Method for purifying heme-binding proteins using mixed-mode chromatography

Abstract

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

Description

Technical Field

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

[0002] Hemolysis is characterized by the destruction of red blood cells and is a hallmark of anemias associated with red blood cell abnormalities such as enzyme deficiencies, hemoglobinopathies, hereditary spherocytosis, paroxysmal nocturnal hemoglobinuria, and acanthocytic anemia, as well as external 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., Streptococcus, Enterococcus, and Staphylococcus), parasitic infections (e.g., Plasmodium), toxins, and trauma (e.g., burns). Hemolysis is also a common condition in patients who have received blood transfusions, particularly massive transfusions, and who use extracorporeal cardiopulmonary support.

[0003] The side effects observed in patients with conditions associated with hemolysis are mainly attributed to the release of iron and iron-containing compounds (such as hemoglobin (Hb) and heme) from erythrocytes. Under physiological conditions, the released hemoglobin is bound by soluble proteins such as haptoglobin and transported to macrophages and hepatocytes. However, when the incidence of hemolysis accelerates and becomes pathological in nature, the buffering capacity of haptoglobin becomes overwhelmed. As a result, hemoglobin is rapidly oxidized to methemoglobin, which in turn releases free heme (comprising protoporphyrin IX and iron). Although heme plays a key role (for example, as a part of essential proteins such as hemoglobin and myoglobin) in several biological processes, free heme is highly toxic. Free heme is the source of redox-active iron, which produces highly toxic reactive oxygen species (ROS) that damage lipid membranes, proteins and nucleic acids. The toxicity of heme is further exacerbated by its ability to embed into lipid membranes, where it causes oxidation of membrane components and promotes cell lysis and death.

[0004] The evolutionary pressure of sustained low-level extracellular Hb / heme exposure has led to compensatory mechanisms that control the adverse 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 Hb-scavenging proteins, haptoglobin and heme-scavenging proteins, hemopexin, and α1-microglobulin. However, although endogenous haptoglobin and hemopexin control the adverse effects of free Hb / heme under physiological steady-state conditions, they play little role 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 (Lin et al., 2016, Molecular Medicine, 22: 22-31), anti-inflammatory and pro-inflammatory activity, inhibition of cell adhesion, and binding to certain divalent metal ions. In addition, hemopexin infusion has been shown to reduce 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 the expected market demand will require high-capacity purification methods. However, previously developed methods for purifying hemopexin from human plasma were limited to small-scale production methods for research purposes such as toxicology studies and Phase I clinical production (see, e.g., WO 2014 / 055552; Tsutsui and Mueller, 1981, Analytical Biochemistry, 121:244-250; and Muller-Eberhard, 1988, Methods in Enzymology, 163:563-565).

[0006] Therefore, there remains a need to develop improved methods for purifying hemopexin. SUMMARY OF THE INVENTION

[0008] In one aspect of the present invention, there is provided a method for purifying hemopexin from a solution containing hemopexin and other proteins, the method comprising:

[0009] (i) providing a solution comprising hemopexin and other proteins, wherein the solution comprises less than about 300 mM NaCl;

[0010] (ii) passing the solution of step (i) through a mixed-mode cation exchange chromatography resin under conditions that promote selective binding of hemopexin to the resin over binding of other proteins to the resin;

[0011] (iii) washing the resin after step (ii) to remove unbound protein;

[0012] (iv) eluting the hemopexin bound to the resin after step (iii); and

[0013] (v) recovering the hemopexin eluted in step (iv).

[0014] In one embodiment, the method further comprises:

[0015] (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; and

[0016] (vii) recovering the unbound fraction containing hemopexin.

[0017] In another aspect of the present invention, a composition comprising a hemopexin recovered by the methods disclosed herein is provided.

[0018] In another aspect of the present invention, a formulation comprising a composition disclosed herein and a pharmaceutically acceptable carrier is provided.

[0019] 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.

[0020] In another aspect of the present invention, a method of treating a disorder associated with hemolysis is provided, the method comprising administering to a subject in need thereof a composition or formulation disclosed herein.

