of about 7.5.

[0098] In one embodiment, the elution buffer further comprises about 100 mM to about 200 mM NaCl. This equates to an elution buffer having a conductivity ranging from about 10 mS / cm (100 mM NaCl) to about 18 mS / cm (200 mM NaCl). In certain embodiments, the elution buffer comprises about 140 to 160 mM NaCl. In one embodiment, the elution buffer comprises about 150 mM NaCl. However, those skilled in the art will recognize that the NaCl concentration of the elution buffer may depend on the protein composition applied to the column and may need to be adjusted beyond the stated limits to achieve the desired recovery and purity of hemopexin eluted from the resin.

[0099] In one embodiment, the elution buffer contains about 20 mM to about 60 mM sodium phosphate (e.g., about 20 mM, 30 mM, 40 mM, 50 mM, 60 mM sodium phosphate). In one embodiment, the extraction buffer contains about 30 mM to about 50 mM sodium phosphate. In one embodiment, the elution buffer contains about 20 mM sodium phosphate. In one embodiment, the elution buffer contains about 30 mM sodium phosphate. In one embodiment, the elution buffer contains about 40 mM sodium phosphate. In one embodiment, the elution buffer contains about 50 mM sodium phosphate. In one embodiment, the elution buffer contains about 60 mM sodium phosphate.

[0100] The conductivity of the elution buffer may suitably be about 16 mS / cm to about 24 mS / cm (e.g., about 16 mS / cm, about 17 mS / cm, about 18 mS / cm, about 19 mS / cm, about 20 mS / cm, about 21 mS / cm, about 22 mS / cm, about 23 mS / cm, or about 24 mS / cm). Thus, in one embodiment, the conductivity of the elution buffer is about 16 mS / cm to about 24 mS / cm, preferably about 17 mS / cm to about 23 mS / cm, preferably about 17 mS / cm to about 22 mS / cm, preferably about 17 mS / cm to about 21 mS / cm, preferably about 17 mS / cm to about 20 mS / cm, or more preferably about 17 mS / cm to about 19 mS / cm. In one embodiment, the conductivity of the elution buffer is about 17 mS / cm to about 21 mS / cm. In one embodiment, the conductivity of the elution buffer is about 17 mS / cm to about 20 mS / cm. In one embodiment, the conductivity of the elution buffer is about 17 mS / cm to about 19 mS / cm. The conductivity of the elution buffer is determined at any suitable temperature, preferably ambient temperature, for example, about 18°C ​​to about 25°C, preferably about 18°C, or preferably about 19°C, or preferably about 20°C, or preferably about 21°C, or preferably about 22°C, or preferably about 23°C, or preferably about 24°C, or preferably about 25°C. In one embodiment, the conductivity is measured at ambient temperature. In one embodiment, the conductivity is measured at a temperature of about 18°C ​​to about 25°C.

[0101] In some embodiments, the volume of elution buffer applied to the mixed-mode cation exchange chromatography resin is advantageously sufficient to completely elute the bound hemopexin from the resin. However, in some cases, it may be sufficient to elute only a fraction of the bound hemopexin from the resin. In one embodiment, the volume of elution buffer applied to the mixed-mode cation exchange chromatography resin is about 3 CV.

[0102] In one embodiment, collection of the hemopexin eluate begins after application of about 0.5 column volumes (CV) of elution buffer to the mixed-mode cation exchange chromatography resin, and the A of the elution peak. 280nm This continues until the signal drops below 50 mAU (path length 2 mm).

[0103] In one embodiment, the recovered hemopexin eluate suitably has a purity (e.g., substantial purity) of at least about 50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%).

[0104] Thus, in one embodiment, the recovered hemopexin eluate preferably comprises at least 50%, preferably at least 51%, preferably at least 52%, preferably at least 53%, preferably at least 54%, preferably at least 55%, preferably at least 56%, preferably at least 57%, preferably at least 58%, preferably at least 59%, preferably at least 60%, preferably at least 61%, preferably at least 62%, preferably at least 63%, preferably at least 64%, preferably at least 65%, preferably at least 66%, preferably at least 67%, preferably at least 68%, preferably at least 69%, preferably at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least The soluble solids have a purity of at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, or preferably 100%.

[0105] In one embodiment, the recovered hemopexin eluate has a purity of about 70% to about 99%.

[0106] In one embodiment, the conductivity of the recovered hemopexin eluate is about 16 mS / cm to about 22 mS / cm (e.g., about 16 mS / cm, about 17 mS / cm, about 18 mS / cm, about 19 mS / cm, about 20 mS / cm, about 21 mS / cm, or about 22 mS / cm). In one embodiment, the conductivity of the recovered hemopexin eluate is about 17 mS / cm to about 21 mS / cm. In one embodiment, the conductivity of the recovered hemopexin eluate is about 17 mS / cm to about 20 mS / cm. In one embodiment, the conductivity of the recovered hemopexin eluate is about 17 mS / cm to about 19 mS / cm. In one embodiment, the conductivity of the recovered hemopexin eluate is about 17 mS / cm. In one embodiment, the conductivity of the recovered hemopexin eluate is about 18 mS / cm. In one embodiment, the conductivity of the recovered hemopexin eluate is about 19 mS / cm. In one embodiment, the conductivity of the recovered hemopexin eluate is about 20 mS / cm. In one embodiment, the conductivity of the recovered hemopexin eluate is about 18 to about 19 mS / cm. The conductivity of the recovered hemopexin eluate is determined at any suitable temperature, preferably ambient temperature, for example, about 18°C ​​to about 25°C, preferably about 18°C, or preferably about 19°C, or preferably about 20°C, or preferably about 21°C, or preferably about 22°C, or preferably about 23°C, or preferably about 24°C, or preferably about 25°C. In one embodiment, the conductivity of the recovered hemopexin eluate is measured at ambient temperature. In one embodiment, the conductivity of the recovered hemopexin eluate is measured at a temperature of about 18°C ​​to about 25°C.

[0107] In one embodiment, the collected hemopexin eluate can be stored for future use. In one embodiment, the collected hemopexin eluate can be stored at a temperature of less than about 23° C. for up to 48 hours. In another embodiment, the collected hemopexin eluate can be stored at a temperature of about 2° C. to about 8° C. for at least 7 days.

[0108] In one embodiment, the recovered hemopexin eluate is further purified, as needed, for example, by concentrating and diafiltering the eluted hemopexin through an ultrafiltration membrane, sterile filtering the concentrated and / or diafiltered hemopexin, and / or further chromatographic purification. In one embodiment, the recovered hemopexin eluate is further purified by ultrafiltration. In one embodiment, the recovered hemopexin eluate is further purified by tangential flow filtration. In another embodiment, the recovered hemopexin eluate is further purified by single-pass tangential flow filtration.

[0109] Mixed-mode anion-exchange chromatography In one embodiment, the method described herein comprises: (vi) passing the recovered hemopexin eluate of step (v) through a mixed-mode anion exchange chromatography resin under conditions that allow impurities in the recovered hemopexin eluate to bind to the resin while allowing hemopexin to pass through the resin as an unbound fraction; (vii) recovering the unbound fraction containing hemopexin; Further includes:

[0110] Those skilled in the art will understand that any mixed-mode anion exchange chromatography resin can be used to further purify hemopexin from the recovered hemopexin eluate, as long as it suitably allows hemopexin from the recovered hemopexin eluate to pass through the resin, while other proteins and impurities in the recovered hemopexin eluate have the ability to bind to the chromatography resin. By evaluating several different mixed-mode anion exchange chromatography resins, the inventors unexpectedly found that a mixed-mode anion exchange chromatography resin containing an N-benzylmethylethanolamine ligand (e.g., Capto Adhere™) is particularly suitable for further purification of hemopexin, including commercial- or industrial-scale production. Thus, in one embodiment, the mixed-mode anion exchange chromatography resin contains an N-benzylmethylethanolamine ligand.

[0111] Optimization of the chromatographic efficiency of the mixed-mode anion exchange chromatography resin is achieved by adjusting variables such as temperature, column length, column bed height, height equivalent to a theoretical plate (HETP), and linear flow rate. One skilled in the art will understand that any adjustments to such variables may alter the binding of impurities to the chromatography column or the elution of hemopexin in the unbound fraction.

[0112] In one embodiment, the column bed height is about 15 cm (ie, 15±2 cm).

[0113] In one embodiment, the column linear flow rate is about 120 cm / hr.

[0114] A solution suitable for equilibrating a mixed-mode anion exchange resin (also referred to herein as an equilibration buffer) may contain a buffering agent at a concentration of about 10 mM to about 200 mM, preferably about 10 to about 60 mM, or more preferably about 40 mM. The pH of the equilibration buffer may range from 5 to about 9, and the conductivity of the equilibration buffer may suitably be less than about 18 mS / cm. In one embodiment, the conductivity of the equilibration buffer is about 16 mS / cm to about 22 mS / cm (e.g., about 16 mS / cm, about 17 mS / cm, about 18 mS / cm, about 19 mS / cm, about 20 mS / cm, about 21 mS / cm, or about 22 mS / cm). In one embodiment, the conductivity of the equilibration buffer is about 17 mS / cm. In one embodiment, the conductivity of the equilibration buffer is about 18 mS / cm. In one embodiment, the conductivity of the equilibration buffer is about 19 mS / cm. In one embodiment, the conductivity of the equilibration buffer is about 20 mS / cm. The conductivity of the equilibration buffer is determined at any suitable temperature, preferably ambient temperature, for example, about 18°C ​​to about 25°C, preferably about 18°C, or preferably about 19°C, or preferably about 20°C, or preferably about 21°C, or preferably about 22°C, or preferably about 23°C, or preferably about 24°C, or preferably about 25°C. In one embodiment, the conductivity of the equilibration buffer is measured at ambient temperature. In one embodiment, the conductivity of the equilibration buffer is measured at a temperature of about 18°C ​​to about 25°C.

[0115] In one embodiment, the mixed-mode anion exchange chromatography resin is equilibrated with an equilibration buffer comprising a pH of about 7.0, preferably about 7.1, preferably about 7.2, preferably about 7.3, preferably about 7.4, preferably about 7.5, preferably about 7.6, preferably about 7.7, preferably about 7.8, preferably about 7.9 or preferably about 8.0.

[0116] In one embodiment, the equilibration buffer has a pH of about 7.5.

[0117] In one embodiment, the equilibration buffer comprises about 100 mM to about 200 mM NaCl (e.g., 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM NaCl).

[0118] Thus, in one embodiment, the equilibration buffer comprises about 100 mM, preferably about 110 mM, preferably about 120 mM, preferably about 130 mM, preferably about 140 mM, preferably about 150 mM, preferably about 160 mM, preferably about 170 mM, preferably about 180 mM, preferably about 190 mM or preferably about 200 mM.

[0119] In another embodiment, the equilibration buffer contains about 150 mM NaCl.

[0120] In one embodiment, the volume of equilibration buffer required for pre-equilibration is 1 column volume (CV) or more. In one embodiment, the volume of equilibration buffer required for pre-equilibration is 3 CV or more. In one embodiment, the volume of equilibration buffer required for pre-equilibration is about 3 CV. In one embodiment, the pH of the eluate from the mixed-mode anion exchange chromatography resin after pre-equilibration is about pH 7.4 to about 7.6 (e.g., pH 7.4, 7.5, or 7.6). In one embodiment, the conductivity of the eluate from the mixed-mode anion exchange chromatography resin after pre-equilibration is about 20 mS / cm.

[0121] The concentration of hemopexin in the recovered hemopexin eluate prior to step (vi) is suitably concentrated, for example, by passing the recovered hemopexin eluate through an ultrafiltration membrane. Thus, in one embodiment, the concentration of hemopexin in the recovered hemopexin eluate is concentrated prior to step (vi). In one embodiment, the concentration of hemopexin in the recovered hemopexin eluate is concentrated by passing the recovered hemopexin eluate through an ultrafiltration membrane. In one embodiment, the concentration of hemopexin in the concentrated hemopexin eluate is about 10 mg / mL to about 30 mg / mL (e.g., about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, or about 30 mg / mL), preferably about 10 mg / mL to about 30 mg / mL, about 15 mg / mL to about 25 mg / mL, or more preferably about 20 mg / mL.

[0122] In one embodiment, the amount of hemopexin passed through the resin in step (vi) is about 20 g to about 50 g per liter of resin (e.g., about 20 g, about 21 g, about 22 g, about 23 g, about 24 g, about 25 g, about 26 g, about 27 g, about 28 g, about 29 g, about 30 g, about 31 g, about 32 g, about 33 g, about 34 g, about 35 g, about 36 g, about 37 g, about 38 g, about 39 g, about 40 g, about 41 g, about 42 g, about 43 g, about 44 g, about 45 g, about 46 g, about 47 g, about 48 g, about 49 g, or about 50 g per liter of resin). In another embodiment, the amount of hemopexin passed through the resin in step (iv) is about 30 g per liter of resin.

[0123] The unbound fraction containing hemopexin recovered in step (vii) may suitably have a conductivity of about 16 mS / cm to about 22 mS / cm (e.g., about 16 mS / cm, about 17 mS / cm, about 18 mS / cm, about 19 mS / cm, about 20 mS / cm, about 21 mS / cm, or about 22 mS / cm). In one embodiment, the conductivity of the recovered unbound fraction is about 17 mS / cm to about 21 mS / cm. In one embodiment, the conductivity of the recovered unbound fraction is about 17 mS / cm to about 20 mS / cm. In one embodiment, the conductivity of the recovered unbound fraction is about 17 mS / cm. In one embodiment, the conductivity of the recovered unbound fraction is about 18 mS / cm. In one embodiment, the conductivity of the recovered unbound fraction is about 19 mS / cm. In one embodiment, the conductivity of the recovered unbound fraction is about 20 mS / cm. The conductivity of the recovered unbound fraction is determined at any suitable temperature, preferably ambient temperature, for example, about 18°C ​​to about 25°C, preferably about 18°C, or preferably about 19°C, or preferably about 20°C, or preferably about 21°C, or preferably about 22°C, or preferably about 23°C, or preferably about 24°C, or preferably about 25°C. In one embodiment, the conductivity of the recovered unbound fraction is measured at ambient temperature. In one embodiment, the conductivity of the recovered unbound fraction is measured at a temperature of about 18°C ​​to about 25°C.

[0124] After the collected hemopexin eluate from step (v) is passed through the resin in step (vi), the resin is washed to ensure that all hemopexin is collected in the unbound hemopexin fraction. Suitable wash solutions and conditions will be known to those skilled in the art. In one embodiment, the wash solution comprises 40 mM sodium phosphate, 150 mM NaCl at pH 7.5. In one embodiment, the volume of wash solution applied to the mixed-mode anion exchange chromatography resin is about 3 CV. In one embodiment, the conductivity of the wash solution is about 16 mS / cm to about 22 mS / cm (e.g., about 16 mS / cm, about 17 mS / cm, about 18 mS / cm, about 19 mS / cm, about 20 mS / cm, about 21 mS / cm, or about 22 mS / cm). In one embodiment, the conductivity of the wash solution is about 17 mS / cm. In one embodiment, the conductivity of the wash solution is about 18 mS / cm. In one embodiment, the conductivity of the wash solution is about 19 mS / cm. In one embodiment, the conductivity of the wash solution is about 20 mS / cm. The conductivity of the wash solution is determined at any suitable temperature, preferably ambient temperature, for example, from about 18°C ​​to about 25°C, preferably about 18°C, or preferably about 19°C, or preferably about 20°C, or preferably about 21°C, or preferably about 22°C, or preferably about 23°C, or preferably about 24°C, or preferably about 25°C. In one embodiment, the conductivity of the wash solution is measured at ambient temperature. In one embodiment, the conductivity of the wash solution is measured at a temperature of from about 18°C ​​to about 25°C.

[0125] In one embodiment, the collection of the unbound fraction containing hemopexin is carried out by 280nm started at 50 mAU or more (measured at the column outlet; path length 2 mm), and A 280nm The process is terminated when the AU drops below 50 mAU (measured at the column outlet; path length 2 mm).

[0126] In one embodiment, the unbound fraction containing hemopexin can be stored for future use. In one embodiment, the unbound fraction containing hemopexin can be stored at less than 23° C. for up to 48 hours. In another embodiment, the unbound fraction containing hemopexin can be stored at about 2° C. to about 8° C. for up to 7 days.

[0127] Virus inactivation When hemopexin is used for clinical or veterinary applications (e.g., for administration to a subject with a condition associated with hemolysis), it may be desirable to reduce the active virus content (i.e., viral titer) and the level of other potential infectious agents (e.g., prions). This may be desirable, for example, when the feedstock (i.e., starting material / solution) containing hemopexin and other proteins is derived from plasma. Methods for reducing viral titer in a solution will be known to those skilled in the art. Examples include pasteurization (e.g., incubating the solution at 60°C for 10 hours in the presence of high concentrations of stabilizers such as glycine (e.g., 2.75M) and sucrose (e.g., 50%), and / or other selected excipients or salts), dry heat treatment, viral filtration (e.g., passing the solution through a nanofilter, e.g., with a 20 nm cutoff), and / or subjecting the solution to treatment with a suitable organic solvent and detergent for a period and under conditions to inactivate the viruses in the solution. Solvent-detergent (SD) inactivation has been used for over 20 years to inactivate enveloped viruses, particularly in plasma-derived products. Therefore, it can be performed using a variety of reagents and methods known in the art (see, for example, U.S. Pat. Nos. 4,540,573 and 4,764,369, incorporated herein by reference). Suitable solvents include tri-n-butyl phosphate (TnBP) and ethers, preferably TnBP (typically at about 0.3%). Suitable surfactants include polysorbate (Tween) 80, polysorbate (Tween) 20, and Triton X-100 (typically at about 0.3%). The selection of processing conditions, including solvent and surfactant concentrations, depends in part on the characteristics of the feedstock. Lower purity feedstocks generally require higher concentrations of reagents and more extreme reaction conditions. A preferred surfactant is polysorbate 80, and a particularly preferred combination is polysorbate 80 and tri-n-butyl phosphate (TnBP).The feedstock is stirred with solvent and detergent reagents at a temperature and for a time sufficient to inactivate any enveloped viruses that may be present. For example, the solvent-detergent treatment is carried out at 23±2°C for approximately 2-24 hours. The solvent-detergent chemicals are then removed, for example, by adsorption onto a chromatographic medium such as a C-18 hydrophobic resin, or by eluting them in the drop-through fraction of an ion-exchange resin under conditions that adsorb the protein of interest.

[0128] The viral inactivation step can be performed at any suitable stage of the methods disclosed herein. In one embodiment, the unbound fraction containing hemopexin is subjected to the viral inactivation step after step (vii). Prior to the viral inactivation step, the unbound fraction containing hemopexin recovered in step (vii) can be suitably concentrated to, for example, minimize the volume of product handled during subsequent processing steps, including viral inactivation. Thus, in one embodiment, the methods described herein further comprise concentrating the unbound fraction containing hemopexin recovered in step (vii). In one embodiment, the unbound fraction containing hemopexin recovered in step (vii) is concentrated prior to the viral inactivation step. Suitable methods for concentrating the hemopexin-containing unbound fraction recovered in step (vii) will be well known to those skilled in the art, and illustrative examples thereof include, for example, UF / DF using an UF / DF membrane such as a Millipore Pellicon 3 cassette fitted with Biomax (PES), a Pellicon 2 cassette (Millipore), or a polyethersulfone or Hydrosart cassette (Sartorius). In one embodiment, the hemopexin-containing unbound fraction recovered in step (vii) is concentrated by ultrafiltration.

[0129] Passing the partially purified hemopexin (i.e., the unbound fraction containing hemopexin) through a mixed-mode anion exchange chromatography resin followed by a viral inactivation step such as a solvent-detergent treatment advantageously avoids solvent and detergent interference with the purification and limits the number of purification steps that need to be performed, which is particularly advantageous for industrial-scale production.

[0130] In one embodiment disclosed herein, the virus inactivation step comprises exposing the unbound fraction comprising hemopexin of step (vii) to a solution comprising a detergent and a solvent.

[0131] In one embodiment, the temperature of the unbound fraction containing hemopexin before the addition of the solution containing the surfactant and solvent is about 21°C to about 25°C (e.g., 21°C, 22°C, 23°C, 24°C, or 25°C).

[0132] In one embodiment, the solvent is tri-n-butyl phosphate (TnBP).

[0133] In one embodiment, the surfactant is polysorbate 80 (PS80).

[0134] In one embodiment, the solvent-detergent treatment comprises exposing the recovered unbound fraction of step (vii) to 1% polysorbate 80 (PS80) and 0.3% tri-n-butyl phosphate (TnBP).

[0135] The unbound fraction containing hemopexin and the solution containing detergent and solvent are incubated for a time and under conditions suitable for achieving viral clearance. For example, the unbound fraction containing hemopexin and the solution containing detergent and solvent are mixed (i.e., agitated) using any method known to those skilled in the art, illustrative examples of which include stirring, shaking, rotation, and rocking. In one embodiment, the unbound fraction containing hemopexin and the solution containing detergent and solvent are mixed by agitation. According to this embodiment, the unbound fraction containing hemopexin and the solution containing detergent and solvent are agitated so that a vortex is generated in about 5% to about 20% of the liquid depth (e.g., about 5%, 10%, 15%, or 20% of the liquid depth). In one embodiment, the unbound fraction containing hemopexin and the solution containing detergent and solvent are agitated with a vortex of about 5% to 10% of the liquid depth.

[0136] In one embodiment, the solution containing the unbound fraction containing hemopexin and the detergent and solvent is incubated at about 23° C. (i.e., 23° C.±2° C.). In another embodiment, the solution containing the unbound fraction containing hemopexin and the detergent and solvent is incubated for about 1 hour to about 24 hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours).

[0137] Viral inactivation, including those described herein, may suitably further comprise adjusting the solution to a low pH. The low pH may be a pH of about 2 to about 4. In one embodiment, low pH viral inactivation is carried out in the presence of caprylate.

[0138] In another example, viral inactivation is achieved by contacting the hemopexin-containing fraction with n-octyl-β-D-glucopyranoside (OG), thereby forming an OG-IgG mixture.

[0139] In a further example, low pH viral inactivation is carried out in the presence of N,N-dimethylmyristylamine N-oxide (TDAO).

[0140] In a further example, viral inactivation can occur by exposing the hemopexin-containing fraction to a solvent-detergent inactivation process. Suitable solvent-detergent treatments will be known to those skilled in the art, and illustrative examples thereof include surfactants, including biodegradable and / or environmentally friendly surfactants. Exemplary biodegradable and / or environmentally friendly surfactants suitable for use in viral inactivation processes, particularly to inactivate lipid-enveloped viruses, include N,N-dimethylmyristylamine N-oxide (TDAO), polysorbate 80 (PS80), polyoxyethylene (10) isooctylcyclohexyl ether (TRITON® X-100 reduced form), and nonionic surfactants prepared from glucose and alcohol (e.g., Simulsol™ formulations). In one embodiment, the surfactant is N,N-dimethylmyristylamine N-oxide (TDAO). In another embodiment, the surfactant is polysorbate 80. In another embodiment, the surfactant is polyoxyethylene (10) isooctylcyclohexyl ether (TRITON® X-100 reduced form). In yet another embodiment, the surfactant is a nonionic surfactant prepared from glucose and alcohol.

[0141] The methods disclosed herein may further include a virus filtration step. For example, a virus filtration membrane with a pore size of about 15 nm to about 20 nm can be used to remove microorganisms and viruses from a solution. Illustrative examples of suitable nanofilters include Planova S20N (Asahi Kasei Corporation), Virosart HC (Sartorius), and Planova 20N (Asahi Kasei Corporation). In one embodiment, the recovered hemopexin is subjected to virus filtration. In one embodiment, the virus filtration involves passing the recovered hemopexin through a virus filter having a pore size of about 15 nm to about 20 nm in diameter.

[0142] In one embodiment, the methods described herein further comprise ultrafiltration / diafiltration of any of the solutions described herein that contain hemopexin. Illustrative examples of suitable ultrafiltration / diafiltration membranes include Pellicon 2 cassettes (Millipore) or polyethersulfone or Hydrosart cassettes (Sartorius).

[0143] Ion exchange chromatography In one embodiment, the method described herein comprises: (ix) passing the virus-inactivated hemopexin solution through an ion exchange chromatography resin under conditions that allow the hemopexin to bind to the resin; (x) optionally washing the resin after step (ix); (xi) eluting the hemopexin bound to the resin in step (ix); (xii) recovering the hemopexin eluted from step (xi); Further includes:

[0144] Ion exchange chromatography is based on the binding of amino acids (e.g., histidine) to positively or negatively charged functional groups, allowing proteins with a net negative charge (i.e., they are captured on a positively charged anion exchange resin) or a net positive charge (i.e., they are captured on a negatively charged cation exchange resin) to be bound to -N+(C2H5)2, -N+(CH3), -COO - or -SO3 - This allows the protein to be retained in a column containing immobilized functional groups such as

[0145] In one embodiment, the ion exchange chromatography resin is a cation exchange chromatography resin (e.g., Eshmuno CPS) or an anion exchange chromatography resin (e.g., Capto Q™). In one embodiment, the ion exchange chromatography resin is a cation exchange chromatography resin. In one embodiment, the ion exchange chromatography resin is an anion exchange chromatography resin.

[0146] Optimization of the chromatographic performance of an ion exchange chromatography resin is achieved by adjusting variables such as pressure, temperature, column length, column bed height, height equivalent to a theoretical plate (HETP), and linear flow rate. One skilled in the art will understand that any adjustment to such variables may alter the selective binding of hemopexin to the resin.

[0147] In one embodiment, the column bed height is about 10 cm to about 25 cm, preferably at least about 10 cm, preferably at least about 11 cm, preferably at least about 12 cm, preferably at least about 13 cm, preferably at least about 14 cm, preferably at least about 15 cm, preferably at least about 16 cm, preferably at least about 17 cm, preferably at least about 18 cm, preferably at least about 19 cm, preferably at least about 20 cm, preferably at least about 21 cm, preferably at least about 22 cm, preferably at least about 23 cm, preferably at least about 24 cm, or more preferably about 25 cm. In one embodiment, the column bed height is about 15 cm (i.e., 15±2 cm).

[0148] In one embodiment, the column linear flow rate is about 100 cm / hr to about 200 cm / hr, preferably about 100 cm / hr to about 150 cm / hr, preferably about 110 cm / hr to about 130 cm / hr, or more preferably about 115 cm / hr to about 125 cm / hr. In one embodiment, the column linear flow rate is about 120 cm / hr.

[0149] The ion exchange chromatography resin is preferably equilibrated in preparation for passing the virus-inactivated hemopexin solution through it in step (ix) described herein. This is preferably achieved by passing a suitable equilibration buffer through the ion exchange chromatography resin prior to step (ix). The pH of the equilibration buffer may preferably be about 5 to 7, more preferably about 6. In one embodiment, the equilibration buffer comprises about 20 mM to about 60 mM of a buffering agent. Suitable buffers will be well known to those skilled in the art, and illustrative examples include sodium phosphate. In one embodiment, the equilibration buffer comprises about 20 mM to about 60 mM (e.g., about 20 mM, 30 mM, 40 mM, 50 mM, 60 mM) sodium phosphate. In one embodiment, the equilibration buffer comprises about 30 mM to about 50 mM sodium phosphate. In one embodiment, the equilibration buffer comprises about 20 mM sodium phosphate. In one embodiment, the equilibration buffer comprises about 30 mM sodium phosphate. In one embodiment, the equilibration buffer comprises about 40 mM sodium phosphate. Solutions suitable for equilibrating the pH of an ion exchange chromatography resin (i.e., a pH equilibration buffer) typically range from 5 to about 9. In one embodiment, the pH equilibration buffer comprises 40 nM sodium phosphate at a pH of 6.0. Additional solutions for equilibrating the conductivity of an ion exchange chromatography resin (i.e., an equilibration buffer) are also contemplated herein, typically comprising a buffering agent at a concentration of about 20 mM to about 100 mM to obtain a conductivity of about 10 mS / cm. In one embodiment, the equilibration buffer comprises 25 mM sodium phosphate, 25 mM sodium acetate, and 38 mM NaCl at a pH of 6.0. In one embodiment, the conductivity of the equilibration buffer for the ion exchange chromatography resin is from about 8 mS / cm to about 12 mS / cm, preferably about 8 mS / cm, preferably about 9 mS / cm, preferably about 10 mS / cm, preferably about 11 mS / cm, or preferably about 12 mS / cm.In one embodiment, the conductivity of the equilibration buffer for the ion exchange chromatography resin is about 8 mS / cm to about 12 mS / cm (e.g., about 8 mS / cm, about 8.5 mS / cm, about 9 mS / cm, about 9.5 mS / cm, about 10 mS / cm, about 10.5 mS / cm, about 11 mS / cm, about 11.5 mS / cm, or about 12 mS / cm). In one embodiment, the conductivity of the equilibration buffer for the ion exchange chromatography resin is about 8 mS / cm to about 10 mS / cm. In one embodiment, the conductivity of the equilibration buffer for the ion exchange chromatography resin is about 8.5 mS / cm to about 9.5 mS / cm. The conductivity of an equilibration buffer for an ion exchange chromatography resin is determined at any suitable temperature, preferably ambient temperature, for example, about 18°C ​​to about 25°C, preferably about 18°C, or preferably about 19°C, or preferably about 20°C, or preferably about 21°C, or preferably about 22°C, or preferably about 23°C, or preferably about 24°C, or preferably about 25°C. In one embodiment, the conductivity of an equilibration buffer for an ion exchange chromatography resin is measured at ambient temperature. In one embodiment, the conductivity of an equilibration buffer for an ion exchange chromatography resin is measured at a temperature of about 18°C ​​to about 25°C.

[0150] In one embodiment, the volume of equilibration buffer required for pre-equilibration is 1 column volume (CV) or more. In one embodiment, the pH of the eluate from the ion exchange chromatography resin after pre-equilibration is about pH 5.9 to about 6.1 (e.g., pH 5.9, 6.0, or 6.1). In one embodiment, the conductivity of the eluate from the mixed-mode anion exchange chromatography resin after pre-equilibration is about 10 mS / cm.

[0151] In one embodiment, prior to step (ix), the pH of the virally inactivated hemopexin solution is adjusted to a value of about pH 5.9 to about 6.2 (eg, pH 5.9, 6.0, 6.1, or 6.2).

[0152] Thus, in one embodiment, prior to step (ix), the pH of the virus-inactivated hemopexin solution is adjusted to a value of about 5.9 to about 6.2, preferably about 5.9, preferably about 6.0, preferably about 6.1, or preferably about 6.2.

[0153] In one embodiment, prior to step (ix), the pH of the virally inactivated hemopexin solution is adjusted to about 6.0.

[0154] In one embodiment, prior to step (ix), the conductivity of the virally inactivated hemopexin solution is adjusted to a value of about 8 mS / cm to about 12 mS / cm, preferably about 8 mS / cm, preferably about 9 mS / cm, preferably about 10 mS / cm, preferably about 11 mS / cm, or preferably about 12 mS / cm. In one embodiment, the conductivity of the virally inactivated hemopexin solution is about 8 mS / cm to about 12 mS / cm (e.g., about 8 mS / cm, about 8.5 mS / cm, about 9 mS / cm, about 9.5 mS / cm, about 10 mS / cm, about 10.5 mS / cm, about 11 mS / cm, about 11.5 mS / cm, or about 12 mS / cm). In one embodiment, the conductivity of the virally inactivated hemopexin solution is about 8 mS / cm to about 10 mS / cm. In one embodiment, the conductivity of the virus-inactivated hemopexin solution is about 8.5 mS / cm to about 9.5 mS / cm. The conductivity of the virus-inactivated hemopexin solution is determined at any suitable temperature, preferably ambient temperature, for example, about 18°C ​​to about 25°C, preferably about 18°C, or preferably about 19°C, or preferably about 20°C, or preferably about 21°C, or preferably about 22°C, or preferably about 23°C, or preferably about 24°C, or preferably about 25°C. In one embodiment, the conductivity of the virus-inactivated hemopexin solution is measured at ambient temperature. In one embodiment, the conductivity of the virus-inactivated hemopexin solution is measured at a temperature of about 18°C ​​to about 25°C.

[0155] In one embodiment, prior to step (ix), the conductivity of the virally inactivated hemopexin solution is adjusted to about 10 mS / cm (ie, 10±2 mS / cm).

[0156] In one embodiment, the amount of hemopexin passed through the resin in step (ix) is about 30 g to about 60 g per liter of resin (e.g., about 30 g, about 31 g, about 32 g, about 33 g, about 34 g, about 35 g, about 36 g, about 37 g, about 38 g, about 39 g, about 40 g, about 41 g, about 42 g, about 43 g, about 44 g, about 45 g, about 46 g, about 47 g, about 48 g, about 49 g, about 50 g, about 51 g, about 52 g, about 53 g, about 54 g, about 55 g, about 56 g, about 57 g, about 58 g, about 59 g, or about 60 g per liter of resin). In another embodiment, the amount of hemopexin passed through the resin in step (ix) is about 40 g per liter of resin.

[0157] Once the virus-inactivated hemopexin solution has been passed through the ion exchange chromatography resin, the flow-through fraction can be collected and stored for future use.

[0158] Bound hemopexin can be eluted from the ion exchange chromatography resin by any means known to those skilled in the art. Prior to eluting the hemopexin from the resin, the resin can optionally be washed with a suitable wash solution or wash buffer under conditions that retain the hemopexin bound to the resin. Suitable wash buffers and wash conditions will be known to those skilled in the art. The concentration of the wash buffer will depend to some extent on the column load, but a typical wash solution has a buffering effect at a pH of about 6 to about 8. In one embodiment, the wash buffer comprises 25 mM sodium phosphate, 25 mM sodium acetate, and 38 mM sodium chloride at pH 6.0. In one embodiment, the volume of wash solution applied to the mixed-mode anion exchange chromatography resin is about 3 CV. The flow-through wash fraction can also be collected and stored for future use, if desired.

[0159] In one embodiment, the bound hemopexin can be eluted from the ion exchange chromatography resin using an elution buffer containing 20 mM sodium phosphate and 0.6 M sodium chloride at pH 7.2. In one embodiment, the volume of elution buffer applied to the ion exchange chromatography resin is about 3 CV. The elution buffer may suitably have a conductivity of about 45 mS / cm to about 60 mS / cm (e.g., about 45 mS / cm, 46 mS / cm, 47 mS / cm, 48 mS / cm, 49 mS / cm, 50 mS / cm, 51 mS / cm, 52 mS / cm, 53 mS / cm, 54 mS / cm, 55 mS / cm, 56 mS / cm, 57 mS / cm, 58 mS / cm, 59 mS / cm, or 60 mS / cm). Thus, in one embodiment, the conductivity of the elution buffer is about 45 mS / cm to about 60 mS / cm. In one embodiment, the conductivity of the elution buffer is between about 47 mS / cm and about 56 mS / cm. In one embodiment, the conductivity of the elution buffer is about 47 mS / cm. In one embodiment, the conductivity of the elution buffer is about 48 mS / cm. In one embodiment, the conductivity of the elution buffer is about 49 mS / cm. In one embodiment, the conductivity of the elution buffer is about 50 mS / cm. In one embodiment, the conductivity of the elution buffer is about 51 mS / cm. In one embodiment, the conductivity of the elution buffer is about 52 mS / cm. In one embodiment, the conductivity of the elution buffer is about 53 mS / cm. In one embodiment, the conductivity of the elution buffer is about 54 mS / cm. In one embodiment, the conductivity of the elution buffer is about 55 mS / cm. In one embodiment, the conductivity of the elution buffer is about 56 mS / cm. The conductivity of the elution buffer is determined at any suitable temperature, preferably ambient temperature, for example, from about 18°C ​​to about 25°C, preferably about 18°C, or preferably about 19°C, or preferably about 20°C, or preferably about 21°C, or preferably about 22°C, or preferably about 23°C, or preferably about 24°C, or preferably about 25°C. In one embodiment, the conductivity of the elution buffer is measured at ambient temperature. In one embodiment, the conductivity of the elution buffer is measured at a temperature of from about 18°C ​​to about 25°C.

[0160] In one embodiment, the collection of eluted hemopexin is280nm is initiated at 50 mAU or more (path length 2 mm), and A 280nm The process is terminated when the AU drops below 50 mAU (measured at the column outlet; path length 2 mm).

[0161] The eluted hemopexin recovered from the ion exchange chromatography resin may preferably have a conductivity of about 35 mS / cm to about 50 mS / cm (e.g., about 35 mS / cm, 36 mS / cm, 37 mS / cm, 38 mS / cm, 39 mS / cm, 40 mS / cm, 41 mS / cm, 42 mS / cm, 43 mS / cm, 44 mS / cm, 45 mS / cm, 46 mS / cm, 47 mS / cm, 48 mS / cm, 49 mS / cm, or 50 mS / cm). Thus, in one embodiment, the conductivity of the eluted hemopexin is about 35 mS / cm to about 50 mS / cm. In one embodiment, the conductivity of the eluted hemopexin is about 35 mS / cm to about 45 mS / cm. In one embodiment, the conductivity of the eluted hemopexin is about 40 mS / cm to about 45 mS / cm. In one embodiment, the conductivity of the eluted hemopexin is about 40 mS / cm. In one embodiment, the conductivity of the eluted hemopexin is about 41 mS / cm. In one embodiment, the conductivity of the eluted hemopexin is about 42 mS / cm. In one embodiment, the conductivity of the eluted hemopexin is about 43 mS / cm. In one embodiment, the conductivity of the eluted hemopexin is about 44 mS / cm. In one embodiment, the conductivity of the eluted hemopexin is about 45 mS / cm. The conductivity of the eluted hemopexin is determined at any suitable temperature, preferably ambient temperature, for example, from about 18°C ​​to about 25°C, preferably about 18°C, or preferably about 19°C, or preferably about 20°C, or preferably about 21°C, or preferably about 22°C, or preferably about 23°C, or preferably about 24°C, or preferably about 25°C. In one embodiment, the conductivity of the eluted hemopexin is measured at ambient temperature. In one embodiment, the conductivity of the eluted hemopexin is measured at a temperature of about 18°C ​​to about 25°C.

[0162] The eluted hemopexin recovered from the ion exchange chromatography resin can be stored for future use. In one embodiment, the eluted hemopexin can be stored at less than 23° C. for up to 24 hours. In another embodiment, the eluted hemopexin can be stored at about 2° C. to about 8° C. for up to 7 days.

[0163] The eluted hemopexin may also be subjected to further purification, if desired, for example, by concentrating and diafiltering the hemopexin through an ultramembrane and / or sterile filtering the concentrated and / or diafiltered hemopexin. In some embodiments, the eluted hemopexin is subjected to virus removal.

[0164] Techniques for size-based virus removal will be known to those of skill in the art, illustrative examples of which include filtration and nanofiltration.

[0165] In one embodiment, viral removal is achieved by nanofiltration.

[0166] In one embodiment, virus removal is achieved by sequentially pre-filtering and filtering through a virus filter. Suitable pre-filters will be known to those skilled in the art, and illustrative examples thereof include nanofilters or other suitable filters (e.g., Sartopore 2XLM 0.1 μm) with a pore size of about 0.1 μm or about 0.2 μm. Other suitable filters will also be known to those skilled in the art, and illustrative examples thereof include Planova BioEX and Virosart HF. In one embodiment, the virus filter has a pore size of less than about 0.2 μm, or preferably less than about 0.1 μm.

[0167] In one embodiment, the prefilter area is sufficient to provide suitable prefiltration without product loss, e.g., 0.6 m per 100 L of eluted hemopexin. 2 Exceeds.

[0168] In one embodiment, the viral filter area is sufficient to maintain throughput and achieve viral clearance, e.g., 1 m per 100 L of eluted hemopexin. 2 is.

[0169] In one embodiment, the pre-filter area is at least 0.6 times the virus filter area.

[0170] In one embodiment, the prefilter and virus filter are contacted with a wash solution prior to virus filtration of the eluted hemopexin. Suitable virus filtration wash solutions will be known to those of skill in the art. In one embodiment, the virus filtration wash solution comprises NaCl and sodium phosphate. In one embodiment, the volume of the virus filtration wash solution applied to the prefilter and virus filter is at least about 6 capsule volumes. In one embodiment, the virus filtration wash solution is applied to the prefilter and virus filter at a pressure selected from one or more or all of 0.3 bar, about 0.5 to about 1 bar, and about 3.0 bar (i.e., 3.0±0.1). Those skilled in the art will understand that different pressures are applied at different stages of filter preparation (i.e., pre-washing).

[0171] In one embodiment, the eluted hemopexin is applied sequentially to the pre-filter and virus filter at a pressure of about 3.0 bar (ie, 3.0±0.1).

[0172] Those skilled in the art will appreciate that the protein load for application to a virus filter will depend on the filter and filter area used. In one embodiment, the protein load of the filter is 1400 g / m 2 is less than.

[0173] In one embodiment, the pre-filter and virus filter are washed after filtration of the eluted hemopexin (i.e., post-wash). In one embodiment, the post-wash solution is a virus filtration solution described elsewhere herein. In one embodiment, the volume of the virus filtration wash solution applied to the pre-filter and virus filter is at least about 3 capsule volumes. In one embodiment, the post-wash solution is applied sequentially to the pre-filter and virus filter at a pressure of about 3.0 bar (i.e., 3.0±0.1).

[0174] The hemopexin-containing filtrate recovered from the prefilter and virus filter can be stored for future use. In one embodiment, the hemopexin-containing filtrate can be stored at less than 23°C for up to 24 hours. In another embodiment, the hemopexin-containing filtrate can be stored at about 2°C to about 8°C for up to 7 days.