[0021] In another aspect of the present invention, there is provided use of a composition or formulation disclosed herein in the preparation of a medicament for treating a disorder associated with hemolysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The mixed-mode anion exchange chromatography resin Capto Adhere is shown to separate hemopexin from other contaminating proteins. Photographs of non-reducing SDS-PAGE of fractions from a stepwise pH elution of mixed-mode anion exchange chromatography screens using (A) Capto Adhere (Cytiva), (B) HEA Hypercel (Pall), (C) PPA Hypercel (Pall), and (D) MEP Hypercel (Pall) are shown. Lanes 1 and 2 = flowthrough, 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.

[0024] Figure 2 The amount of protein (%; y-axis) in the solution extracted from the Fraction IV-4 paste is shown across the pH range (pH; x-axis) from pH 4 to pH 8. Line represented by t = transferrin, line represented by a = albumin, line represented by h = haptoglobin, line represented by H = hemopexin.

[0025] Figure 3 The consistency of FIV-4 paste extraction between different batches is shown. Graphical representation of hemopexin concentration (g / L; y-axis) and FIV-4 paste batches. Hemopexin concentration measured by reverse phase HPLC.

[0026] Figure 4 Shows that by using an extraction buffer with a higher pH, the yield of hemopexin from FIV-4 paste can be increased. Graphical representation of hemopexin concentration (g / L; y-axis) and FIV-4 paste batches (x-axis) after extraction at pH 6.2 (black bars) and 7.5 (grey bars).

[0027] Figure 5 Shown is the development and optimization of Capto MMC chromatography as an effective capture step for commercial purification of hemopexin. A series of photographs showing the 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, eluted with a stepwise NaCl gradient. Lane 1 = protein marker, lane 2 = load, lane 3 = drop-through, lane 4 = wash, lane 5 = 50mM NaCl, lane 6 = 150mM, lane 7 = 300mM NaCl, lane 8 = 500mM NaCl, lane 9 = 1M NaCl, lane 9 = hemopexin control.

[0028] Figure 6 Optimization of the loading and elution conditions for the Capto MMC chromatogram is shown. Photographic display of non-reducing SDS-PAGE analysis of fractions from a Capto MMC chromatogram loaded at pH 6.5, 200 mM NaCl. Lane 1 = protein marker (molecular weight standard), lane 2 = hemopexin standard, lane 3 = extract, lane 4 = drop-through, lane 5 = wash fraction, pH 6.5, 200 mM NaCl, lane 6 = wash fraction, pH 7.0, lane 7 = elution fraction, pH 7.5, 150 mM NaCl, lane 8 = elution fraction, pH 7.5, 500 mM NaCl, lane 9 = elution fraction, pH 7.5, 1 M NaCl.

[0029] Figure 7 Shown is the concentration of hemopexin (g / L; y-axis) in the unbound fraction from a Capto MMC column using different loading amounts (g / L resin; x-axis).

[0030] Figure 8 shows the purity of the hemopexin product obtained from a three-step chromatography workflow (Capto MMC, Capto Adhere, and Eshmuno CPS) after different loading conditions were applied to the Capto MMC step. (A) Photographic representation of non-reducing SDS-PAGE analysis of the Eshmuno CPS eluate of the hemopexin product obtained from different Capto MMC loading conditions. (B) Graphical representation of the purity (%) of the hemopexin measured by RP-HPLC at different stages of the workflow using Capto MMC loaded under different conditions. Circles represent the Capto MMC eluate, triangles represent the Capto Adhere eluate, and squares represent the Eshmuno CPS eluate.

[0031] Figure 9 Representative chromatogram of Capto MMC mixed-mode cation exchange chromatography showing protein concentration as UV absorbance (AU; y-axis) versus time (minutes; x-axis). The peak containing hemopexin is labeled.

[0032] Figure 10 The development and optimization of conditions for further purification of hemopexin by Capto Adhere chromatography is shown. The recovery (%; y-axis) of various proteins recovered 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 represented by t = transferrin, bars represented by a = albumin, bars represented by h = haptoglobin, and bars represented by H = hemopexin.

[0033] Figure 11 shows the development and optimization of conditions for further purification of hemopexin by Capto Adhere chromatography. Recovery (%; y-axis) of (A) hemopexin and (B) transferrin from the eluate of Capto MMC applied to a Capto Adhere mixed-mode anion exchange chromatography column loaded at various pH and NaCl conditions (x-axis).

[0034] Figure 12The purity of hemopexin obtained from Capto Adhere mixed-mode anion exchange chromatography of Capto MMC eluate is shown. 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.