[0175] Concentration and dialysis In one embodiment, the method described herein further comprises exposing the eluted hemopexin recovered in step (xii) to ultrafiltration and / or diafiltration. In one embodiment, the eluted hemopexin recovered in step (xii) is exposed to ultrafiltration and / or diafiltration to increase the concentration of the eluted hemopexin to about 50 mg / mL to about 120 mg / mL (e.g., about 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, 55 mg / mL, 56 mg / mL, 57 mg / mL, 58 mg / mL, 59 mg / mL, 60 mg / mL, 61 mg / mL, 62 mg / mL, 63 mg / mL, 64 mg / mL, 65 mg / mL, 66 mg / mL, 67 mg / mL, 68 mg / mL, 69 mg / mL, 70 mg / mL, 71 mg / mL, 72 mg / mL, 73 mg / mL, 74 mg / mL, 75 mg / mL, 76 mg / mL, 77 mg / mL, 78 mg / mL, 79 mg / mL, 80 mg / mL, 81 mg / mL, 82 mg / mL, 83 mg / mL, 84 mg / mL, 85 mg / mL, 86 mg / mL, 87 mg / mL, 88 mg / mL, 89 mg / mL, 90 mg / mL, 91 mg / mL, 92 mg / mL, 93 mg / mL, 94 mg / mL, 95 mg / mL, 96 mg / mL, 97 mg / mL, 98 mg / mL, 99 mg / mL, 100 mg / mL, 101 mg / mL, 102 mg / mL, 103 mg / mL, 104 mg / mL, 105 mg / mL, 106 mg / g / mL, 61mg / mL, 62mg / mL, 63mg / mL, 64mg / mL, 65mg / mL, 66mg / mL, 67mg / mL, 68mg / mL, 69mg / mL, 70mg / mL, 7 1mg / mL, 72mg / mL, 73mg / mL, 74mg / mL, 75mg / mL, 76mg / mL, 77mg / mL, 78mg / mL, 79mg / mL, 80mg / mL, 81mg / mL , 82mg / mL, 83mg / mL, 84mg / mL, 85mg / mL, 86mg / mL, 87mg / mL, 88mg / mL, 89mg / mL, 90mg / mL, 91mg / mL, 92mg / mL mL, 93mg / mL, 94mg / mL, 95mg / mL, 96mg / mL, 97mg / mL, 98mg / mL, 99mg / mL, 100mg / mL, 101mg / mL, 102mg / mL, Adjust to the following value: 103mg / mL, 104mg / mL, 105mg / mL, 106mg / mL, 107mg / mL, 108mg / mL, 109mg / mL, 110mg / mL, 111mg / mL, 112mg / mL, 113mg / mL, 114mg / mL, 115mg / mL, 116mg / mL, 117mg / mL, 118mg / mL, 119mg / mL or 120mg / mL).

[0176] Thus, in one embodiment, the concentration of eluted hemopexin is from about 50 mg / mL to about 120 mg / mL, preferably about 50 mg / mL, preferably about 51 mg / mL, preferably about 52 mg / mL, preferably about 53 mg / mL, preferably about 54 mg / mL, preferably about 55 mg / mL, preferably about 56 mg / mL, preferably about 57 mg / mL, preferably about 58 mg / mL, preferably about 59 mg / mL, preferably about 60 mg / mL, preferably about 61 mg / mL, preferably about 62 mg / mL, preferably about 63 mg / mL, preferably about 64 mg / mL. mg / mL, preferably about 65 mg / mL, preferably about 66 mg / mL, preferably about 67 mg / mL, preferably about 68 mg / mL, preferably about 69 mg / mL, preferably about 70 mg / mL, preferably about 71 mg / mL, preferably about 72 mg / mL, preferably about 73 mg / mL, preferably about 74 mg / mL, preferably about 75 mg / mL, preferably about 76 mg / mL, preferably about 77 mg / mL, preferably about 78 mg / mL, preferably about 79 mg / mL, preferably about 80 mg / mL, preferably about 81 mg / mL, preferably about 82 mg / mL, preferably about 83 mg / mL, preferably about 84 mg / mL, preferably about 85 mg / mL, preferably about 86 mg / mL, preferably about 87 mg / mL, preferably about 88 mg / mL, preferably about 89 mg / mL, preferably about 90 mg / mL, preferably about 91 mg / mL, preferably about 92 mg / mL, preferably about 93 mg / mL, preferably about 94 mg / mL, preferably about 95 mg / mL, preferably about 96 mg / mL, preferably about 97 mg / mL, preferably about 98 mg / mL, preferably about 99 mg / mL, is about 100 mg / mL, preferably about 101 mg / mL, preferably about 102 mg / mL, preferably about 103 mg / mL, preferably about 104 mg / mL, preferably about 105 mg / mL, preferably about 106 mg / mL, preferably about 107 mg / mL, preferably about 108 mg / mL, preferably about 109 mg / mL, preferably about 110 mg / mL, preferably about 111 mg / mL, preferably about 112 mg / mL, preferably about 113 mg / mL, preferably about 114 mg / mL, preferably about 115 mg / mL, preferably about 116 mg / mL,Preferably, it is adjusted to a value of about 117 mg / mL, preferably about 118 mg / mL, preferably about 119 mg / mL, or preferably about 120 mg / mL.

[0177] In one embodiment, the concentration of the eluted hemopexin is adjusted to about 100 mg / mL.

[0178] The methods described herein may suitably include adjusting or increasing the concentration of hemopexin in the filtrate. Suitable methods for adjusting the concentration of hemopexin in the filtrate will be well known to those skilled in the art, and illustrative examples thereof include diafiltration.

[0179] In one embodiment, the concentration of hemopexin in the filtrate is about 50 mg / mL to about 120 mg / mL, preferably about 50 mg / mL, preferably about 51 mg / mL, preferably about 52 mg / mL, preferably about 53 mg / mL, preferably about 54 mg / mL, preferably about 55 mg / mL, preferably about 56 mg / mL, preferably about 57 mg / mL, preferably about 58 mg / mL, preferably about 59 mg / mL, preferably about 60 mg / mL, preferably about 61 mg / mL, preferably about 62 mg / mL, preferably about 63 mg / mL, preferably about 64 mg / mL mL, preferably about 65 mg / mL, preferably about 66 mg / mL, preferably about 67 mg / mL, preferably about 68 mg / mL, preferably about 69 mg / mL, preferably about 70 mg / mL, preferably about 71 mg / mL, preferably about 72 mg / mL, preferably about 73 mg / mL, preferably about 74 mg / mL, preferably about 75 mg / mL, preferably about 76 mg / mL, preferably about 77 mg / mL, preferably about 78 mg / mL, preferably about 79 mg / mL, preferably about 80 mg / mL, preferably about 81 mg / mL, preferably about 82 mg / mL, preferably about 83 mg / mL, preferably about 84 mg / mL, preferably about 85 mg / mL, preferably about 86 mg / mL, preferably about 87 mg / mL, preferably about 88 mg / mL, preferably about 89 mg / mL, preferably about 90 mg / mL, preferably about 91 mg / mL, preferably about 92 mg / mL, preferably about 93 mg / mL, preferably about 94 mg / mL, preferably about 95 mg / mL, preferably about 96 mg / mL, preferably about 97 mg / mL, preferably about 98 mg / mL, preferably about 99 mg / mL, about 100 mg / mL, preferably about 101 mg / mL, preferably about 102 mg / mL, preferably about 103 mg / mL, preferably about 104 mg / mL, preferably about 105 mg / mL, preferably about 106 mg / mL, preferably about 107 mg / mL, preferably about 108 mg / mL, preferably about 109 mg / mL, preferably about 110 mg / mL, preferably about 111 mg / mL, preferably about 112 mg / mL, preferably about 113 mg / mL, preferably about 114 mg / mL, preferably about 115 mg / mL, preferably about 116 mg / mL,Preferably, it is adjusted to a value of about 117 mg / mL, preferably about 118 mg / mL, preferably about 119 mg / mL, or preferably about 120 mg / mL.

[0180] In one embodiment, the concentration of hemopexin in the filtrate is adjusted to about 100 mg / mL.

[0181] In another aspect disclosed herein, there is provided a method for purifying hemopexin from a solution containing hemopexin and other proteins, comprising: (i) providing a solution containing hemopexin and other proteins, wherein the solution comprises about 225 mM NaCl, about 40 mM sodium phosphate, a pH of about 6.4 + / - 1 and a conductivity of about 27 + / - 1 mS / cm; (ii) passing the solution of step (i) through a mixed-mode cation exchange chromatography resin under conditions that promote the selective binding of hemopexin to the resin over the binding of other proteins to the resin; (iii) washing the resin after step (ii) to remove unbound proteins; (iv) eluting the hemopexin bound to the resin after step (iii) with an elution buffer comprising about 150 mM NaCl, about 40 mM sodium phosphate and a pH of about 7.5+ / -1; (v) recovering the hemopexin eluted in step (iv); (vi) passing the recovered hemopexin eluate of step (v) through a mixed-mode anion exchange chromatography resin under conditions that allow impurities in the recovered hemopexin eluate to bind to the resin while allowing hemopexin to pass through the resin as an unbound fraction; (vii) recovering the unbound fraction containing hemopexin; (viii) optionally exposing the recovered unbound fraction of step (vii) to a virus inactivation step to obtain a virus-inactivated hemopexin solution; A method is provided which includes:

[0182] In another aspect disclosed herein, there is provided a method for purifying hemopexin from a solution containing hemopexin and other proteins, comprising: (i) providing a solution containing hemopexin and other proteins, the solution comprising about 225 mM NaCl, about 40 mM sodium phosphate, a pH of about 6.4 + / - 1 and a conductivity of about 23 to about 25 mS / cm; (ii) passing the solution of step (i) through a mixed-mode cation exchange chromatography resin under conditions that promote the selective binding of hemopexin to the resin over the binding of other proteins to the resin; (iii) washing the resin after step (ii) to remove unbound proteins; (iv) eluting the hemopexin bound to the resin after step (iii) with an elution buffer comprising about 150 mM NaCl, about 40 mM sodium phosphate, a pH of about 7.5 + / - 1 and a conductivity of about 18 to about 21 mS / cm; (v) recovering the hemopexin eluted in step (iv); (vi) passing the recovered hemopexin eluate of step (v) through a mixed-mode anion exchange chromatography resin under conditions that allow impurities in the recovered hemopexin eluate to bind to the resin while allowing hemopexin to pass through the resin as an unbound fraction; (vii) recovering the unbound fraction containing hemopexin; (viii) concentrating the unbound fraction recovered from step (vii); (ix) optionally exposing the concentrated unbound fraction of step (viii) to a virus inactivation step to obtain a virus-inactivated hemopexin solution; A method is provided which includes:

[0183] The processes described herein may be suitably and conveniently carried out batchwise or continuously.

[0184] composition In one aspect of the present invention, a composition is provided comprising hemopexin recovered by the methods disclosed herein. In one embodiment, the composition comprises a hemopexin content of at least 80% of the total protein (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total protein).

[0185] In another embodiment, the composition comprises a hemopexin content of at least 90% of total protein. In another embodiment, the composition comprises a hemopexin content of at least 95%. In another embodiment, the composition comprises a hemopexin content of at least 97%. In yet another embodiment, the composition comprises a hemopexin content of at least 98%.

[0186] Compositions containing hemopexin recovered by the methods of the present invention disclosed herein are substantially free of other components normally associated with hemopexin (e.g., other plasma-derived proteins). Thus, in one embodiment, a composition containing hemopexin contains other components normally associated with hemopexin (i.e., impurities) at less than 20% of the total protein, preferably less than 10% of the total protein, and more preferably less than 5% of the total protein. Those skilled in the art will appreciate that the level of impurities present in compositions of the present invention may depend on the intended use of the composition. For example, if the composition is to be administered to a human subject in need thereof (i.e., for clinical use), it is desirable for the composition to contain less than 5% (of the total protein) of impurities. Conversely, if the protein is to be used in vitro, it is acceptable for the composition to contain more than 5% (of the total protein) of impurities.

[0187] In another aspect disclosed herein, a formulation is provided comprising a composition comprising the hemopexin described herein and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers, diluents and / or excipients will be known to those skilled in the art, and illustrative examples of these include solvents, dispersion media, antifungal and antibacterial agents, surfactants, isotonic and absorption agents, etc.

[0188] The formulation can also be formulated by adding suitable stabilizers, such as amino acids, carbohydrates, salts, and surfactants (or combinations thereof). In certain embodiments, the stabilizer comprises a mixture of a sugar alcohol and an amino acid. The stabilizer may comprise a mixture of a sugar (e.g., sucrose or trehalose), a sugar alcohol (e.g., mannitol or sorbitol), and an amino acid (e.g., proline, glycine, and arginine). In one embodiment, the formulation comprises an amino acid such as arginine. In other embodiments, the formulation comprises a divalent metal ion at a concentration of up to 100 mM and a complexing agent as described in U.S. Patent No. 7,045,601. In various embodiments, the pH is preferably about 6.5-7.5, and the osmolality is at least 240 mosmol / kg.

[0189] The formulation can also be sterilized by filtration before dispensing and long-term storage. Preferably, the formulation will substantially retain its original stability characteristics for at least 2 months, 4 months, 6 months, 8 months, 10 months, 12 months, 18 months, 24 months, 36 months, or more. For example, a formulation stored at 2-8°C or 25°C typically retains substantially the same molecular size distribution as measured by HPLC-SEC when stored for 6 months or more. Certain embodiments of the pharmaceutical formulation can be stable for at least 6 months, 12 months, 18 months, 24 months, 36 months, or even more when stored at 2-8°C and / or room temperature, and may be suitable for commercial pharmaceutical use.

[0190] The compositions or formulations described herein can be formulated into any of many possible dosage forms, such as injectable formulations.Formulation and subsequent administration (dosage) are within the skill of those skilled in the art.Dosage will depend on the subject's responsiveness to treatment, but will continue as long as desired effects (e.g., reduction of free Hb / heme levels) are desired.Those skilled in the art can easily determine the optimal dosage, dosing method and repetition rate.

[0191] In one embodiment disclosed herein, the formulation has a volume of at least 5 mL and contains at least 5 mg / mL (e.g., 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 150 mg / mL, or 200 mg / mL) hemopexin. In another embodiment, the pharmaceutical formulation has a volume of at least 5 mL and contains at least 20 mg / mL hemopexin. In certain embodiments, the formulation has a volume of at least 5 mL and comprises hemopexin at a concentration of about 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 150 mg / mL, or 200 mg / mL. In another aspect, a container is provided that contains at least 5 mL of a formulation comprising hemopexin, wherein the concentration of hemopexin in the formulation is at least 20 mg / mL.

[0192] In one preferred embodiment, the formulation comprises about 15 mM citrate phosphate buffer, about 150 mM NaCl, the pH is preferably about 7.2, and the concentration of hemopexin is about 100 mg / mL.

[0193] In one embodiment, the composition or formulation contains a hemopexin content of about 95 mg / mL to about 110 mg / mL. In one embodiment, the composition or formulation contains a hemopexin content of about 100 mg / mL to about 110 mg / mL. In one embodiment, the composition or formulation contains a hemopexin content of about 103 mg / mL to about 106 mg / mL. In one embodiment, the composition or formulation contains a hemopexin content of about 103 mg / mL to about 104 mg / mL. In one embodiment, the composition or formulation contains a hemopexin content of about 106 mg / mL to about 107 mg / mL. In one embodiment, the composition or formulation contains a hemopexin content of about 100 mg / mL. In one embodiment, the composition or formulation contains a hemopexin content of about 103 mg / mL. In one embodiment, the composition or formulation contains a hemopexin content of about 106 mg / mL.

[0194] In one embodiment, the composition or formulation comprises a heme-binding activity of about 1000 μM to about 2000 μM. In one embodiment, the composition or formulation comprises a heme-binding activity of about 1300 μM to about 1900 μM. In one embodiment, the composition or formulation comprises a heme-binding activity of about 1500 μM to about 1800 μM. In one embodiment, the composition or formulation comprises a heme-binding activity of about 1600 μM to about 1800 μM. In one embodiment, the composition or formulation comprises a heme-binding activity of about 1700 μM.

[0195] In one embodiment, the composition or formulation comprises at least about 80% heme-specific binding activity of the total protein. In one embodiment, the composition or formulation comprises at least about 85% heme-specific binding activity of the total protein. In one embodiment, the composition or formulation comprises at least about 90% heme-specific binding activity of the total protein. In one embodiment, the composition or formulation comprises at least about 91% heme-specific binding activity of the total protein. In one embodiment, the composition or formulation comprises at least about 92% heme-specific binding activity of the total protein. In one embodiment, the composition or formulation comprises at least about 93% heme-specific binding activity of the total protein. In one embodiment, the composition or formulation comprises at least about 94% heme-specific binding activity of the total protein. In one embodiment, the composition or formulation comprises at least about 95% heme-specific binding activity of the total protein. In one embodiment, the composition or formulation comprises at least about 96% heme-specific binding activity of the total protein. In one embodiment, the composition or formulation comprises at least about 97% heme-specific binding activity of the total protein. In one embodiment, the composition or formulation comprises at least about 98% heme-specific binding activity of the total protein. In one embodiment, the composition or formulation comprises at least about 99% heme-specific binding activity of the total protein. In one embodiment, the composition or formulation comprises about 100% heme-specific binding activity of the total protein.

[0196] In one embodiment, the composition or formulation comprises a CD91 dissociation constant (KD) of about 0.50 μM to about 2.0 μM. In one embodiment, the composition or formulation comprises a CD91 dissociation constant (KD) of about 1.00 μM to about 1.5 μM. In one embodiment, the composition or formulation comprises a CD91 dissociation constant (KD) of about 1.05 μM to about 1.15 μM. In one embodiment, the composition or formulation comprises a CD91 dissociation constant (KD) of about 1.10 μM to about 1.20 μM. In one embodiment, the composition or formulation comprises a CD91 dissociation constant (KD) of about 1.13±0.06 μM. In one embodiment, the composition or formulation comprises a CD91 dissociation constant (KD) of about 1.06±0.05 μM.

[0197] In one embodiment, the composition or formulation comprises a transferrin content of less than about 0.50 mg / mL. In one embodiment, the composition or formulation comprises a transferrin content of less than about 0.40 mg / mL. In one embodiment, the composition or formulation comprises a transferrin content of less than about 0.30 mg / mL. In one embodiment, the composition or formulation comprises a transferrin content of less than about 0.25 mg / mL. In one embodiment, the composition or formulation comprises a transferrin content of less than about 0.20 mg / mL. In one embodiment, the composition or formulation comprises a transferrin content of less than about 0.15 mg / mL. In one embodiment, the composition or formulation comprises a transferrin content of less than about 0.12 mg / mL. In one embodiment, the composition or formulation comprises a transferrin content of less than about 0.11 mg / mL.

[0198] In one embodiment, the composition or formulation comprises an albumin content of less than about 0.05 mg / mL. In one embodiment, the composition or formulation comprises an albumin content of less than about 0.025 mg / mL. In one embodiment, the composition or formulation comprises an albumin content of less than about 0.020 mg / mL. In one embodiment, the composition or formulation comprises an albumin content of less than about 0.015 mg / mL. In one embodiment, the composition or formulation comprises an albumin content of less than about 0.010 mg / mL. In one embodiment, the composition or formulation comprises an albumin content of less than about 0.009 mg / mL. In one embodiment, the composition or formulation comprises an albumin content of less than about 0.0085 mg / mL. In one embodiment, the composition or formulation comprises an albumin content of less than about 0.008 mg / mL.

[0199] In one embodiment, the composition or formulation comprises a haptoglobin content of less than about 0.05 mg / mL. In one embodiment, the composition or formulation comprises a haptoglobin content of less than about 0.04 mg / mL. In one embodiment, the composition or formulation comprises a haptoglobin content of less than about 0.03 mg / mL. In one embodiment, the composition or formulation comprises a haptoglobin content of less than about 0.025 mg / mL. In one embodiment, the composition or formulation comprises a haptoglobin content of less than about 0.02 mg / mL. In one embodiment, the composition or formulation comprises a haptoglobin content of less than about 0.015 mg / mL. In one embodiment, the composition or formulation comprises a haptoglobin content of less than about 0.010 mg / mL. In one embodiment, the composition or formulation comprises a haptoglobin content of less than about 0.005 mg / mL.

[0200] In one embodiment, the composition or formulation comprises an apo-A1 content of less than about 0.10 mg / mL. In one embodiment, the composition or formulation comprises an apo-A1 content of less than about 0.09 mg / mL. In one embodiment, the composition or formulation comprises an apo-A1 content of less than about 0.08 mg / mL. In one embodiment, the composition or formulation comprises an apo-A1 content of less than about 0.07 mg / mL. In one embodiment, the composition or formulation comprises an apo-A1 content of less than about 0.06 mg / mL. In one embodiment, the composition or formulation comprises an apo-A1 content of less than about 0.05 mg / mL.

[0201] In one embodiment, the composition or formulation comprises a high molecular weight (HMW) hemopexin aggregate content of less than about 1.0% of total protein. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.9% of total protein. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.8% of total protein. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.7% of total protein. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.6% of total protein. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.5% of total protein. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.4% of total protein. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of about 0.4% to about 0.5% of total protein.

[0202] In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 1.0% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.9% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.8% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.7% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.6% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.5% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.4% of total protein as determined by size-exclusion high performance liquid chromatography, hi one embodiment, the composition or formulation comprises an HMW hemopexin aggregate content of about 0.4% to about 0.5% of total protein as determined by size-exclusion high performance liquid chromatography.

[0203] In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 90% of total protein. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 91% of total protein. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 92% of total protein. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 93% of total protein. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 94% of total protein. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 95% of total protein. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 96% of total protein. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 97% of total protein. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 98% of total protein. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 99% of the total protein.

[0204] In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 90% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 91% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 92% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 93% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 94% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 95% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 96% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 97% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 98% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 99% of total protein as determined by size-exclusion high-performance liquid chromatography.

[0205] In one embodiment, the composition or formulation comprises a low molecular weight (LMW) impurity content of less than about 1.0% of the total protein. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.9% of the total protein. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.8% of the total protein. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.7% of the total protein. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.6% of the total protein. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.5% of the total protein. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.4% of the total protein. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.3% of the total protein.

[0206] In one embodiment, the composition or formulation comprises a low molecular weight (LMW) impurity content of less than about 1.0% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.9% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.8% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.7% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.6% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.5% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.4% of total protein as determined by size-exclusion high-performance liquid chromatography. In one embodiment, the composition or formulation comprises an LMW impurity content of less than about 0.3% of total protein as determined by size-exclusion high performance liquid chromatography.

[0207] In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 80% of total protein. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 82% of total protein. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 84% of total protein. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 86% of total protein. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 88% of total protein. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 90% of total protein. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 92% of total protein. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 94% of total protein. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 96% of total protein. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 98% of total protein. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 99% of total protein.

[0208] In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 80% of total protein as determined by reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 82% of total protein as determined by reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 84% of total protein as determined by reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 86% of total protein as determined by reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 88% of total protein as determined by reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 90% of total protein as determined by reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 92% of total protein as determined by reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 94% of total protein as determined by reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 96% of total protein as determined by reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 98% of total protein as determined by reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 99% of total protein as determined by reducing SDS-PAGE.

[0209] In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 80% of total protein as determined by non-reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 82% of total protein as determined by non-reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 84% of total protein as determined by non-reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 86% of total protein as determined by non-reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 88% of total protein as determined by non-reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 90% of total protein as determined by non-reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 92% of total protein as determined by non-reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 94% of total protein as determined by non-reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 96% of total protein as determined by non-reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 98% of total protein as determined by non-reducing SDS-PAGE. In one embodiment, the composition or formulation comprises a hemopexin purity content of at least about 99% of total protein as determined by non-reducing SDS-PAGE.

[0210] In one embodiment, the protein content of the composition or formulation comprises an isoelectric point (pI) of about 5.0 to about 6.5. In one embodiment, the protein content of the composition or formulation comprises an isoelectric point (pI) of about 5.2 to about 6.4. In one embodiment, the protein content of the composition or formulation comprises an isoelectric point (pI) of about 5.4 to about 6.3. In one embodiment, the protein content of the composition or formulation comprises an isoelectric point (pI) of about 5.5 to about 6.2. In one embodiment, the protein content of the composition or formulation comprises an isoelectric point (pI) of about 5.6 to about 6.1. In one embodiment, the protein content of the composition or formulation comprises an isoelectric point (pI) of about 5.7 to about 6.0. In one embodiment, the protein content of the composition or formulation comprises a minimum isoelectric point (pI) of about 5.4 to about 5.6. In one embodiment, the protein content of the composition or formulation comprises a minimum isoelectric point (pI) of about 5.5. In one embodiment, the protein content of the composition or formulation comprises a major isoelectric point (pI) of about 5.8 to about 5.9. In one embodiment, the protein content of the composition or formulation comprises a major isoelectric point (pI) of about 5.8 to about 5.85. In one embodiment, the protein content of the composition or formulation comprises a maximum isoelectric point (pI) of about 6.00 to about 6.10. In one embodiment, the protein content of the composition or formulation comprises a maximum isoelectric point (pI) of about 6.00 to about 6.05. In one embodiment, the protein content of the composition or formulation comprises a maximum isoelectric point (pI) of about 6.03. In one embodiment, the protein content of the composition or formulation comprises a maximum isoelectric point (pI) of about 6.04.

[0211] In one embodiment, the protein content of the composition or formulation comprises an isoelectric point (pI) of about 5.0 to about 6.5, as determined by capillary isoelectric focusing (cIEF). In one embodiment, the protein content of the composition or formulation comprises an isoelectric point (pI) of about 5.2 to about 6.4, as determined by cIEF. In one embodiment, the protein content of the composition or formulation comprises an isoelectric point (pI) of about 5.4 to about 6.3, as determined by cIEF. In one embodiment, the protein content of the composition or formulation comprises an isoelectric point (pI) of about 5.5 to about 6.2, as determined by cIEF. In one embodiment, the protein content of the composition or formulation comprises an isoelectric point (pI) of about 5.6 to about 6.1, as determined by cIEF. In one embodiment, the protein content of the composition or formulation comprises an isoelectric point (pI) of about 5.7 to about 6.0, as determined by cIEF. In one embodiment, the protein content of the composition or formulation comprises a minimum isoelectric point (pI) of about 5.4 to about 5.6, as determined by cIEF. In one embodiment, the protein content of the composition or formulation comprises a minimum isoelectric point (pI) of about 5.5, as determined by cIEF. In one embodiment, the protein content of the composition or formulation comprises a major isoelectric point (pI) of about 5.8 to about 5.9, as determined by cIEF. In one embodiment, the protein content of the composition or formulation comprises a major isoelectric point (pI) of about 5.8 to about 5.85, as determined by cIEF. In one embodiment, the protein content of the composition or formulation comprises a maximum isoelectric point (pI) of about 6.00 to about 6.10, as determined by cIEF. In one embodiment, the protein content of the composition or formulation comprises a maximum isoelectric point (pI) of about 6.00 to about 6.05, as determined by cIEF. In one embodiment, the protein content of the composition or formulation comprises a maximum isoelectric point (pI) of about 6.03 as determined by cIEF. In one embodiment, the protein content of the composition or formulation comprises a maximum isoelectric point (pI) of about 6.04 as determined by cIEF.

[0212] In one embodiment, the composition or formulation comprises a protease activity level of less than about 5 nKat / L. In one embodiment, the composition or formulation comprises a protease activity level of less than about 4 nKat / L. In one embodiment, the composition or formulation comprises a protease activity level of less than about 3 nKat / L.

[0213] In one embodiment, the composition or formulation comprises a prekallikrein activity level of less than about 30 IU / mL. In one embodiment, the composition or formulation comprises a prekallikrein activity level of less than about 25 IU / mL. In one embodiment, the composition or formulation comprises a prekallikrein activity level of less than about 20 IU / mL.

[0214] In one embodiment, the composition or formulation comprises a tri(n-butyl)phosphate (TnBP) content of less than about 10 μg / mL. In one embodiment, the composition or formulation comprises a TnBP content of less than about 8 μg / mL. In one embodiment, the composition or formulation comprises a TnBP content of less than about 7 μg / mL. In one embodiment, the composition or formulation comprises a TnBP content of less than about 6 μg / mL. In one embodiment, the composition or formulation comprises a TnBP content of less than about 5 μg / mL. In one embodiment, the composition or formulation comprises a TnBP content of less than about 4 μg / mL. In one embodiment, the composition or formulation comprises a TnBP content of less than about 3 μg / mL. In one embodiment, the composition or formulation comprises a TnBP content of less than about 2 μg / mL.

[0215] In one embodiment, the composition or formulation comprises a PS80 content of less than about 20 mg / mL. In one embodiment, the composition or formulation comprises a PS80 content of less than about 18 mg / mL. In one embodiment, the composition or formulation comprises a PS80 content of less than about 16 mg / mL. In one embodiment, the composition or formulation comprises a PS80 content of less than about 14 mg / mL. In one embodiment, the composition or formulation comprises a PS80 content of less than about 12 mg / mL. In one embodiment, the composition or formulation comprises a PS80 content of less than about 10 mg / mL. In one embodiment, the composition or formulation comprises a PS80 content of less than about 8 mg / mL.

[0216] Also contemplated herein are compositions or formulations that include a combination of any two or more of the features described herein. As a non-limiting example, a composition or formulation described herein may include a combination of any two or more of the following features: (a) a hemopexin content of about 95 mg / mL to about 110 mg / mL; (b) a heme-binding activity of about 1000 μM to about 2000 μM; (c) heme-specific binding activity of at least about 80% of the total protein; (d) a CD91 dissociation constant (KD) of about 0.50 μM to about 2.0 μM; (e) a transferrin content of less than about 0.50 mg / mL; (f) an albumin content of less than about 0.05 mg / mL; (g) a haptoglobin content of less than about 0.05 mg / mL; (h) an apo-A1 content of less than about 0.10 mg / mL; (i) high molecular weight (HMW) hemopexin aggregate content of less than about 1.0% of total protein as determined by size-exclusion high-performance liquid chromatography; (j) a hemopexin monomer content of at least about 90% of the total protein as determined by size-exclusion high-performance liquid chromatography; (k) a low molecular weight (LMW) impurity content of less than about 1.0% of total protein as determined by size-exclusion high-performance liquid chromatography; (l) a hemopexin purity content of at least about 80% of total protein as determined by reducing SDS-PAGE or as determined by non-reducing SDS-PAGE; (m) an isoelectric point (pI) of about 5.0 to about 6.5 as determined by capillary isoelectric focusing (cIEF); (n) protease activity levels less than about 5 nKat / L; (o) prekallikrein activity levels less than about 30 IU / mL; (p) a tri(n-butyl) phosphate (TnBP) content of less than about 10 μg / mL; and (q) A PS80 content of less than about 20 mg / mL.

[0217] In one embodiment, a composition or formulation described herein comprises a combination of any two or more of the following features: (a) a hemopexin content of about 95 mg / mL to about 110 mg / mL; (b) a heme-binding activity of about 1600 μM to about 1800 μM; (c) heme-specific binding activity of at least about 97% of the total protein; (d) a CD91 dissociation constant (KD) of approximately 1.10 μM to approximately 1.20 μM; (e) a transferrin content of less than about 0.25 mg / mL; (f) an albumin content of less than about 0.009 mg / mL; (g) a haptoglobin content of less than about 0.03 mg / mL; (h) an apo-A1 content of less than about 0.06 mg / mL; (i) high molecular weight (HMW) hemopexin aggregate content of less than about 0.6% of total protein; (j) a hemopexin monomer content that is at least about 99% of the total protein; (k) a low molecular weight (LMW) impurity content of less than about 0.4% of the total protein; (l) a hemopexin purity content of at least about 88% of total protein; (m) an isoelectric point (pI) of about 5.4 to about 6.3; (n) protease activity levels less than about 3 nKat / L; (o) prekallikrein activity levels less than about 20 IU / mL; (p) a tri(n-butyl) phosphate (TnBP) content of less than about 5 μg / mL; and (q) A PS80 content of less than about 18 mg / mL.

[0218] In one embodiment, a composition or formulation described herein comprises (i) a hemopexin content of about 95 mg / mL to about 110 mg / mL, (ii) a hemopexin monomer content of at least about 99% of total protein, and (ii) a heme-specific binding activity of at least about 97% of total protein. In another embodiment, the composition or formulation further comprises (i) a transferrin content of less than about 0.25 mg / mL, and (ii) a haptoglobin content of less than about 0.03 g / L.

[0219] In one embodiment, the compositions or formulations described herein further comprise no detectable amounts of apolipoprotein A1 and / or albumin. In one embodiment, the compositions or formulations described herein further comprise (i) an albumin content of less than about 0.009 mg / mL, and (ii) an apo-A1 content of less than about 0.06 mg / mL.

[0220] In one embodiment, the compositions or formulations described herein further comprise no detectable protease activity. Suitable methods for determining protease activity will be familiar to those skilled in the art, and illustrative examples thereof are described, for example, in Zhang et al. (eds., Assay Guidance Manual [Internet]. Bethesda, MD: Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004), the entire contents of which are incorporated herein by reference.

[0221] In one embodiment, a composition or formulation described herein is suitable for pharmaceutical administration after storage at 2°C to 8°C and / or ambient (e.g., room temperature) temperature for 12 months.

[0222] Treatment method In another aspect of the present invention, there is provided a method of treating a condition associated with hemolysis, comprising administering to a subject in need thereof a composition or formulation of the present invention disclosed herein.

[0223] The term "subject" as used herein refers to animals, including primates (lower or higher primates). Higher primates include humans. While the present invention has particular application targeting conditions in humans, it will be understood by those skilled in the art that non-human animals will also benefit from the compositions and methods disclosed herein. Thus, those skilled in the art will understand that the present invention has both human and veterinary applications. For convenience, "animal" includes livestock and companion animals such as cattle, horses, sheep, pigs, camelids, goats, donkeys, dogs, and cats. With respect to horses, this includes horses used in the horse racing industry as well as horses used in the entertainment or livestock industries.

[0224] The compositions or formulations described herein that contain hemopexin can be administered to a subject by any suitable route. Illustrative examples of suitable administration routes include intravenous, subcutaneous, intraarterial or infusion. In one embodiment, the compositions or formulations described herein are administered intravenously.

[0225] If necessary, the methods of treatment described herein may further include administering a second therapeutic agent. The second therapeutic compound may be co-administered to the subject sequentially (before or after administration of the compositions or formulations disclosed herein) or simultaneously. In one embodiment, the second therapeutic agent is an iron chelator (e.g., deferoxamine or deferiprone).

[0226] In another aspect disclosed herein, there is provided a use of a composition or formulation comprising hemopexin as described herein in the manufacture of a medicament for treating a condition associated with hemolysis. In one embodiment, the composition or formulation described herein is formulated for use in humans.

[0227] The compositions and formulations described herein are particularly suitable for treating subjects with hemolysis-related conditions, including those associated with a risk of hemoglobin / heme-mediated toxicity. Hemolysis-related conditions, including those associated with a risk of hemoglobin / heme-mediated toxicity, are known in the art. In one embodiment, the condition is selected from acute hemolytic conditions and / or chronic hemolytic conditions. In one embodiment, the condition is selected from the group consisting of hemolytic anemia, transfusion-induced hemolysis, hemolytic-uremic syndrome, autoimmune disease, malaria infection, trauma, blood transfusion, open heart surgery using cardiopulmonary bypass, and burns, including the treatment of hemoglobinemia or hemoglobinuria associated with hemolysis after burns. In one embodiment, the condition is selected from the group consisting of sickle cell anemia, hereditary spherocytosis, hereditary elliptocytosis, thalassemia, congenital dyserythroid anemia, paroxysmal nocturnal hemoglobinuria, systemic lupus erythematosus, and chronic lymphocytic leukemia.

[0228] Those skilled in the art will understand that the invention described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the invention includes all such variations and modifications that fall within the spirit and scope of the invention. The invention also includes all of the steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of any two or more of said steps or features. [Example]

[0229] Specific embodiments of the present invention will now be described with reference to the following examples, which are for illustrative purposes only and are not intended to limit the scope of the generality set forth hereinabove. [Example]

[0230] Screening of mixed-mode chromatography resins for hemopexin purification. Initial investigations found that the isoelectric points of hemopexin and the major contaminating proteins in FIV-4 were all very similar, ranging from approximately pI 4.8 to 6.3. This indicated that ion-exchange chromatography was unlikely to provide a robust purification step for hemopexin. As an alternative, mixed-mode chromatography resins were investigated, taking advantage of their ability to allow separation by hydrophobic interactions as well as charge. In initial resin screening studies, several mixed-mode cation and anion-exchange resins were screened in a high-throughput format to evaluate their utility for hemopexin purification.

[0231] Mixed-mode cation exchange The mixed-mode cation exchange resins Capto MMC (Cytiva), Eshmuno HCX (Merck), Nuvia cPrime (Bio-Rad), and Toyopearl MX-TRP 650M (Tosoh Corporation) were screened in this study.

[0232] The FIV-4 paste was resuspended in acetate buffer, pH 4.5, and loaded onto a mixed-mode cation-exchange column equilibrated with the same buffer. Unbound material was washed off with the same buffer, and bound proteins were eluted with a stepwise NaCl gradient.

[0233] Moderate selectivity for hemopexin was not observed with either the Eshmuno HCX or Toyopearl MX-TRP 650M resins, with the majority of all proteins eluting in the unbound fraction. In contrast, no proteins eluted from Capto MMC and Nuvia cPrime, indicating strong retention of all proteins. Given the expectation (based on pI) that hemopexin should be one of the more strongly retained proteins, these resins were identified as potentially suitable for a suitable hemopexin capture step. Further development of these resins was performed to determine whether sufficient selectivity could be achieved for effective purification (Example 4).

[0234] Mixed-mode anion exchange Mixed-mode anion exchange resins were screened in this study: Capto Adhere (Cytiva), MEP Hypercel (Sartorius), HEA Hypercel (Sartorius), and PPA Hypercel (Sartorius).

[0235] FIV-4 paste was resuspended in phosphate buffer at pH 7.5 and loaded onto a mixed-mode anion-exchange column equilibrated with the same buffer. Unbound material was washed off with the same buffer, and bound protein was eluted with a stepwise NaCl gradient. In a second experiment, the paste was resuspended in 140 mM NaCl / 30 mM KCl at pH 7.5, applied to the anion-exchange column, and eluted with a stepwise decreasing pH gradient.

[0236] The mixed-mode resins MEP Hypercel, HEA Hypercel, and PPA Hypercel showed poor selectivity, with hemopexin co-eluting with contaminating proteins in several fractions (Figure 1).

[0237] When loaded onto Capto Adhere resin in the presence of salt, hemopexin was found in reasonably high purity in the unbound fraction. This clearly represents a useful purification step; however, when loading paste extracts, binding of all other proteins would result in low hemopexin capacity, and the column would rapidly become saturated with contaminants. This suggests that Capto Adhere chromatography is best used as a second purification step once most contaminating proteins have been removed.

[0238] Further studies performed to optimize conditions for downstream purification using Capto Adhere are discussed in Example 5.

[0239] Additional cation / anion exchange resins Initial trials will focus on purifying hemopexin from extracted FIV-4 paste using cation exchange resins Eshmuno CPX, Eshmuno S, Eshmuno COO, and Fractogel EMD COO. - Screening of the cation exchange resins (M) was performed. Both Eshmuno CPX and Eshmuno COO resins showed binding selectivity for hemopexin, but the recovery rates were low, with 40% and 20% of hemopexin present in the flow-through fraction, respectively. Therefore, these cation exchange resins were not considered suitable for the first purification step.

[0240] The feasibility of the anion exchange chromatography resins Eshmuno Q, Fractogel TMAE Hicap (M), and Fractogel DMAE (M) was investigated for their potential use as a polishing step. The tests were performed using hemopexin, which contains only traces of contaminating proteins, as expected after partial purification. Of these, Eshmuno Q gave favorable results, but further development of this resin was discontinued in favor of cation exchange resins.

[0241] After screening the cation exchange chromatography resins Eshmuno COO, Eshmuno CPS, Eshmuno CPX, and Fractogel SE HC(M), Eshmuno CPS was identified as a viable option for the polishing step. It was determined that the final stage of the hemopexin purification process likely resulted in substantially pure hemopexin, with some residual contaminating transferrin. Therefore, further development studies used a surrogate feed mixture of 5 mg / mL hemopexin and 0.5 mg / mL transferrin. These further studies demonstrated that at pH 6.0 and a conductivity of 10 mS / cm, Eshmuno CPS resin had a high binding capacity for hemopexin (approximately 40 mg / mL resin) and was able to remove approximately 80% of the contaminating transferrin.

[0242] This chromatography step was expected to be well positioned as the final polishing step in the hemopexin purification process. In the overall process design, this likely resulted in a feed solution for this column containing 1% polysorbate 80 (PS80) and 0.3% tri(n-butyl) phosphate (TnBP) after viral inactivation. Using the above conditions and a partially purified intermediate as the load material, the solvent / detergent reagents were shown to not affect the separation properties of Eshmuno CPS. The solvent / detergent reagents eluted in the flow-through during the loading and washing steps, and the concentrations of PS80 and TnBP in the hemopexin product were low. This demonstrated that Eshmuno CPS resin is a viable option for both pure polishing and removal of solvent / detergent from the product.

[0243] Eshmuno CPS chromatography resin, not tested in the initial screening of cation exchange resins using paste extracts, demonstrated high binding selectivity for hemopexin and was therefore reevaluated for its potential use as an initial purification step. When the extracted paste was used as the load material, consistent breakthrough of hemopexin was observed during loading, resulting in a loss of approximately 40% of hemopexin regardless of the load conductivity (5 and 10 mS / cm). This may indicate competition for binding sites with other components in the crude extract and / or the presence of different forms of hemopexin, such as heme complexes. Nevertheless, further development of Eshmuno CPS resin as an initial purification step was not pursued due to low recovery rates.

[0244] Eshmuno CPS chromatography resin was further optimized for use as a polishing step after viral inactivation. These studies are described in Example 7. [Example]

[0245] Evaluation of extraction conditions of fraction IV-4 paste for hemopexin purification The first step in the purification process of hemopexin from FIV-4 paste is resuspension in an aqueous buffer. Because several different proteins are present in FIV-4 paste, certain extraction conditions may preferentially solubilize hemopexin while leaving some or all of the contaminating proteins in the insoluble fraction. Solubilization conditions were required that maximize the amount of hemopexin extracted from FIV-4 paste and provide the protein in a matrix compatible with clarification and further downstream purification. From a practical standpoint, it is preferable to perform solubilization in relatively small volumes to facilitate large-scale production. With these goals in mind, several extraction buffers and conditions were investigated to optimize the extraction process steps. These tests are briefly described below.