[0035] Figure 13 Optimization of the hemopexin loading limit of a Capto Adhere mixed-mode anion exchange chromatography column is shown. Photographic representation of non-reducing SDS-PAGE analysis of unbound fractions from Capto Adhere chromatography. Lane labels indicate the amount of protein loaded per ml of resin.

[0036] Figure 14 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) measured by turbidimetry or RP-HPLC from eluates from Capto Adhere loaded under various pH and NaCl conditions (x-axis).

[0037] Figure 15 Representative chromatograms from a Capto Adhere mixed-mode anion exchange column showing protein recovery as UV absorbance (AU; y-axis) versus time (minutes; x-axis) Hemopexin is contained in the unbound fraction.

[0038] Figure 16 The kinetics of viral inactivation during solvent detergent incubation of Capto MMC eluates are shown. Titers (log 10 TCID 50 Graphical representation of volume (mL; y-axis) and time (minutes; x-axis).

[0039] Figure 17Purification of hemopexin by Eshmuno CPS chromatography is shown. Photographic representation of non-reducing SDS-PAGE analysis of fractions of SD-treated Capto Adhere eluate under various loading conditions. Lane 1 = Marker 12MW standard; Lane 2 = Thawed Capto MMC eluate; Lane 3 = Unbound fraction from Capto Adhere; Lane 4 = Eshmuno CPS feed; Lane 5 = Unbound fraction from Eshmuno CPS; Lane 6 = Eshmuno CPS 100mM NaCl eluate; Lane 7 = Eshmuno CPS 200mM NaCl eluate; Lane 8 = pH 8.0, 200mM NaCl eluate; and Lane 9 = pH 7.5, 150mM NaCl eluate.

[0040] Figure 18 The binding capacity of Eshmuno CPS resin is shown.Graphic representation of the amount of hemopexin (mg / mL; y-axis) in the unbound fraction from a 5 mL Eshmuno CPS column with different loadings (mg / mL resin; x-axis).

[0041] Figure 19 shows the robustness of the loading conditions of the Eshmuno CPS column. (A) Graphical representation of the recovery (%; y-axis) of hemopexin and transferrin at various pH and conductivity conditions (x-axis) as quantified by immunoturbidimetry. (B) Photographic representation of non-reducing SDS-PAGE analysis of eluates from an Eshmuno CPS column using different 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.

[0042] Figure 20 The effect of pH and conductivity on virus filtration is shown. Filter flux (L / m 2 ; y-axis) and time (minutes; x-axis).

[0043] Figure 21 shows the effect of the prefilter to virus filter area ratio on virus filtration. (A) Filter flux (L / m2) at prefilter to virus filter surface area ratios of 0.75:1 and 0.22:1 using a Sartopore 2XLM prefilter. 2 (B) Filter flux (L / m2) at prefilter to virus filter surface area ratios of 0.25:1 and 1.6:1 using a Virosart Max prefilter. 2 ; y-axis) and time (minutes; x-axis).

[0044] Figure 22 shows the effectiveness of the Asahi BioEX filtration step in removing Minor Virus of Mouse (MVM) from purified hemopexin. (A) Filter flux (L / m 2 ; y-axis) versus time (minutes; x-axis). (B) Viral titer of purified hemopexin spiked with MVM when filtered using AsahiBioEx filters.

[0045] Figure 23 Shown is the partitioning of the heme-hemopexin complex and hemopexin on a Capto MMC column. Graphical representation of absorbance (mAu; y-axis) and volume (mL; x-axis) of a 1:1 mixture of hemopexin and heme-hemopexin complex during chromatography on Capto MMC.

[0046] Figure 24 is a flow chart of a method for purifying hemopexin according to embodiments disclosed herein.

[0047] Figure 25 Shown are a series of photographic representations of non-reducing SDS-PAGE analysis of fractions from several batches of Fraction V paste (A), (B) and (C) hemopexin purification at a small laboratory scale.

[0048] FIG26 shows hemopexin recovery and purity following an efficient, streamlined, laboratory-scale batch of a hemopexin purification method according to embodiments disclosed herein. (A) Graphical representation of the step recovery (%; y-axis) for each chromatographic step (x-axis) of the efficient, streamlined, laboratory-scale batch. (B) Photographic representation of a non-reducing SDS-PAGE analysis of a process intermediate.

[0049] Figure 27 Characterization of hemopexin drug substance is shown. 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 composite of the two original gels.