[0246] Development of an initial FIV-4 paste extract for laboratory-scale batches Initial extraction studies conducted over a pH range from pH 4 to pH 8 demonstrated that only small amounts of hemopexin and haptoglobin were solubilized below pH 5.5, while large amounts of albumin and transferrin were present in the extraction solution across all pH levels (Figure 2). Within the pH range examined, no conditions were identified that promoted preferential extraction of hemopexin.

[0247] Hemopexin extraction was demonstrated to be most effective and yield high at neutral or near-neutral pH. Hemopexin was not preferentially extracted under these conditions, and most contaminating proteins were also extracted under these conditions. Efficient resuspension of FIV-4 paste was demonstrated with paste:buffer ratios ranging from 1:5 to 1:20 and mixing times of 1 to 4 hours, with comparable hemopexin intermediates produced across these condition combinations.

[0248] Following initial extraction process development testing, laboratory-scale batch production using the second generation Hemopexin process (Example 13) was performed utilizing the following extraction conditions: pH 6.5 with 250 mM NaCl, a paste:buffer ratio of 1:10, and a mixing time of 2 hours.

[0249] FIV-4 Paste Batch Consistency During initial experiments, the hemopexin concentration of extracted FIV-4 paste solutions was observed to vary by more than 50%, and this variation was hypothesized to be due to batch variation of FIV-4 paste.

[0250] To investigate this, the consistency of FIV-4 paste extraction and depth filtration was examined for 10 batches of FIV-4 paste. Experiments were performed using 0.2 kg of paste from each batch solubilized in 40 mM phosphate, 180 mM NaCl, pH 6.2 at a paste:buffer ratio of 1:10 for 2 hours at room temperature. Extracts were collected at 625 g / m 2 Filter area: 0.04 m pre-coated with Celpure C1000 2 The extract was clarified by filtration through a 3M 90LP depth filter with a filter area of ​​1000 ml and post-washed with 1 filter volume (Example 3). The hemopexin concentration of the extract was measured by reverse phase HPLC before and after depth filtration.

[0251] When analyzed by reverse-phase HPLC, hemopexin concentrations in the extracted pastes varied from 0.6 to 0.76 g / L (Figure 3). Performance through the depth filtration process steps was consistent across the batches tested, with hemopexin recoveries ranging from approximately 80 to 95% regardless of the method of quantification.

[0252] Optimization of FIV-4 paste extraction for pilot-scale production Despite early studies showing maximum extraction of hemopexin at pH 7.5, extractions for laboratory testing were performed at pH 6.2 to closely match the equilibration conditions for the first chromatographic step. To improve yields at pilot scale, tests were conducted to determine whether extraction at pH 7.5 extracted enough hemopexin to justify the extra step of adjusting the pH after clarification.

[0253] Hemopexin extraction was performed using 10 batches of FIV-4 paste at pH 6.2 and 7.5. Extractions were performed at room temperature for 1 hour in 40 mM phosphate, 180 mM NaCl at the indicated pH, at a paste:buffer ratio of 1:10. Increasing the extraction pH to 7.5 demonstrated that approximately 20% more hemopexin was solubilized, regardless of paste batch (Figure 4). This was considered a significant increase in yield.

[0254] As part of the Capto MMC chromatography process development, robustness testing of loading conditions was performed and a NaCl concentration of 225 mM was identified as the target loading condition (Example 4).

[0255] Following these tests, the extraction buffer conditions were changed to 225 mM NaCl to match the conductivity to the Capto MMC equilibrium and to pH 7.5 to maximize yield. These conditions were used for the remaining lab-scale and pilot-scale batches (Examples 13 and 14).

[0256] Prior to pilot-scale batch production, solubilization of FIV-4 paste in smaller volumes of extraction buffer was investigated to minimize tank size in a manufacturing environment. Hemopexin extractions performed at 1:2.5 and 1:5 paste:buffer ratios demonstrated comparable amounts of hemopexin extracted, ranging from 5.5 to 6.6 g / kg paste. Initially, concerns were raised that residual ethanol in the paste would result in lower recoveries from the first chromatographic purification step, Capto MMC resin, at lower solubilization rates. Submission of clarified extract pastes from 1:2.5, 1:5, and 1:10 extractions to Capto MMC chromatography demonstrated that the lower ratios did not affect purity and recovery, demonstrating the lack of significant impact of residual ethanol and the suitability of the extract at ratios as low as 1:2.5. Tests performed using a 1:1 extraction paste:buffer ratio demonstrated lower recoveries of hemopexin and protein than the Capto MMC chromatography step.

[0257] At lower extraction ratios, larger post-filter press wash volumes were required to maximize protein recovery, with up to three times the press volume required at an extraction ratio of 1:2.5.

[0258] Optimization of FIV-4 paste extraction for manufacturing scale At a manufacturing scale, paste is likely frozen prior to use; in this case, solubilization directly from frozen paste allows for simpler paste handling. To assess this, the solubilization efficiency of hemopexin was evaluated after extraction of frozen paste, thawing the paste at 2–8°C, and resuspending it in buffer at room temperature (approximately 22°C) and 4°C. The results demonstrated that extraction efficiency using frozen paste was slightly higher than that obtained using thawed paste, regardless of the temperature of the extraction buffer. Because the paste was stored at 4°C for several days before extraction, this suggests that there was some degradation of hemopexin in the FIV-4 paste under these storage conditions. Recovery by depth filtration was demonstrated to be consistent regardless of the temperature of the paste and extraction buffer.

[0259] As part of further process optimization for manufacturing scale, the NaCl concentration of the extraction buffer was modified to achieve a conductivity for the clarified extract paste comparable to that of the equilibration buffer for the Capto MMC column. Performing FIV-4 paste extraction in a buffer containing 400 mM NaCl resulted in a clarified extract paste process intermediate with a conductivity of 27 mS / cm, comparable to the Capto MMC equilibration buffer. This eliminates the need for conductivity adjustment after depth filtration and clarification.

[0260] Final process extraction method The FIV-4 paste containing hemopexin is resuspended in an extraction buffer of 40 mM sodium phosphate, 400 mM sodium chloride, pH 7.5±0.1 at a paste:buffer ratio of 1:2.5 w / w. The paste is broken into small clumps and slowly added to the extraction buffer. The extract is stirred at room temperature for at least 120 minutes before clarification, and for at least 60 minutes after all visible clumps have dissociated. [Example]

[0261] Evaluation of fraction IV-4 paste clarification conditions for hemopexin purification Development of an efficient filtration process was necessary to remove suspended particulate matter and prevent column fouling prior to downstream chromatographic purification process steps. Following the development of initial conditions for solubilization of hemopexin from FIV-4 paste, clarification process development was performed along with additional extraction testing to ensure compatibility of extraction and clarification conditions. Depth filtration and clarification testing was performed with the understanding that a filter press would be utilized for depth filtration at a manufacturing scale. These tests are briefly described below.

[0262] Early developments in depth filtration and clarification Several different depth filtration agents from manufacturers 3M and Pall were screened for optimal throughput and clarification of extracted FIV-4 paste. Fraction IV-4 paste was resuspended in phosphate buffer (250 mM NaCl, pH 6.5) at a paste:buffer ratio of 1:10. Extracts were clarified using 47 mm disc filters, 60 mm disc filters, or small cartridges under a constant pressure of 2 bar.

[0263] The initial pass standard for filter performance was at least 200 L / m 2The filter area throughput and turbidity were set at less than 80 NTU. The NTU specification was based on initial testing that found this level of turbidity after 0.22 μm filtration of the extract, with no apparent column fouling by this material.

[0264] Dual-layer cartridge filters and sandwich filters produced some of the highest levels of clarity, but throughput performance was very low due to cartridge clogging by residual filter aid from the original FIV-4 paste. A two-step filtration process involving a first coarse filter, such as a 3M 30SP filter sheet, followed by a second, finer-grade depth filter sheet has been shown to produce a highly clear product intermediate at high throughput. However, in a manufacturing setting, a two-step or sandwich filter filtration process is undesirable.

[0265] When used as a single layer filter, 3M Zeta Plus 90SP filter material was selected to produce a product intermediate with reasonable clarity and throughput.

[0266] The best throughput and clarification were achieved when the depth filters were precoated with filter aid. Addition of filter aid to the extracted paste feed was less efficient, with reduced clarification and lower throughput observed compared to pre-coating the filters with filter aid (premixed aid). There was little difference in depth filtration performance using the two different filter aids, Celpure C1000 and Celpure C300.

[0267] Filter press capacity test: 0.11m 2 The filter area of ​​ / kg paste was identified and a frame depth of 4 cm provided sufficient capacity to perform depth filtration of the extracted paste solution.

[0268] A relatively high flow rate was required in the filter press to produce a uniform coating of filter aid and filter cake. At lower flow rates, it was observed that the filter aid would accumulate at the bottom of the filter frame, reducing filter efficiency and causing inlet line fouling. Tests performed showed that a flow rate of 6.25 L / m 2 The flow rate of filter area / min was confirmed to result in a uniform coating of filter aid on the filter sheet.

[0269] Following the early development of the depth filtration process, 625 g / m 2 Filter area: 0.11 m pre-coated with Celpure C1000 2 Further tests were carried out at an extraction ratio of 1:10 using a 3M 90LP depth filter with a filter area of ​​1 / kg paste and a post-wash of 1 filter volume.

[0270] Depth filtration and clarification optimization Variability in recovery during the depth filtration step was observed in laboratory-scale batches, with one particular batch recovering only 50-60% of the hemopexin over the course of the step (Example 13). Investigation of the cause indicated that recovery was significantly reduced when filtration was performed without a post-wash step. Lower extract protein concentrations, alternative extraction buffers, or high-salt post-washes had little or no effect on product recovery. Taken together, this indicates that losses during the depth filtration step are unlikely to be due to protein binding to the filter and are most likely due to insufficient post-wash volume.

[0271] 0.012m 2 A 20 × 20 cm filter press with a filter area of ​​1 / L extract was used to determine the optimal post-depth filter wash volume for extraction with 1:10 paste:buffer, and the results showed that approximately twice the press volume was required to recover all of the protein.

[0272] The FIV-4 paste extraction conditions were optimized to reduce the volume so that it was more suitable for large-scale manufacturing, and the extraction was performed at a paste:buffer ratio of 1:2.5. When the extraction was performed at a ratio of 1:2.5, the filter area used was adjusted according to the amount of paste used so that the solids load per unit area was the same as for a 1:10 extraction. This was 9 kg paste / m 2 Filter area or 0.031m 2 / L extract. Filtration efficiency for the 1:2.5 extraction ratio was not substantially different from the 1:10 extraction, but a larger post-wash volume of 3 times the press volume was required to recover most of the protein.

[0273] Estimation of hemopexin concentration in clarified extracts Measurement of the hemopexin concentration of the clarified extract paste solution is required to calculate the appropriate loading onto the Capto MMC chromatography resin. At the extract paste solution stage, hemopexin accounts for a low percentage of the total protein content. Therefore, immunoturbidimetry or reverse-phase HPLC was used during early development to obtain hemopexin concentration. During production, it was not feasible to implement complex or time-consuming methods to obtain the concentration for loading onto the Capto MMC resin; therefore, the OD of the extract solution was used. 280nm developed a simple in-process test method.

[0274] OD from FIV-4 Paste Batch Consistency Test (Example 4) 280nm The data were compared to the hemopexin concentration of each batch as determined by reverse phase HPLC. On average, the hemopexin concentration was 0.05 (OD 280nm It was found that the α-value can be estimated by dividing by 13 (Table 1).

[0275] Final Clarification Method FIV-4 extract paste solution was added at 1 ml per 9 kg of paste using a frame depth of 4 cm. 2The clarification is carried out with 3M 90SP Zeta Plus depth filtration media in a filter press with a filter area of ​​6.25 L / m. 2 / min flow rate, filter area 1m 2 The FIV-4 extract paste solution is then pre-washed with 1 press volume of extraction buffer containing 625 g of Celpure 1000 filter aid per press volume. Filtration of the FIV-4 extract paste solution is then performed at 6.25 L / m. 2 The depth-filtered extract is then filtered at a flow rate of 1 / min, which typically generates a pressure of less than 1.5 bar (maximum 2 bar) during filtration. The filter press is post-washed with 3 press volumes of Capto MMC equilibration buffer. The resulting depth-filtered extract is pH adjusted to pH 6.4 ± 0.1 with 0.5 M HCl and the conductivity is verified to be 26-28 mS / cm. The depth-filtered extract is then filtered through a 33 cm filter. 2 Further clarification is performed using a Millipak 200 0.2 μm cartridge or equivalent with an area of ​​1 / L to produce a clarified extract paste. [Example]

[0276] Mixed-mode chromatography using Capto MMC resin Two mixed-mode resins, Capto MMC and Nuvia cPrime, were optimized and compared for use as the first purification step in the hemopexin manufacturing process described herein. Initial testing demonstrated little difference in the yield and purity of hemopexin purified by Capto MMC and Nuvia cPrime resins. However, after several tests to optimize the performance of these two resins, Capto MMC resin demonstrated slightly better purity than Nuvia cPrime resin. The tests conducted during the development and optimization of Capto MMC resin are summarized below.

[0277] Development and optimization of Capto MMC chromatography Initial testing showed that Capto MMC resin binds very strongly to all proteins at low pH (Example 1). We hypothesized that if hemopexin were among the more strongly bound proteins, we could establish conditions under which Capto MMC could be an effective capture step.

[0278] An initial evaluation of binding conditions was performed over a pH range of 5.0 to 7.0, with the goal of maximizing hemopexin binding while minimizing contaminant binding. At each pH, ​​elution conditions were evaluated at a range of NaCl concentrations (50 mM, 150 mM, 300 mM, 500 mM, and 1 M). The highest contaminant levels were observed in the unbound fraction for loading at pH 7.0. While some hemopexin did not bind to the Capto MMC resin and was observed in the unbound fraction, approximately 50% of the loaded hemopexin was eluted at higher NaCl concentration combinations, with purity levels of approximately 30% (Figure 5).

[0279] Based on the results of initial studies, it was determined that optimal binding conditions required further manipulation of either pH, NaCl concentration, or both. Therefore, studies were conducted to examine binding conditions between pH 6.0 and 7.5 over a range of NaCl concentrations, along with elution conditions between pH 7.0 and pH 7.5.

[0280] Loading the hemopexin starting material in 200 mM NaCl at either pH 6.0 or pH 6.5 and eluting at pH 7.5 resulted in high recoveries of hemopexin, greater than 90%. However, higher purity (approximately 90%) was observed when loaded at pH 6.5 compared to 58% purity at pH 6.0. Under the loading conditions of pH 6.5, 200 mM NaCl, hemopexin appeared to be nearly the only protein bound, with most other proteins eluting in the unbound fraction (Figure 6). Clearly, these loading and elution conditions are highly suitable for hemopexin purification using Capto MMC resin.

[0281] After establishing the binding and elution conditions for hemopexin purification, the hemopexin binding capacity of the Capto MMC resin and the appropriate loading amount were determined. The clarified extract paste was loaded onto a 5 mL Capto MMC column for a contact time of 7.5 minutes, resulting in a total hemopexin load of 250 mg per mL of resin. Fractions were collected during the loading step and analyzed for hemopexin concentration by immunoturbidimetry.

[0282] After loading approximately 4 mg of hemopexin per mL of resin, a small amount of hemopexin was observed in the unbound fraction. This amount remained fairly constant until approximately 20 mg of hemopexin per mL of resin was loaded, after which the amount of hemopexin breakthrough increased significantly (Figure 7). This indicates that a certain proportion of hemopexin does not bind to Capto MMC under the current conditions, and the remaining hemopexin could be captured, albeit with some loss, up to a capacity of approximately 20 mg per mL of resin.

[0283] This is consistent with the existence of two populations of hemopexin with different physicochemical characteristics, and raises the question of the existence of a heme-hemopexin complex (Example 10).

[0284] The optimal hemopexin load for the Capto MMC chromatography process step was further investigated by loading various amounts of clarified extract paste and quantifying the eluted hemopexin. The eluted amount generally corresponded to the inverse of that seen in the unbound fraction. Recovery rates remained fairly consistent at 70-75% until the hemopexin load exceeded approximately 15 mg per mL of resin, decreasing to approximately 65% ​​hemopexin recovery at a load of 20 mg per mL of resin. Based on this, the optimal load was determined to be 14 mg per mL of resin, with 20 mg per mL of resin considered the maximum acceptable load.

[0285] Again, there is a constant recovery of the initial 70-75%, indicating the presence of a second population of hemopexin with weaker binding properties (Example 10).

[0286] In other abbreviated tests, the effect on column retention volume was evaluated at various load contact times. These tests showed that a 3-minute contact time produced very similar results to those outlined above for a 7.5-minute contact time. While this allows for more rapid loading of the feed, it was determined that a 15-cm bed height and 7.5-minute contact time would allow the entire method to be run at a linear velocity of 120 cm / hr, which is reasonably achievable with most instrumentation and avoids overpressure issues that can occur at higher flow rates. A 15-cm bed height is also within the range recommended for ease of column packing on a commercial scale.

[0287] Loading Conditions To more specifically define the optimal loading conditions and determine the acceptable operating range of these conditions, robustness tests of Capto MMC loading conditions were performed.

[0288] Capto MMC loading conditions were investigated in the pH range of 6.0-6.6 and NaCl concentrations of 160-250 mM, and FIV-4 extraction was performed in Capto MMC equilibration buffer.

[0289] Through the Capto MMC chromatography step, hemopexin purity and recovery were unaffected over the pH range of 6.2 to 6.6 at NaCl concentrations of 160 to 250 mM. However, material processed at pH 6.0 had poorer purity and recovery characteristics, lower at 160 mM NaCl than at 200 mM NaCl.

[0290] To determine the effect of Capto MMC loading parameters on the quality of the final product, material was processed through a predicted purification process using various Capto MMC loading conditions.

[0291] Following processing through the Capto MMC chromatography step at each loading condition (except pH 6.6, 250 mM NaCl), the hemopexin product was processed through Capto Adhere chromatography, solvent / detergent treatment, and Eshmuno CPS chromatography under optimal conditions for these steps (Examples 5–7). Increased hemopexin purity was observed after processing through the Capto Adhere and Eshmuno CPS chromatography steps, with all conditions demonstrating comparable final purities of approximately 96% hemopexin by RP-HPLC (Figure 8). All loading conditions demonstrated comparable protein contaminant profiles in the Eshmuno CPS eluate process intermediates, with only a faint transferrin band observed by non-reducing SDS-PAGE. This demonstrates the robustness of the downstream processing steps of Capto Adhere and Eshmuno CPS chromatography, capable of producing comparable final products even under suboptimal Capto MMC loading conditions.

[0292] Loading condition data defined the target Capto MMC chromatographic loading conditions as pH 6.4 and 225 mM NaCl, demonstrating that a range of pH 6.2-6.6 and 200-250 mM NaCl was acceptable.

[0293] Capto MMC eluate stability During development, Capto MMC eluate was required as a feedstock in the evaluation of other chromatography resins. This could be expedited by purifying large quantities of material that were stored and used in these development studies. During commercial manufacturing, situations may arise where product intermediates require storage. Therefore, understanding the stability of stored product intermediates is of considerable importance.

[0294] To ensure that the stored material was of sufficient quality, a brief stability study was performed and is summarized below.

[0295] Fraction 4-IV paste was extracted 1:10 into 40 mM phosphate, 250 mM NaCl, pH 6.50, and clarified by 0.22 μm filtration. The clarified extract was purified by Capto MMC chromatography, equilibrated in the same buffer, and eluted with 40 mM phosphate, 150 mM NaCl, pH 7.5.

[0296] Partially purified hemopexin was stored at 4°C and room temperature for up to 7 days. Samples were taken at the start of the study (T=0), after 2 days, and after 7 days and analyzed for protein concentration, purity, monomer content, heme-binding activity, and charge heterogeneity. To confirm that samples could be frozen without affecting the results, samples were subjected to one freeze-thaw episode and analyzed as described above. This allowed for the use of frozen T=0 controls at each time point.

[0297] The results showed no significant changes in hemopexin under any of the storage conditions (see Table 2). This indicates that Capto MMC eluate can be stored at room temperature or below for up to 7 days without affecting product quality. Alternatively, the product can be frozen at least once with no adverse effects.

[0298] Capto MMC Chromatography Process The clarified extract paste solution (Example 3) is loaded onto a mixed-mode Capto MMC chromatography column equilibrated with 5 CV of equilibration buffer (40 mM sodium phosphate, 225 mM NaCl, pH 6.4). The product is loaded to a target load of 14 g of hemopexin per L of resin. After product loading, the column is further washed with 3 CV of equilibration buffer to remove unbound proteins. Hemopexin is eluted with 3 CV of elution buffer (40 mM sodium phosphate, 150 M NaCl, pH 7.5). Collection of the Capto MMC eluate begins 5 minutes into the elution buffer application phase and continues until the UV absorbance returns to baseline. An exemplary chromatographic profile is shown in Figure 9. The concentration of the eluted hemopexin is typically 3-6 mg / mL, with a purity of 70-85%. After product collection, the Capto MMC column is regenerated with 40 mM phosphate, 1 M NaCl, pH 7.5 buffer. [Example]

[0299] Development and optimization of Capto Adhere chromatography Initial chromatographic screening studies identified Capto Adhere as a promising resin for hemopexin purification (Example 3). In these studies, hemopexin eluted from this resin with reasonable purity in the unbound fraction when loaded at pH 7.5 containing NaCl and KCl. Because hemopexin was found in the unbound fraction where most other proteins bound to the column, this resin appeared to be best suited for situations with low contaminating protein loads. Therefore, further studies were conducted to optimize Capto Adhere conditions as a second step following partial purification with Capto MMC. The studies performed during the development and optimization of the Capto Adhere resin are summarized below.

[0300] Initial chromatographic screening studies showed that the drop-through fraction of Capto Adhere contained primarily hemopexin with small amounts of transferrin when operated under loading conditions of 140 mM NaCl, 30 mM KCl (pH 7.5). The effects of various loading salt concentrations and pH conditions were investigated to optimize binding of contaminating proteins.

[0301] Before partially purified hemopexin (i.e., Capto MMC eluate) became available, the first test was performed using the clarified extract paste as the starting material. Loading conditions of pH 6.5, 7.0, and 7.5, combined with NaCl concentrations of 100, 150, and 250 mM, were examined, and the concentrations of the major protein species in the unbound fraction were measured by immunoturbidimetry. The results showed that the highest hemopexin purity and recovery were observed with the loading conditions of pH 7.5 and 100 mM NaCl. While most conditions were able to remove albumin very effectively, many other species remained (Figure 10). It is suspected that these other species remained present due to a combination of the high contaminating protein load and potential overloading of the column. Further testing using the clarified extract as the starting material was not performed.

[0302] Because the Capto Adhere chromatography step appeared to be the most suitable second purification step, further development of loading conditions was performed using hemopexin after partial purification using Capto MMC resin. In this study, the Capto MMC eluate was adjusted to pH 7.5 with a range of NaCl concentrations and applied to a miniature Capto Adhere column equilibrated with the same buffer. Because the feed for this experiment consisted of partially purified material consisting primarily of hemopexin and transferrin, only these two proteins were quantified in the unbound fraction by immunoturbidimetry and SDS-PAGE was performed to assess purity. This experiment was performed as a rapid study, with a 1-minute load contact time for most conditions. The 150 mM NaCl condition was then repeated at pH 7.5 and 8.0 with an 8-minute contact time.

[0303] Very high hemopexin recovery was observed (92-97%) at NaCl load concentrations of 100-200 mM NaCl, while the highest removal of contaminating proteins (primarily transferrin) was achieved at 150 mM for an 8-minute contact time and 200 mM for a 1-minute contact time (Figure 11). These results were reinforced by SDS-PAGE analysis, which demonstrated very high hemopexin purity at 100-200 mM NaCl (Figure 12).

[0304] The previous process step, the elution step of Capto MMC chromatography, utilized a buffer with 150 mM NaCl and pH 7.5, which was well within the optimal conditions for Capto Adhere described above. Therefore, if the same conditions were used for Capto Adhere loading, the process could be streamlined. For this reason, 150 mM NaCl and pH 7.5 were selected as the most appropriate loading conditions for Capto Adhere.

[0305] The binding capacity of Capto Adhere resin was determined using partially purified hemopexin (Capto MMC eluate) and a 7.5-minute contact time (120 cm / hr for a 15 cm bed height column) under the loading conditions determined above. A 250 mg hemopexin load per mL of resin (corresponding to approximately 60 mg of contaminating protein per mL of resin) was applied to the resin. Breakthrough of haptoglobin, albumin, and transferrin was measured in the unbound fraction by immunoturbidimetry, and purity was assessed by SDS-PAGE.

[0306] Analysis showed that haptoglobin began to appear in the unbound fraction after a hemopexin load of 36 mg hemopexin per mL of resin. This was supported by SDS-PAGE analysis, which demonstrated excellent hemopexin purity in fractions up to this load ( FIG. 13 ). For simplicity and to provide a margin for error, it was decided that an appropriate load limit for Capto Adhere chromatography would be set at 30 g hemopexin / L resin. Transferrin breakthrough in Capto Adhere chromatography was nearly immediate, meaning that any residual levels of transferrin in the product after Capto Adhere chromatography would require further purification steps.

[0307] Loading Conditions To determine the robustness of the Capto Adhere loading conditions, the pH and conductivity of the Capto MMC eluate was adjusted and loaded onto Capto Adhere under a range of loading conditions: pH 7.0 to pH 8.0 and NaCl concentrations of 100 to 200 mM.

[0308] Hemopexin recovery on the Capto Adhere column was comparable for all loading conditions, and nearly complete removal of albumin and haptoglobin was observed by SDS-PAGE (Figure 14). Transferrin recovery, as measured by RP-HPLC, ranged from 9 to 25%, with higher recovery observed at a 200 mM NaCl concentration level, indicating that higher conductivity reduces contaminant binding (Figure 14). These data indicate that the optimal loading conditions for the Capto Adhere chromatography step are pH 7.0-8.0 with NaCl concentrations of 100 mM-150 mM (approximately 15.8-21 mS / cm) to ensure maximum transferrin removal. However, given the demonstrated ability of an additional cation exchange step to remove transferrin (Example 7), extending the NaCl (conductivity) range to 200 mM (approximately 25.6 mS / cm) is also feasible.

[0309] During the manufacture of laboratory- and pilot-scale batches, the Capto MMC eluate was adjusted from pH 7.2–7.3 to pH 7.5 and approximately 18.5–20 mS / cm before being loaded onto the Capto Adhere resin. Capto Adhere robustness testing demonstrated that the pH and conductivity of the unadjusted MMC eluate did not affect the purification capacity of the Capto Adhere resin or the recovery of hemopexin. Therefore, the Capto MMC eluate could be loaded without pH and conductivity adjustment to simplify the process in a manufacturing environment. Small-scale runs demonstrated this to be the case, with no impact on the chromatographic profile, purity, or hemopexin recovery.

[0310] Additional processing runs were conducted to evaluate the feasibility of running Capto MMC and Capto Adhere chromatography columns in series. In contrast to the pooled Capto MMC eluate, the serial processing runs exhibited higher contaminating protein levels as evidenced by non-reducing SDS-PAGE gels, with stronger transferrin and haptoglobin bands. A lower hemopexin recovery of 33% was observed for the serial processing run using hemopexin recovered from the clarified extract paste, compared to 47% when operated under current conditions. Although serial operation of Capto MMC and Capto Adhere columns was not considered suitable for use in the manufacturing process under current conditions, it may be feasible with further optimization.

[0311] Capto Adhere Chromatography Process The Capto MMC eluate from the previous step is typically pH 7.2 and has a conductivity of approximately 18-19 mS / cm. While this differs slightly from the equilibration conditions for Capto Adhere, adjustment of the pH and conductivity of the Capto MMC eluate is not necessary. The product is loaded onto a Capto Adhere mixed-mode chromatography column equilibrated with 5 CV of 40 mM sodium phosphate, 150 mM NaCl, pH 7.5. The product is loaded to a target load of 30 g hemopexin / L resin, and the unbound hemopexin fraction is collected with an additional 3 CV wash of equilibration buffer. An exemplary chromatographic profile is shown in Figure 15. The eluted hemopexin is typically greater than 90% pure and has a concentration of 1-3 mg / mL. After product collection, the Capto Adhere column is regenerated with 40 mM phosphate, 1 M NaCl, pH 7.5 buffer. [Example]

[0312] Confirmation of virus inactivation by solvents and detergents Virus inactivation by solvent and detergent treatment Solvent-detergent (SD) treatment with 1% polysorbate 80 (PS80) and 0.3% tri-n-butyl phosphate (TnBP) is one of two specialized virus removal / inactivation steps for the hemopexin process. This treatment disrupts the membranes of enveloped viruses and is a well-defined, reliable method for virus inactivation in the plasma industry. The effectiveness of SD treatment for hemopexin was demonstrated in Capto MMC eluates using pseudorabies virus (PRV) as a model. In this study, a reduction of approximately 5.7 log saturation was achieved after 60 minutes of incubation without damaging the hemopexin intermediate. 10 A reduction in virus titer of 100 mg / kg / day was achieved (Figure 16).

[0313] Stability studies with Capto MMC-purified hemopexin maintained in SD solution for 24 hours at 25°C showed no change in product purity or aggregation state and negligible change in heme-binding activity, which, taken together with the clearance data, indicates that SD treatment is a viable strategy for virus reduction in hemopexin processes.

[0314] However, when SD treatment is performed with the Capto MMC eluate (as in the spiking / exclusion study), there is the possibility of SD interference with the subsequent Capto Adhere purification step. Additionally, it is unlikely that SD will be removed by this step. To avoid these issues, SD treatment was performed on the material after Capto Adhere treatment. Given the increased protein purity after Capto Adhere, it is unlikely that viral inactivation will be adversely affected by this change. This would also limit the number of purification steps that need to be performed in the post-VI grade area during commercial manufacturing.

[0315] Solvent and surfactant treatment process The Capto Adhere eluate is filtered through a 0.22 μm filter into a jacketed vessel and heated to 21–25°C. A stock solvent and detergent solution containing 20% ​​w / w polysorbate 80 (PS80) and 6% w / w tri-n-butyl phosphate (TnBP) is slowly added to the Capto Adhere eluate over 5–10 min to a target concentration of 1.0% w / w PS80 and 0.3% w / w TnBP. The solvent-detergent-treated bulk is agitated using an agitation speed and impeller size that allows vortexing to approximately 10% of the vessel depth without causing aeration. After at least 15 min, the agitation speed is reduced to approximately 5% of the vessel depth without causing aeration. Incubation begins when the solution temperature reaches 23°C and continues for 2–24 h with agitation, maintaining the temperature at 23 ± 2°C. [Example]

[0316] Ion exchange chromatography using Eshmuno CPS resin Four ion-exchange chromatography resins, Capto Q, Capto DEAE, Capto S, and Eshmuno CPS, were investigated for the removal of solvent and detergent process reagents from hemopexin process intermediates. Capto Q chromatography resin, used in a previous hemopexin process (as previously described in WO 2014 / 055552), was demonstrated to bind hemopexin and contaminating proteins (e.g., transferrin, haptoglobin, and albumin). Elution of hemopexin was observed at NaCl concentrations between 50 and 100 mM, although transferrin was shown to co-elute with hemopexin. Further investigations over a pH range of 7.0 to 8.0 did not demonstrate further reduction of transferrin contamination.

[0317] Capto DEAE chromatography resin was observed to bind hemopexin and contaminating proteins, although a slight loss of approximately 5% hemopexin was observed in the drop-through fraction. Transferrin was observed to co-elute with hemopexin over a range of NaCl concentrations, haptoglobin eluted above 150 mM NaCl, and albumin eluted above 200 mM NaCl. Capto S chromatography resin demonstrated poor binding potential for hemopexin, which was present in the drop-through fraction.

[0318] A chromatography resin screening study identified Eshmuno CPS ion exchange resin as a suitable resin for use in the chromatographic polishing step after the solvent and detergent treatment process step (Example 1). Eshmuno CPS resin exhibited high binding capacity for hemopexin, efficiently removed transferrin, and demonstrated superior purification compared to Capto Q, Capto DEAE, and Capto S resins.

[0319] Development and optimization of Eshmuno CPS chromatography Initial evaluation of Eshmuno CPS resin with SD-treated Capto Adhere eluate was performed using equilibration and loading conditions of pH 6.0 and 10 mS / cm conductivity (Example 1). Elution of bound hemopexin was performed using a step gradient of 100 mM, 200 mM, and 300 mM NaCl. Under these conditions, purification of hemopexin was demonstrated, with 99% of transferrin recovered in the drop-through fraction, while 98% of hemopexin was recovered in the 300 mM NaCl elution. The final purity of hemopexin after Eshmuno CPS chromatography is shown by SDS-PAGE analysis (Figure 17).

[0320] Regarding the simplification of the hemopexin process operation, elution of hemopexin from Eshmuno CPS resin in hemopexin formulation buffer offers the advantages of shortening diafiltration time during UF / DF and reducing the number of required buffers. Small-scale processing results in elution of 99.6% of the loaded hemopexin in formulation buffer (0.9 mM citric acid, 14.1 mM sodium phosphate, 150 mM NaCl, pH 7.2), demonstrating the feasibility of direct elution into hemopexin formulation buffer. In subsequent tests on virus filtration, a 0.6 M NaCl concentration was found to allow for higher filter throughput (Example 8). Considering that all contaminating proteins are found in the unbound fraction in Eshmuno CPS chromatography, elution at this NaCl concentration likely allows for a simpler transition to the virus filtration step.

[0321] The binding capacity for Eshmuno CPS resin was determined by using the Capto Adhere eluate as the feedstock and loading the column to the breakthrough point. Binding conditions were pH 6.0 and 10 mS / cm, as described above. Breakthrough was observed after loading 46 g of hemopexin per L of resin (FIG. 18). As a result, the target load for hemopexin on Eshmuno CPS resin was set at 40 g hemopexin / L resin, approximately 90% of the breakthrough point, which minimized the risk of overloading and loss of hemopexin throughout the process steps.

[0322] The ability of Eshmuno CPS resin to remove polysorbate 80 was demonstrated by loading a sample of 1% polysorbate 80. Analysis of the drop-through, elution, and regeneration fractions demonstrated 100% recovery of the loaded polysorbate 80 in the drop-through fraction, demonstrating that polysorbate 80 is not retained by Eshmuno CPS resin.

[0323] Loading Conditions To determine the robustness of the loading conditions, transferrin was spiked into solvent- and detergent-treated Capto Adhere eluates and loaded onto an Eshmuno CPS column at pH 5.8–6.2 and conductivity 8–12 mS / cm. Transferrin removal on the Eshmuno CPS column was optimal at loading conditions of pH 6.0–6.2 and conductivity 10–12 mS / cm, but acceptable results were observed even at 8 mS / cm. At a loading condition of pH 5.8, transferrin was detected by RP-HPLC in the Eshmuno CPS eluate at conductivity levels of 8 and 10 mS / cm (Figure 19). Therefore, the recommended loading conditions for the Eshmuno CPS chromatography step are pH 6.0–6.2 and conductivity 8.0–12 mS / cm.

[0324] Eshmuno CPS chromatography process steps The solvent / detergent-treated Capto Adhere eluate is pH adjusted to pH 6.0 ± 0.1 with 0.5 M acetic acid and diluted with PFW or WFI to a target conductivity of 10 mS / cm. The conditioned product is loaded onto an Eshmuno CPS ion exchange chromatography column equilibrated with 5 CV of 25 mM sodium phosphate, 25 mM sodium acetate, 38 mM NaCl, pH 6.0. The conditioned product is loaded to a target load of 40 g hemopexin / L resin. After product loading, the column is further washed with 3 CV of equilibration buffer to remove SD and unbound proteins. Hemopexin is eluted with 3 CV of 20 mM sodium phosphate, 0.6 M NaCl, pH 7.2. Collection of the Eshmuno CPS eluate begins when the UV absorbance is above 50 mAU (2 mm path length) and continues until the UV absorbance reaches baseline (A for 2 mm path length). 280nm Continue until the elution returns to <50 mAU. The eluted hemopexin is typically greater than 95% pure and has a concentration of 6-10 mg / mL. After collection of the product, the Eshmuno CPS column is regenerated with 40 mM phosphate, 1 M NaCl pH 7.5 buffer. [Example]

[0325] Optimizing viral filtration for the hemopexin process Viral filtration is one of two specialized virus removal / inactivation steps in the hemopexin process. The virus filtration step is a well-defined, robust method for virus reduction in the plasma industry, removing viruses based on size differences. As previously described in WO 2014 / 055552, the first-generation hemopexin process virus filtration method using Planova BioEX virus filters served as the basis for further development of the virus filtration step. Literature has shown that various factors affect nanofiltration, including solution pressure, protein concentration, conductivity, and pH, and the impact of each of these factors on virus filtration throughput was investigated. The development testing performed on the virus filtration step is summarized below.

[0326] Viral filtration development and optimization Initial testing has shown that higher pressures result in greater filter throughput, 113 L / m at 2 bar. 2 compared to 152 L / m in 1.5 hours at 3 bar. 2 However, a higher decay rate was also observed for filtration at 3 bar. When the data were fitted to an exponential decay model, the maximum throughput was 371 L / m at 3.0 bar, in contrast to the throughput data at 1.5 hours. 2 at a lower pressure of 2 bar, compared to 473 L / m 2It was shown that the throughput was 3 bar. The lower throughput at 3 bar is consistent with the "trapping" theory of particle retention in virus filters, where particles are retained in pockets at high pressure but allowed to diffuse out of these pockets at low pressure; it is for this reason that higher pressures are preferred for viral clearance. Although the results of this test indicate that the throughput at 3 bar is lower than that at 2 bar, the throughput achieved was acceptable for commercial production of hemopexin. This, combined with the time savings achieved at 3 bar and the more favorable pressure for viral clearance, led to the decision to utilize 3 bar for all subsequent testing.

[0327] Developmental studies to optimize viral filtration throughput were performed using a Sartopore 2XLM prefilter coupled to a BioEX filter at approximately the pH and conductivity of the Eshmuno CPS eluate.

[0328] For Eshmuno CPS eluates, no substantial differences in filtration throughput and flux decay were observed across the pH range of 6.5 to 7.5. Based on this finding, it was determined that adjusting the pH of the eluate prior to viral filtration would provide little or no throughput benefit.

[0329] For the initial hemopexin process, as previously described in WO 2014 / 055552, the conductivity of pure hemopexin material for virus filtration was equivalent to 600 mM NaCl. Early development testing of the second-generation process also demonstrated superior throughput at higher conductivities. Confirmatory testing identified that moderate to high conductivities resulted in increased throughput, with CPS eluates at conductivity values ​​of 37 mS / cm (600 mM NaCl) and 54 mS / cm (1 M NaCl) demonstrating comparable throughputs 13% greater than those at 18 mS / cm (150 mM NaCl) (Figure 20). Flux decay rates did not vary significantly with conductivity despite changes in throughput, indicating that increasing conductivity neither increases filter permeability nor reduces fouling rates, but allows solution access to a larger available area or a greater number of pores.

[0330] During pilot-scale production, low viral filtration throughput was observed, only 45–70 L / m 2 Filter fouling occurred after the prefilter surface area was investigated as a possible cause, showing that throughput improved significantly when larger prefilter areas were used. In this experiment, a prefilter area:virus filter area ratio of 0.75:1 resulted in an approximately 30% increase in throughput compared to a ratio of 0.22:1 (Figure 21A). Flux decay was also lower with larger prefilter areas, indicating that greater differences were evident at higher loads. To account for differences in flux decay and sample volume limitations, data from subsequent experiments were fit to an exponential curve and extrapolated to determine the theoretical maximum throughput.

[0331] When the above experiment was repeated using a different prefilter, Virosart Max (Sartorius), the throughput was further improved (Figure 21B). Curve fitting of the filtration data revealed a throughput of 551 L / m 2A maximum throughput of 1040 L / m can be achieved with this prefilter at a filter area:virus filter area ratio of 0.25:1, and 1040 L / m at a ratio of 1.67:1. 2 This is clearly superior to the maximum throughput achieved with the Sartopore XLM and would allow for the use of a smaller Virosart Max prefilter.

[0332] Evaluation of alternative virus filters as a substitute for the Asahi Kasei BioEX showed that the Sartorius Stedim Virosart HF filter outperformed the BioEX in the same experiment at 1044 L / m 2 Slightly higher than 1068L / m 2 The Virosart HF filter was identified as a suitable substitute, with a maximum throughput of 1000 sachets per 1000 ml. The Sartorius Stedim Virosart HC and Merck-Millipore Viresolve Pro filters were not suitable substitutes, as both had lower maximum throughputs than the BioEx filter. BioEX remains the preferred filter, having undergone initial viral validation testing for use in the hemopexin process and been utilized in producing material for the hemopexin Phase 1 trial mentioned elsewhere herein (and described in WO 2014 / 055552). However, the throughput of the Virosart HF filter strongly suggests that it may be used as a substitute for the BioEX filter, if desired.

[0333] Virus spike addition experiment The effectiveness of the Asahi BioEX filtration process was evaluated by its ability to remove the model virus MVM (minute virus of mice) from purified hemopexin. MVM (a worst-case model virus for filtration due to its small size) was spiked into Eshmuno CPS eluate and filtered at 0.0003 ml. 2The samples were filtered through Asahi BioEx filters with a filter area of ​​7.2 log. Excellent viral clearance was achieved throughout the filtration process via evaluation of relevant sample fractions under worst-case combined conditions for viral breakthrough. 10 A final reduction in viral titer of greater than or equal to 100 mg / mL was also achieved for a representative combined pool (Figure 22), demonstrating that the BioEX filter provides suitable viral clearance for implementation in the hemopexin process.