[0050] Figure 28 FIG2 is a flow chart of the process steps prior to virus inactivation of a hemopexin purification method according to embodiments disclosed herein. Process intermediates are shown in bold text.

[0051] Figure 29 FIG2 is a flow chart of post-viral inactivation process steps of a hemopexin purification method according to embodiments disclosed herein. Process intermediates are shown in bold text.

[0052] Figure 30 is a flow chart of pre- and post-viral inactivation process steps of a hemopexin purification method according to embodiments disclosed herein.

[0053] Detailed description

[0054] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of stated elements or integers or groups of elements or integers but not the exclusion of any other elements or integers or groups of elements or integers.

[0055] Reference in this specification to any prior publication (or information derived therefrom) or any known content is not and should not be taken as an acknowledgement or approval or any form of suggestion that the prior publication (or information derived therefrom) or known content forms part of the common general knowledge in the field of the new attempt to which this specification relates.

[0056] It must be noted that, as used in this specification, the singular forms "a," "an," and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a resin" includes a single resin, as well as two or more resins; reference to "the composition" includes a single composition, as well as two or more compositions; and so on.

[0057] In the absence of any indication to the contrary, the "%" content mentioned in this specification should be understood to mean % w / w (weight / weight). For example, a solution comprising a hemopexin content of at least 80% of the total protein refers to a composition comprising a hemopexin content of at least 80% w / w of the total protein.

[0058] As used herein, "about" as applied to one or more values refers to a value similar to the reference value. In certain embodiments, unless otherwise specified or obvious from the context (unless such a number exceeds 100% of the possible value), the term "about" refers to a range of values that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in either direction (greater than or less than). In specific embodiments, the term "about" refers to ±10% of the stated value.

[0059] The present invention is based, at least in part, on the discovery that hemopexin can be purified from human plasma on a commercial scale. Thus, in one aspect of the invention, there is provided a method for purifying hemopexin from a solution containing hemopexin and other proteins, the method comprising:

[0060] (i) providing a solution comprising hemopexin and other proteins, wherein the solution comprises less than about 300 mM NaCl;

[0061] (ii) passing the solution of step (i) through a mixed-mode cation exchange chromatography resin under conditions that promote selective binding of hemopexin to the resin over binding of the other proteins to the resin;

[0062] (iii) washing the resin after step (ii) to remove unbound protein;

[0063] (iv) eluting the hemopexin bound to the resin after step (iii); and

[0064] (v) recovering the hemopexin eluted in step (iv).

[0065] Hemopexin (Hx) has been described as a 60-kD plasma β-1B glycoprotein consisting of a single 439-amino acid long peptide chain forming two domains connected by an interdomain linker. It has the highest known affinity for heme (K) of all characterized heme-binding proteins. d <1 pM) and binds heme at an equimolar ratio between the two Hx domains in the pocket formed by the interdomain linker.

[0066] Hemopexin represents a major line of defense against heme toxicity, at least in part due to its ability to bind heme with high affinity and act as a specific carrier of heme from the bloodstream to the liver. Hemopexin has also been reported to possess serine protease activity and several other functions, such as anti-inflammatory and pro-inflammatory activities, the ability to inhibit cell adhesion, and the binding of certain divalent metal ions.