[0334] Virus filtration process The Eshmuno CPS eluate was passed sequentially through a 0.1 μm prefilter and a Planova BioEX filter at a pressure of 3.0 ± 0.1 bar, with a filter size of 1 m per 100 L of Eshmuno CPS eluate. 2 The pre-filter area should be at least 0.6 times the virus filter area. The filter is post-washed with 3x BioEX capsule volumes of virus filter wash buffer, which is pooled with the bulk filtrate to form the BioEX filtrate. [Example]

[0335] Concentration and diafiltration of BioEX filtrate The concentration and diafiltration process steps for the earlier hemopexin process, as previously described in WO2014 / 055552, were used without modification for this process. The concentration and diafiltration process steps were successfully implemented into the hemopexin process, and no further development or optimization of the process steps was undertaken. [Example]

[0336] Identification and quantification of heme-hemopexin complexes in FIV-4 paste During development of the Capto MMC chromatography process, poor recovery of hemopexin was observed, with a significant portion of hemopexin observed in the unbound fraction. This appeared to vary depending on the batch of paste used. The fact that this occurred at low column loads and optimal binding conditions (Example 6) led to the hypothesis that a population of hemopexin exists with distinct physicochemical characteristics. Initial studies demonstrating a lack of binding to heme agarose and an evaluation of the literature suggested that this population consisted of a heme-hemopexin complex.

[0337] In one key study, a 50% mixture of hemopexin and heme-hemopexin complex was applied to Capto MMC under the conditions established above, and the protein was monitored by absorbance at 280 nm and the heme-hemopexin complex at 411 nm. The results clearly showed pure hemopexin eluting in the bound fraction and the heme-hemopexin complex eluting in the unbound fraction (Figure 23).

[0338] Although the heme-hemopexin complex was shown not to bind to Capto MMC, its presence in the extracted paste was not demonstrated. To address this, the heme-hemopexin complex was isolated from the unbound fraction of the extracted paste by Capto MMC purification. It was found that the heme-hemopexin complex did not bind to Capto Adhere under the process operating conditions used for hemopexin purification, making this step possible to purify the complex from the Capto MMC unbound fraction. In this experiment, when Capto Adhere was loaded at a low volume, most contaminating proteins remained bound to the Capto Adhere resin, and the purified heme-hemopexin complex was collected in the drop-through fraction. The resulting purified heme-hemopexin complex contained significant amounts of transferrin by SDS-PAGE, but the absorbance spectrum showed a peak at 414 nm for the heme-hemopexin complex and no peak for holo-transferrin at 475 nm. This clearly demonstrates the presence of heme-hemopexin complexes in the Capto MMC unbound fraction.

[0339] These results demonstrate that the amount of heme-hemopexin complex can be estimated using Capto Adhere Impres chromatography with small injection volumes. Under these conditions, hemopexin and the heme-hemopexin complex are eluted as a single broad peak in the unbound fraction, while all other proteins are retained. Because the extinction coefficients of hemopexin at 280 nm and the heme-hemopexin complex at 414 nm are nearly identical, the proportion of heme-hemopexin complex is estimated by the ratio of the peak area at 414 nm to the peak area at 280 nm.

[0340] Initial analysis showed that approximately 15% of the hemopexin in the clarified extract paste was present as a heme-hemopexin complex, but this was found to vary between batches of paste.

[0341] A second assay for measuring heme-hemopexin complex content was developed using a combination of RP-HPLC and size-exclusion HPLC (SE-HPLC) with detection at 412 nm. Size-exclusion HPLC of the extracted paste was able to identify a reasonably well-resolved peak for the heme-hemopexin complex, which could be integrated and quantified against a standard curve. Free hemopexin concentration was determined by RP-HPLC, and from two data sets, the portion of hemopexin in complex with heme could be determined. Using this assay, the percentage of heme-hemopexin complex in nine different batches of paste was quantified, showing considerable variation in the amount of complex, ranging from 3.7 to 17.2% of total hemopexin, depending on the paste batch.

[0342] Taken together, the data from the above studies indicate that the amount of complex can vary considerably between batches of paste. Because the heme-hemopexin complex is not recovered in the hemopexin purification process, a clear understanding of the amount of complex is necessary to fully understand process recovery. [Example]

[0343] Process Description The final process developed involves purifying hemopexin from FIV-4 paste using three chromatography steps. After paste solubilization and clarification, hemopexin is purified using Capto MMC and Capto Adhere chromatography, followed by viral inactivation via solvent / detergent treatment. The product is then polished using an Eshmuno CPS salt-tolerant cation exchange column and nanofiltered for viral removal, followed by diafiltration into formulation buffer.

[0344] Examples 2-10 outline much of the development of each of these steps, although some further optimization and simplification was undertaken following extensive laboratory and pilot-scale purification efforts.

[0345] An overview of the final process including all optimizations is shown in Figure 24. [Example]

[0346] Process Reproducibility After initial development and optimization, the purification process was run three times to assess reproducibility. This test was performed before all optimization and process streamlining was complete, but most process parameters were established at this stage and only minor changes were made thereafter. The process was performed essentially as outlined in Figure 24, except that the extraction was performed in 180 mM NaCl (pH 6.2) at a paste:buffer ratio of 1:10, and the Capto MMC column was equilibrated in the same buffer.

[0347] Three batches were produced on a small laboratory scale from three batches of paste (Batches 1, 2, and 3). Batches 1 and 2 were derived from the batches adsorbed for Berinert / Beriplex production. Process intermediates and the final purified product were assayed for hemopexin concentration by immunoturbidimetry and RP-HPLC, and for purity by SDS-PAGE. The final purified product was further analyzed for activity by heme binding.

[0348] The results showed that the process was completely reproducible, regardless of the batch of paste used, and that hemopexin purity and specific activity were nearly identical across the three batches (Table 3 and Figure 25). In all cases, purity was very high, exceeding 99% by SDS-PAGE and densitometry, and heme-binding activity ranged from 99.6 to 104.9% of total protein. While there was some variability in these activity values, they remained extremely high and likely reflect assay variability rather than process or batch inconsistencies.

[0349] One possible source of yield and recovery variability is the amount of heme-hemopexin complex present in the starting material. The hemopexin in this complex is included in the quantification of the starting material, but the complex is removed during the Capto MMC chromatography step of the process. The amount of complex varies between batches of paste, which can lead to batch-to-batch variability in yield and recovery.

[0350] Comparing the recoveries across each processing step, it is clear that most of the process loss occurs during the Capto MMC chromatography step. This is somewhat expected due to the removal of the heme / hemopexin complex during this step. Despite this, reasonably consistent recoveries of 61-71% were observed throughout this step. Recoveries at all other steps were high and consistent.

[0351] Taken together, this data indicates that process yields and recoveries were reasonably consistent across several batches of paste. Product quality was very high and consistent in all cases, with no significant differences evident in pastes subjected to Beriplex / Berinert adsorption. [Example]

[0352] Laboratory-scale large-scale manufacturing Laboratory-scale batch production was performed to obtain baseline data during Hemopexin process development and demonstrate process feasibility. Four laboratory-scale batches were produced at a 3 kg FIV-4 paste scale and processed to Hemopexin drug substance. Several modifications to the Hemopexin process and optimization of process conditions were performed on these batches.

[0353] Initial lab-scale batches Initial lab-scale batches were performed with an FIV-4 paste:buffer extraction ratio of 1:10. The extract was clarified by depth filtration and purified by chromatography using Capto MMC and Capto Adhere resins. Following two initial chromatography steps, the product was treated with solvents and detergents for viral inactivation, and the solvent and detergent reagents were removed via chromatography using Capto Q resin. The resulting process intermediate was subjected to viral filtration using a BioEX filter (lab-scale batch 2 only) before UF / DF into formulation buffer to yield hemopexin DS.

[0354] Two laboratory-scale batches have been successfully manufactured up to the drug substance stage.

[0355] Overall, the quality of the hemopexin purified from these initial laboratory-scale batches was extremely high, with heme specific activity exceeding 90%, hemopexin monomer content exceeding 95%, and contaminating proteins reduced to below detectable levels (Table 4). The quality of the final product was also very consistent between the two batches.

[0356] Optimized lab-scale batch After optimizing some process conditions, additional batches were produced to evaluate the complete process on a laboratory scale. Conditions were optimized for the FIV-4 extraction step (Example 4), and the filter frame depth was increased to 4 cm to allow for higher solids loading. Loading conditions were improved for the Capto MMC chromatography step (Example 4), the Capto Q chromatography step was replaced with Eshmuno CPS (Example 7), and various buffer conditions were optimized. A summary of the modified process conditions is shown in Table 5.

[0357] An optimized laboratory-scale batch was successfully manufactured to the drug substance stage, achieving a 73% hemopexin recovery throughout the process. Because a different batch of FIV-4 paste was used in this batch, the improved recovery over previous batches may be due to differences in the amount of hemopexin and heme / hemopexin complex present. It is also possible that recovery improved due to more efficient recovery from the clarification step through the use of a more appropriate frame size.

[0358] The resulting hemopexin drug substance had a heme-binding specific activity of 97%, a monomer content of greater than 99%, and the contaminating proteins albumin, transferrin, and haptoglobin were reduced to undetectable levels (Table 6). Overall, the optimized laboratory-scale batches demonstrated that the modified conditions resulted in good process recovery and a highly pure hemopexin product.

[0359] Streamlined lab-scale batches Following pilot-scale batch production (Example 14), further optimization was performed to streamline the process at scale. Increasing the extraction buffer NaCl concentration eliminated the need for conductivity adjustment prior to Capto MMC chromatography (Example 2). It was also discovered that Capto Adhere chromatography could be performed without adjusting the pH and conductivity of the Capto MMC eluate (Example 5).

[0360] To confirm that the process changes did not affect hemopexin purity and yield, a streamlined lab-scale batch was produced. The lab-scale batch was produced up to the Eshmuno CPS eluate stage, and hemopexin was eluted from this column at a lower NaCl concentration to provide material for viral filtration conductivity testing. The modified process conditions are summarized in Table 7.

[0361] An overall process recovery of 55% and a yield of 0.18 g / LPEQ were observed for the streamlined batch, which, although lower, is consistent with the yield and recovery of previous batches. Because a different batch of paste was used for this study, the amount of hemopexin and heme / hemopexin complex may have also played a role. Overall, it is unlikely that the streamlined conditions resulted in a decrease in recovery.

[0362] By SDS-PAGE analysis, hemopexin purity was extremely high after Capto MMC chromatography, with no visible contaminant bands after subsequent chromatography steps (Figure 26). Consistent with previous lab-scale batches, very high purity was achieved after Eshmuno CPS chromatography. [Example]

[0363] Pilot-scale batch manufacturing Pilot-scale batches were performed to demonstrate that the process was scalable and capable of producing consistent product intermediates and hemopexin drug substance. Additionally, these pilot-scale batches helped identify potential process issues that may arise in large-scale manufacturing.

[0364] Using the same batch of FIV-4 paste as the starting material, two pilot-scale batches were produced at a 20 kg FIV-4 paste scale. The process followed the parameters outlined in Figure 44, with the following exceptions: the paste was thawed at 4°C for 2.5 days before being extracted with a pH 6.4 buffer containing 225 mM NaCl. Due to limitations in maximum pump speed, the extract was split in half and clarified using a filter area appropriate for half the paste mass before the filtrate was recombined. Due to limited column hardware availability, the Capto Adhere and Eshmuno CPS columns were not large enough to accommodate all of the Capto MMC eluate. The Capto MMC column was loaded to capacity, which meant that only approximately 65% ​​of the Capto MMC eluate was carried through the remainder of the process, with the remainder discarded. Although wasteful, this allowed product quality to be assessed within capacity at every chromatographic step.

[0365] Viral filtration throughput was surprisingly low in both pilot-scale batches, only 47 and 70 L / m for batches 1 and 2, respectively. 2 This led to further development testing focused on filtration conditions, as discussed above in Example 10. Following this development testing, two additional large-scale batches were run under similar conditions to the pilot scale, using this material to verify virus filter throughput under optimized conditions. Essentially, these conditions were 2 650cm with BioEx virus filter 2 These included the use of a Sartopore XLM prefilter and a feed condition of 37 mS / cm conductivity. Under these conditions, all available material filtered easily with only moderate attenuation. The maximum throughput was 521 L / m for the first of these batches. 2 , and 232 L / m for the second one. 2The reason for the difference in the two throughputs remains unclear, but they are clearly a significant improvement over what was seen in the two earlier pilot-scale batches.

[0366] Pilot-scale intermediate and process recoveries Comparing the two pilot-scale batches, comparable recoveries were observed for all steps except for Capto Adhere chromatography (Table 11). The low recovery observed throughout this step in Batch B is presumably due to insufficient mixing of the product prior to sampling, leading to inaccurate concentration measurements. Similar errors were observed in the calculation of Eshmuno CPS recovery, where a value of 130% was calculated for the same batch. The similarity of overall process recoveries for the two batches also contradicts such variations in process recovery.

[0367] Approximately 80% recovery of hemopexin was observed for both batches throughout the depth filtration and clarification steps, and approximately 80% recovery of transferrin, albumin, and haptoglobin was observed for the first batch (Batch A) (Tables 12 and 13). This is consistent with a loss of product volume throughout the process, likely due to insufficient post-cleaning of the filter press. In the second pilot-scale batch, Batch B, variability in recovery of contaminant proteins was observed throughout the depth filtration and clarification steps, ranging from 60 to 90%. Sampling variability is suspected in this case.

[0368] The majority of contaminating proteins were removed in the Capto MMC step, with approximately 85% of total hemopexin recovered (Tables 11 and 12). At least a portion of the hemopexin loss is believed to be due to the presence of heme-hemopexin complexes in the clarified extract, which are not expected to bind to Capto MMC (Example 10). Analysis of the clarified extract used in the pilot-scale batch indicated that up to 20% of total hemopexin may be present as heme complexes. This could easily explain the majority of the hemopexin lost in this step.

[0369] Minimal protein or hemopexin loss was observed for the Eshmuno CPS chromatography, viral filtration, UF / DF and sterile filtration process steps, with approximately 100% recovery observed.

[0370] Overall process recovery of hemopexin was approximately 50-60%. As outlined above in Example 10, FIV-4 paste contains a portion of hemopexin in a complex with heme, which varies between paste batches. Because heme-hemopexin complexes are undesirable in the final product and are removed during the Capto MMC step of the process, it is more relevant to understand the recovery of free hemopexin. Considering the concentration of the complex, recovery of free hemopexin was approximately 70% for both pilot-scale batches, demonstrating high recovery of usable product.

[0371] In summary, the data outlined above demonstrate that reasonably consistent recoveries were achieved throughout each step of the process, and throughout the entire process when performed at pilot manufacturing scale.

[0372] This process produced 50 and 51 grams of purified hemopexin from two pilot-scale batches derived from the equivalent of 13 kg of FIV-4 paste (corrected for material discarded prior to Capto Adhere), equivalent to 0.15 grams of purified hemopexin per liter of plasma input.

[0373] Intermediate product quality at pilot scale The purity of the process intermediates was as expected for both pilot-scale batches, with most contaminating proteins removed in the Capto MMC step and the remaining transferrin almost completely removed in subsequent steps. The purity profiles of each process intermediate were nearly identical across the two batches.

[0374] Protease activity differed significantly between the two batches of extracted paste (Table 8). The high value seen in Batch A is presumably due to the presence of filter aid in the sample, resulting in protease activation during the time between sampling and analysis. This sample in Batch B was filtered immediately after sampling to prevent further activation by this mechanism. Nevertheless, in both batches, protease activity became very low after clarification and remained low for the remainder of the process.

[0375] Prekallikrein activator was found to be less than 20 IU / mL in the extracted paste and remained at this level throughout the process regardless of batch (Table 9).

[0376] Taken together, these data demonstrate that the process produces hemopexin of consistent purity at each step. Proteinaceous contaminants, proteases and PKA, are consistently removed and are at very low levels in the final drug substance, even at hemopexin concentrations of 100 mg / mL.

[0377] Characterization of hemopexin drug substance The pilot-scale process produced highly pure, active hemopexin that met or exceeded all quality standards as defined by the QTPP for Phase 1 manufacturing (Table 10). This is strongly indicated by the very high values ​​for specific activity, percent monomer, and purity by non-reducing SDS-PAGE (Figure 27). Specific proteinaceous contaminants, albumin, haptoglobin, transferrin, and apolipoprotein A1, as well as TnBP and polysorbate 80, were reduced to extremely low levels. Prekallikrein activator activity was reduced to acceptable levels, and residual protease activity was very low.

[0378] The two pilot-scale batches produced very similar Hemopexin DS with very similar results for all quality parameters. As the same batch of paste was used for both pilot-scale batches, this is a strong indication of the reproducibility of the process itself. [Example]

[0379] Manufacturing-scale purification process Step 1: FIV-4 paste extraction Hemopexin-containing fraction IV-4 (FIV-4) paste was resuspended in 40 mM sodium phosphate, 400 mM NaCl, pH 7.5 ± 0.1 (conductivity approximately 36 to approximately 38 mS / cm) at a paste:buffer ratio of 1:2.5 w / w by stirring at room temperature for approximately 20 to approximately 120 minutes before clarification.

[0380] Step 2 - Depth filtration and clarification of the extracted paste The FIV-4 extract paste solution was clarified by depth filtration to remove insoluble and particulate matter, and then further clarified through a 0.22 μm filter. Briefly, the FIV-4 extract paste solution was depth filtered through EK1P filter media using a STAX single-use disposable cartridge. The FIV-4 extract paste solution was then filtered at a flow rate of 6.25 L / m. 2The filter press was post-washed with up to 2.0 press volumes of Capto MMC equilibration buffer (40 mM sodium phosphate, 225 mM NaCl pH 6.4; conductivity ∼23 to ∼25 mS / cm) until the protein concentration was below 0.2 g / L. Following depth filtration, the pH of the depth-filtered extract was adjusted to pH 6.4 ± 0.1 with 0.5 M HCl, and the conductivity was adjusted to 27 mS / cm with 5 M NaCl or water for injection (WFI), as appropriate. The depth-filtered extract was then further clarified using a 0.5 μm or smaller filter, such as a Durapore® 0.2 μm filter. After filtration, the clarified extract solution was stored overnight at <23°C or at 2–8°C for up to 48 hours before proceeding with Capto MMC chromatography.

[0381] Step 3 - Mixed-mode chromatography using Capto MMC resin The clarified extract solution (27±2 mS / cm) obtained from the previous step was loaded onto a pre-equilibrated mixed-mode Capto MMC chromatography column to a target load of 14 g hemopexin / L resin. The hemopexin concentration of the clarified extract solution was determined by OD 280nm The loading capacity was estimated using a ratio of 1 / 3, and this value was used to calculate the loading capacity. After loading the product, the Capto MMC column was washed with equilibration buffer (40 mM sodium phosphate, 225 mM NaCl, pH 6.4; conductivity ∼23 to ∼25 mS / cm), after which the bound hemopexin was eluted. Upon completion of eluate collection, the Capto MMC column was regenerated.

[0382] Hemopexin was eluted from the column with 40 mM sodium phosphate, 150 mM NaCl, pH 7.5 (conductivity ∼18–∼19 mS / cm). Eluted hemopexin concentrations were typically 3–6 mg / mL, with purities of 70–85%. The Capto MMC eluate was stored below 23°C overnight or at 2–8°C for up to 48 hours before further purification by Capto Adhere chromatography.

[0383] Step 4 - Mixed-mode chromatography using Capto Adhere resin The Capto MMC eluate from the previous step (typically pH 7.2, conductivity approximately 18-19 mS / cm) was loaded onto a pre-equilibrated mixed-mode Capto Adhere chromatography column (Cytiva) to a target load of 30 g hemopexin / L resin. The unbound hemopexin fraction was collected, along with an additional 3-CV wash with equilibration buffer (40 mM sodium phosphate, 150 mM NaCl, pH 7.5, conductivity approximately 18-19 mS / cm) to collect the remaining hemopexin. Upon completion of the wash step, the Capto Adhere column was regenerated. The eluted hemopexin concentration was typically 1-3 mg / mL, with a purity greater than 90%. The Capto Adhere eluate was transferred directly to solvent / detergent treatment. If necessary, the eluate can be stored overnight at <23°C or for up to 7 days at 2-8°C before solvent / detergent treatment.

[0384] Step 5 - Capto Adhere Pooling and Concentration The flow-through from the Capto Adhere column (Capto Adhere effluent; conductivity ∼18 mS / cm) was concentrated by tangential flow filtration through a Millipore PES 10 kDa BioMax filter equilibrated with Capto Adhere equilibration buffer (40 mM sodium phosphate, 150 mM NaCl pH 7.5, conductivity ∼18–∼19 mS / cm) to reduce the volume for subsequent S / D treatment and Eshmuno CPS column loading steps. The Capto Adhere concentrate, with a hemopexin content of ∼20 mg / mL, was then stored at <23°C overnight or at 2–8°C for up to 7 days before solvent / detergent treatment.

[0385] Step 6 - Virus inactivation with solvents and detergents A solvent / detergent (SD) treatment step was used as the first viral reduction step in the hemopexin purification process. Briefly, a suspension of tri-n-butyl phosphate (TnBP) and polysorbate 80 (PS80) was added to the concentrated Capto Adhere effluent / eluate to achieve target concentrations of 0.3% w / w and 1.0% w / w, respectively, in the final product solution. The product solution containing TnBP and PS80 was incubated at 21–25°C for 4–24 h.

[0386] Step 7 - Ion Exchange Chromatography using Eshmuno CPS Resin The resulting solvent / detergent-treated solution (conductivity approximately 18-19 mS / cm) from the previous step was pH-adjusted to pH 6.0 ± 0.1 with 0.5 M acetic acid and diluted with PFW or WFI to a target conductivity of approximately 8.5-9.5 mS / cm. The adjusted product was loaded onto a pre-equilibrated Eshmuno CPS ion-exchange chromatography column to a target load of 40 g hemopexin / L resin. After loading the product, the column was further washed with equilibration buffer (25 mM sodium phosphate, 25 mM sodium acetate, 38 mM NaCl, pH 6.0; conductivity approximately 8.5-9.5 mS / cm) to remove SD and unbound proteins. Hemopexin was eluted with 20 mM sodium phosphate, 0.6 M NaCl (pH 7.2) (conductivity approximately 50-52 mS / cm), and the column was regenerated with a 1 M NaCl wash.

[0387] Step 8 - Nanofiltration for virus removal using Planova BioEX This step constitutes the second viral reduction step in the hemopexin manufacturing process. Briefly, the Eshmuno CPS eluate was filtered through a 0.1 μm prefilter and a 1 ml per 100 L Eshmuno CPS eluate. 2 The solution was filtered sequentially through Planova BioEX filters with a filter area of ​​1000 x 1000 mm.

[0388] The viral filtrate can be stored overnight below 23°C or for up to 7 days at 2-8°C before being processed by ultrafiltration and diafiltration.

[0389] Step 9 - Concentration and diafiltration of the BioEX filtrate The BioEX filtrate was concentrated to a membrane with a nominal molecular weight cutoff of 10 kDa or less and a membrane area of ​​0.01 m per L of BioEX filtrate. 2 The buffer was exchanged into formulation buffer (14.1 mM disodium phosphate, 0.9 mM citric acid, 150 mM NaCl, pH 7.2, conductivity approximately 15.5 mS / cm) using an ultrafiltration system (Millipore Pellicon 3, Biomax (PES)). The material was concentrated to a hemopexin protein concentration of 100 mg / mL.

[0390] Step 10 - Sterile filtration The bulk of Hemopexin UF was sterile filtered into a suitable sterile container using a sterile 0.22 μm sterilizing-grade filter at a pressure of less than 3 bar. This filtration is preferably performed aseptically in a laminar flow cabinet using sterile tubing and connectors. The sterile Hemopexin solution can be stored at 2-8°C or frozen at -80°C. [Example]

[0391] Conductivity In this study, solution conductivity throughout the hemopexin purification process was measured at ambient temperature (approximately 18°C ​​to 23°C) using a Thermo Fisher Orion Star A212 conductivity meter. Briefly, the purification process shown in Figure 30 was performed at a 1.5 kg scale using Fraction IV-4 plasma fraction, which was filtered using a filter press before loading onto Capto MMC resin. Two conductivity measurements were taken at each step, with simultaneous readings using two different probes: probe PD (with a cell constant of 0.4750) and recalibrated probe PP (with a cell constant of 0.4200). For buffers and steps requiring conductivity adjustment, multiple conductivity readings were taken.

[0392] Table 14 shows the range of buffer conductivities, and Table 15 shows the range of in-process conductivities.

[0393] The results showed comparable conductivity measurements with different cell constants. The conductivity measurements recommended for production were shown to be comparable through laboratory-scale experiments.

[0394] conclusion The strategy for developing a hemopexin purification process involved screening multiple candidate resins in both high-throughput and laboratory-scale configurations. From these screening studies, lead candidate resins were optimized for hemopexin purity, binding capacity, recovery, and operational simplicity. Viral clearance was achieved by nanofiltration using a solvent / detergent treatment, after optimizing the nanofiltration conditions.

[0395] Following this strategy, we developed a commercially viable method for the purification of hemopexin from Fraction IV-4 paste, which involves three chromatographic purification steps: Capto MMC, Capto Adhere, and Eshmuno CPS, with two viral inactivation steps: solvent / detergent treatment and nanofiltration.

[0396] Because the step with the lowest binding capacity is Capto MMC chromatography, process capacity is best expressed as a function of the size of this column, allowing for scale considerations. This process yields approximately 8-10 grams of pure hemopexin per liter of Capto MMC column volume, making it a high-capacity process. For a 250 L column size, this allows for processing of approximately 450 kg of FIV-4 paste per run.

[0397] Because the heme-hemopexin complex is not purified in this process, a proper estimation of process recovery requires knowledge of the amount of complex in the FIV-4 paste. This can be estimated by size exclusion chromatography or by Capto Adhere chromatography monitored at 414 nm. When the concentration of the complex is omitted, the recovery of active (unconjugated) hemopexin throughout the entire process was 70-72%. If recovery is calculated based on the total amount of hemopexin present (including the complex), recovery will vary depending on the concentration of the complex.

[0398] During development studies, yields ranged from 0.15 to 0.18 g / L PEQ. Similarly, yields per plasma equivalent volume varied depending on the amount of conjugate in the starting material.

[0399] The quality of hemopexin purified by this process is very high, typically measuring approximately 99% purity by non-reducing SDS-PAGE and approximately 99% monomer content. Protein activity is greater than 95%, and all trace contaminants are present at very low levels.

[0400] The process has been demonstrated to be highly reproducible and has been shown to be suitable for large-scale manufacturing by successful production of two pilot-scale manufacturing batches.

[0401] Overall, this process is well suited for the commercial production of high quality hemopexin, for example, as discussed in Example 16.

[0402] Table 1

[0403] Table 2

[0404] Table 3

[0405] Table 4

[0406] Table 5

[0407] Table 6

[0408] Table 7

[0409] Table 8

[0410] Table 9

[0411] Table 10

[0412] Table 11

[0413] Table 12

[0414] Table 13

[0415] Table 14

[0416] Table 15

Claims

1. 1. A method for purifying hemopexin from a solution containing hemopexin and other proteins, comprising: (i) providing a solution containing hemopexin and other proteins, wherein the solution contains less than about 300 mM sodium chloride (NaCl); (ii) passing the solution of step (i) through a mixed-mode cation exchange chromatography resin under conditions that promote the selective binding of hemopexin to the resin over the binding of other proteins to the resin; (iii) washing the resin after step (ii) to remove unbound proteins; (iv) eluting the resin-bound hemopexin after step (iii); (v) recovering the hemopexin eluted in step (iv); A method comprising:

2. The mixed mode cation exchange chromatography resin may be represented by formula (I) or (II): 【Chemistry 1】 The method of claim 1 having the structure:

3. 3. The method of claim 1 or claim 2, wherein the recovered hemopexin eluate of step (v) has a purity of at least about 50%.

4. 4. The method of claim 3, wherein the recovered hemopexin eluate of step (v) has a purity of about 70% to about 99%.

5. The method of any one of claims 1 to 4, wherein the solution of step (i) has a pH of about 6.2 to about 6.

6.

6. 6. The method of claim 5, wherein the solution of step (i) has a pH of about 6.

4.

7. 7. The method of any one of claims 1 to 6, wherein the solution in step (i) comprises about 160 mM to about 250 mM NaCl.

8. 8. The method of claim 7, wherein the solution in step (i) comprises about 200 mM to about 250 mM NaCl.

9. 9. The method of claim 8, wherein the solution in step (i) comprises about 225 mM NaCl.

10. The solution of step (i) is (a) a pH of about 6.2 to about 6.6; (b) about 20 mM to about 60 mM phosphate buffer; (c) about 160 mM to about 250 mM NaCl; The method according to any one of claims 1 to 9, comprising:

11. The solution of step (i) is (a) a pH of about 6.4; (b) about 40 mM phosphate buffer; (c) about 225 mM NaCl; The method of claim 10, comprising:

12. 12. The method of any one of claims 1 to 11, wherein the amount of hemopexin passed through the resin in step (ii) is from about 1 mg to about 40 mg per mL of resin.

13. 13. The method of any one of claims 1 to 12, wherein the solution of step (i) has a conductivity of from about 23 mS / cm to about 28 mS / cm.

14. 14. The method of claim 13, wherein the solution of step (i) has a conductivity of about 23 mS / cm to about 25 mS / cm.

15. The method of any one of claims 1 to 14, wherein the solution is a human plasma fraction.

16. 15. The method of claim 14, wherein the solution of step (i) is derived from Cohn fraction IV.

17. The solution of step (i) is Cohn fraction IV ４ 17. The method of claim 16, derived from

18. The solution of step (i) comprises: (a) resuspending Cohn fraction IV in an extraction buffer to obtain resuspended Cohn fraction IV; (b) passing the resuspended Cohn Fraction IV of step (a) through a filter; and (c) recovering the filtered Cohn Fraction IV extract from step (b).

18. The method of claim 16 or claim 17, wherein the compound is prepared by

19. 20. The method of claim 18, wherein step (a) comprises resuspending Cohn Fraction IV in extraction buffer at a Cohn Fraction IV:extraction buffer ratio of about 1:2 to about 1:

20.

20. 20. The method of claim 19, wherein step (a) comprises resuspending Cohn Fraction IV in extraction buffer at a Cohn Fraction IV:extraction buffer ratio of about 1:2.

5.

21. The method of any one of claims 18 to 20, wherein the extraction buffer has a pH of about 6 to about 8.

22. 22. The method of claim 21, wherein the extraction buffer has a pH of about 6.2 to about 7.

5.

23. 23. The method of claim 22, wherein the extraction buffer has a pH of about 7.

5.

24. 24. The method of any one of claims 18 to 23, wherein the extraction buffer comprises about 20 mM to about 500 mM NaCl.

25. 25. The method of claim 24, wherein the extraction buffer comprises about 400 mM NaCl.

26. 26. The method of claim 25, wherein the extraction buffer comprises about 40 mM sodium phosphate and about 400 mM NaCl.

27. 27. The method of any one of claims 18 to 26, wherein the filtered Cohn Fraction IV extract of step (c) is passed through a fine filter having a pore size of about 0.5 μm or less to obtain a clarified Cohn Fraction IV extract.

28. 28. The method of claim 27, wherein the pH of the clarified Cohn Fraction IV extract is adjusted to a value of about 6.2 to about 6.

6.

29. 29. The method of claim 28, wherein the pH of the clarified Cohn Fraction IV extract is adjusted to about 6.

4.

30. 30. The method of any one of claims 27 to 29, wherein the conductivity of the clarified Cohn Fraction IV extract is adjusted to a value of from about 24 mS / cm to about 30 mS / cm.

31. 31. The method of claim 30, wherein the conductivity of the clarified Cohn Fraction IV extract is adjusted to a value of about 26 mS / cm to about 28 mS / cm.

32. 32. The method of any one of claims 1 to 31, wherein the conductivity of the recovered hemopexin eluate of step (v) is from about 18 to about 19 mS / cm.

33. (vi) passing the recovered hemopexin eluate of step (v) through a mixed-mode anion exchange chromatography resin under conditions that allow impurities in the recovered hemopexin eluate to bind to the resin while allowing hemopexin to pass through the resin as an unbound fraction; (vii) recovering the unbound fraction containing hemopexin; The method of any one of claims 1 to 32, further comprising:

34. 34. The method of claim 33, wherein the mixed-mode anion exchange chromatography resin from step (vi) is equilibrated with a buffer comprising a pH of about 7.0 to about 8.

0.

35. 35. The method of claim 33 or claim 34, wherein the equilibration buffer has a pH of about 7.

5.

36. 36. The method of any one of claims 33 to 35, wherein the equilibration buffer comprises about 100 mM to about 200 mM NaCl.

37. 37. The method of claim 36, wherein the equilibration buffer comprises about 150 mM NaCl.

38. 38. The method of any one of claims 33 to 37, further comprising exposing the recovered unbound fraction of step (vii) to a virus inactivation step to obtain a virus-inactivated hemopexin solution.

39. 39. The method of claim 38, wherein the virus inactivation step comprises exposing the recovered unbound fraction of step (vii) to a solution comprising a detergent and a solvent.

40. 40. The method of claim 39, wherein the solvent is tri-n-butyl phosphate (TnBP).

41. 41. The method of claim 39 or claim 40, wherein the surfactant is polysorbate 80 (PS80).

42. 42. The method of claim 40 or claim 41, wherein the solvent and detergent treatment comprises exposing the recovered unbound fraction of step (vii) to 1% polysorbate 80 (PS80) and 0.3% tri-n-butyl phosphate (TnBP).

43. (ix) passing the virally inactivated hemopexin solution through an ion exchange chromatography resin under conditions that allow the hemopexin to bind to the resin; (x) optionally washing the resin after step (ix); (xi) eluting the hemopexin bound to the resin in step (ix); (xii) recovering the eluted hemopexin from step (xi); The method of any one of claims 38 to 42, further comprising:

44. 44. The method of claim 43, wherein the ion exchange chromatography resin is a cation exchange chromatography resin or an anion exchange chromatography resin.

45. 45. The method of claim 43 or claim 44, wherein prior to step (ix), the pH of the virally inactivated hemopexin solution is adjusted to a value of about 6.0 to about 6.

2.

46. 46. ​​The method of claim 45, wherein prior to step (ix), the pH of the virally inactivated hemopexin solution is adjusted to about 6.

0.

47. 47. The method of any one of claims 43 to 46, wherein prior to step (ix), the conductivity of the virally inactivated hemopexin solution is adjusted to a value of from about 8 mS / cm to about 12 mS / cm.

48. 47. The method of any one of claims 43 to 46, wherein prior to step (ix), the conductivity of the virus-inactivated hemopexin solution is adjusted to about 10 mS / cm.

49. 49. The method of any one of claims 43 to 48, further comprising exposing the eluted hemopexin recovered in step (xii) to diafiltration to adjust the concentration of the eluted hemopexin to a value of about 50 mg / mL to about 120 mg / mL.

50. 50. The method of claim 49, wherein the concentration of the eluted hemopexin is adjusted to about 100 mg / mL.

51. 51. The method of any one of claims 1 to 50, wherein the recovered hemopexin is subjected to viral filtration.

52. 52. The method of claim 51, wherein the viral filtration comprises passing the recovered hemopexin through a viral filter having a pore size of about 15 nm to about 20 nm in diameter.

53. A composition comprising hemopexin recovered by the method of any one of claims 1 to 52.

54. 1. A composition comprising: (a) a hemopexin content of about 95 mg / mL to about 110 mg / mL; (b) a heme-binding activity of about 1000 μM to about 2000 μM; (c) a heme-specific binding activity of at least about 80% of the total protein; (d) a CD91 dissociation constant (KD) of about 0.50 μM to about 2.0 μM; (e) a transferrin content of less than about 0.50 mg / mL; (f) an albumin content of less than about 0.05 mg / mL; (g) a haptoglobin content of less than about 0.05 mg / mL; (h) an apo-A1 content of less than about 0.10 mg / mL; (i) a high molecular weight (HMW) hemopexin aggregate content of less than about 1.0% of total protein as determined by size-exclusion high performance liquid chromatography; (j) a hemopexin monomer content of at least about 90% of total protein as determined by size exclusion high performance liquid chromatography; (k) a low molecular weight (LMW) impurity content of less than about 1.0% of total protein as determined by size exclusion high performance liquid chromatography; (l) a hemopexin purity content of at least about 80% of total protein as determined by reducing or non-reducing SDS-PAGE; (m) an isoelectric point (pI) of about 5.0 to about 6.5 as determined by capillary isoelectric focusing (cIEF); (n) a protease activity level of less than about 5 nKat / L; (o) a prekallikrein activity level of less than about 30 IU / mL; (p) a tri(n-butyl) phosphate (TnBP) content of less than about 10 μg / mL; and (q) a PS80 content of less than about 20 mg / mL A composition comprising a combination of any two or more of the features selected from the group consisting of:

55. 1. A composition comprising: (a) a hemopexin content of about 95 mg / mL to about 110 mg / mL; (b) a heme-binding activity of about 1600 μM to about 1800 μM; (c) a heme-specific binding activity of at least about 97% of the total protein; (d) a CD91 dissociation constant (KD) of about 1.10 μM to about 1.20 μM; (e) a transferrin content of less than about 0.25 mg / mL; (f) an albumin content of less than about 0.009 mg / mL; (g) a haptoglobin content of less than about 0.03 mg / mL; (h) an apo-A1 content of less than about 0.06 mg / mL; (i) a high molecular weight (HMW) hemopexin aggregate content of less than about 0.6% of total protein; (j) a hemopexin monomer content of at least about 99% of the total protein; (k) a low molecular weight (LMW) impurity content of less than about 0.4% of the total protein; (l) a hemopexin purity content of at least about 88% of total protein; (m) an isoelectric point (pI) of about 5.4 to about 6.3; (n) a protease activity level of less than about 3 nKat / L; (o) a prekallikrein activity level of less than about 20 IU / mL; (p) a tri(n-butyl) phosphate (TnBP) content of less than about 5 μg / mL; and (q) a PS80 content of less than about 18 mg / mL A composition comprising a combination of any two or more of the features selected from the group consisting of:

56. 1. A composition comprising: (i) a hemopexin content of about 95 mg / mL to about 110 mg / mL; (ii) a hemopexin monomer content of at least about 99% of the total protein; and (ii) a heme-specific binding activity of at least about 97% of the total protein A composition comprising:

57. (i) a transferrin content of less than about 0.25 mg / mL; and (ii) a haptoglobin content of less than about 0.03 g / L 57. The composition of claim 56, further comprising:

58. 58. The composition of claim 56 or claim 57, further comprising no detectable amounts of apolipoprotein A1 and / or albumin.

59. (i) an albumin content of less than about 0.009 mg / mL; and (ii) an apo-A1 content of less than about 0.06 mg / mL 58. The composition of claim 56 or claim 57, further comprising:

60. 60. The composition of any one of claims 56 to 59, further comprising no detectable protease activity.

61. 61. A formulation comprising the composition of any one of claims 53 to 60 and a pharmaceutically acceptable carrier.

62. 62. The formulation of claim 61, comprising about 15 mM citrate phosphate buffer, about 150 mM NaCl, a pH of about 7.2, and a hemopexin concentration of about 100 mg / mL.

63. 63. A composition according to any one of claims 53 to 60 or a formulation according to claim 61 or 62, which is suitable for pharmaceutical administration after storage at 2°C to 8°C and / or at ambient temperature for 12 months.

64. 64. A method for treating a condition associated with hemolysis, comprising administering to a subject in need thereof a composition according to any one of claims 53 to 60 and 63 or a formulation according to any one of claims 61 to 63.

65. 65. The method of claim 64, wherein the condition is an acute hemolytic condition or a chronic hemolytic condition.

66. 66. The method of claim 65, wherein the condition is selected from the group consisting of hemolytic anemia, transfusion-induced hemolysis, hemolytic uremic syndrome, autoimmune disease, malarial infection, trauma, blood transfusion, open heart surgery using cardiopulmonary bypass, and burn injury, hemoglobinemia and hemoglobinuria with post-burn hemolysis.

67. 66. The method of claim 65, wherein the condition is selected from the group consisting of sickle cell anemia, hereditary spherocytosis, hereditary elliptocytosis, thalassemia, congenital dyserythroid anemia and paroxysmal nocturnal hemoglobinuria, systemic lupus erythematosus, and chronic lymphocytic leukemia.

68. 64. Use of a composition according to any one of claims 53 to 60 and 63 in the manufacture of a medicament for treating a condition associated with hemolysis.

69. 69. The use of claim 68, wherein the condition is selected from the group consisting of hemolytic anemia, transfusion-induced hemolysis, hemolytic uremic syndrome, autoimmune diseases, malarial infections, trauma, blood transfusion, open cardiac surgery using cardiopulmonary bypass, and burn injury, hemoglobinemia and hemoglobinuria with post-burn hemolysis.

70. 69. The use of claim 68, wherein the condition is selected from the group consisting of sickle cell anemia, hereditary spherocytosis, hereditary elliptocytosis, thalassemia, congenital dyserythroid anemia and paroxysmal nocturnal hemoglobinuria, systemic lupus erythematosus and chronic lymphocytic leukemia.

71. A composition according to any one of claims 53 to 60 and 63 or a formulation according to any one of claims 61 to 63 for use in treating a condition associated with hemolysis.

72. 72. The composition for use of claim 71, wherein the condition is selected from the group consisting of hemolytic anemia, transfusion-induced hemolysis, hemolytic uremic syndrome, autoimmune diseases, malarial infection, trauma, blood transfusion, open heart surgery using cardiopulmonary bypass, and burn injury, hemoglobinemia and hemoglobinuria with post-burn hemolysis.

73. 72. The composition for use of claim 71, wherein the condition is selected from the group consisting of sickle cell anemia, hereditary spherocytosis, hereditary elliptocytosis, thalassemia, congenital dyserythroid anemia and paroxysmal nocturnal hemoglobinuria, systemic lupus erythematosus and chronic lymphocytic leukemia.

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