[0067] Extraction and clarification

[0068] Any suitable material comprising hemopexin can be used to prepare the solution used according to the methods described herein. Suitable materials are known to those skilled in the art, and its illustrative examples include plasma fractions, such as various supernatants and precipitates obtained from the plasma fractionation process. In these processes, plasma is usually sequentially subjected to various physical purification methods (e.g., precipitation, filtration, and adsorption) to produce an intermediate product rich in certain proteins. Separating individual plasma proteins by fractionation process can be achieved by utilizing the following facts: different plasma proteins have different solubility, which depends on, for example, pH, temperature, and ionic strength, and (for example) different adsorption properties on different types of solid carriers. Suitable industrial-scale plasma fractionation process will be known to those skilled in the art, and its illustrative examples include using cold ethanol precipitation, following schemes such as Cohn / Oncley fractionation process or Kistler / Nitschmann fractionation process. Typical fractionation procedures are reviewed in Schultze and Heremans (Molecular Biology of human proteins. Vol. 1: Nature and Metabolism of Extracellular Proteins (Elsevier Publishing Company 1966), pp. 236-317). Illustrative examples of ethanol fractionation procedures, 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 entire contents of which are incorporated herein by reference. Illustrative examples of suitable fractions include those derived from Cohn fractions or Kistler-Nitschmann fractions or similar fractions, which are obtained from cold ethanol fractionation of blood-derived plasma. Also contemplated are fractions obtained from plasma fractionation procedures that do not include ethanol, illustrative examples of which include affinity purification (e.g., affinity chromatography or immunoaffinity) and other methods 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 comprising hemopexin is selected from the group consisting of plasma, cryo-poor plasma, IgG-depleted plasma, or Cohn fractions or Kistler-Nitchmann fractions or similar fractions obtained from cold ethanol fractionation of blood-derived plasma. In embodiments, the sample comprising hemopexin is selected from the group consisting of cryo-supernatant, 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 obtained from immunoglobulin purification methods are also contemplated herein. It will be understood by those skilled in the art that the solution comprising hemopexin may comprise other proteins, such as haptoglobin, transferrin, and heme-hemopexin complexes. In some embodiments, when proteins such as haptoglobin, transferrin, and / or the heme-hemopexin complex are present in a solution comprising hemopexin, it may be desirable to remove these proteins, such as by chromatographic separation, prior to performing the methods described herein.

[0069] In an embodiment, the solution comprising hemopexin is a human plasma fraction.

[0070] As described elsewhere herein, the methods disclosed herein can be used for commercial / industrial scale purification of hemopexin. When using the plasma fraction as starting material, on a commercial / industrial scale, the methods described herein can suitably include using the plasma fraction obtained from at least about 500 kg of plasma. Therefore, in embodiments, the plasma fraction is from at least about 500 kg of plasma, preferably from at least about 5,000 kg, preferably from at least about 7,500 kg, preferably from at least about 10,000 kg or preferably from at least about 15,000 kg of plasma. In another embodiment, on a commercial / industrial scale, the methods described herein can suitably include using a batch of Fraction IV-4 pastes from 21,000 kg of plasma, and optionally, multiple batches (2 or more, 3 or more, 4 or more, etc.) are combined into a single batch of starting material.

[0071] Those skilled in the art will understand that plasma for 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.

[0072] In an embodiment, the solution comprising hemopexin is derived from the Cohn fraction or an equivalent fraction from another plasma fractionation process. In an embodiment, the solution comprising hemopexin is a Cohn Fraction IV supernatant, a Cohn Fraction IV precipitate, or an equivalent from another plasma fractionation. In an embodiment, the solution comprising hemopexin is derived from a Fraction IV4 precipitate.

[0073] When the solution comprising hemopexin is derived from a precipitate (e.g., a fraction IV4 precipitate), the precipitate may be suitably stored prior to purification of the hemopexin according to the methods disclosed herein. Suitable storage conditions are known to those skilled in the art, illustrative examples of which include freezing the hemopexin-containing precipitate at -20°C, -80°C, or using liquid nitrogen prior to re-dissolving. Thus, in some embodiments, the precipitate comprising hemopexin is frozen Cohn fraction IV. In particularly preferred embodiments, the solution comprising hemopexin is derived from a frozen fraction IV4 precipitate.

[0074] Those skilled in the art will appreciate that prior to practicing the methods disclosed herein, the frozen precipitate containing hemopexin is thawed. Thawing of such frozen precipitate can be performed at any temperature, preferably at a temperature in the range of about 2 to 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.

[0075] It will be understood that when the starting material is provided in the form of a precipitate (e.g., a Fraction IV4 precipitate), the precipitate must be dissolved or resuspended to provide a suitable starting solution containing hemopexin for use in the methods described herein. In embodiments, the solution containing hemopexin is prepared by the following steps: (a) resuspending the starting material containing hemopexin in an extraction buffer to obtain a solution of resuspended or dissolved hemopexin, (b) passing the resuspended hemopexin solution of step (a) through a filter, and (c) recovering the solution containing hemopexin from step (b). In embodiments, the starting material containing hemopexin is Cohn Fraction IV. In one embodiment, Cohn Fraction IV is Cohn Fraction IV4. In one embodiment, Cohn Fraction IV is a Cohn Fraction IV4 precipitate.

[0076] The extraction buffer used to resuspend the hemopexin-containing starting material may comprise any suitable reagent or combination of reagents capable of solubilizing or resuspending the hemopexin present in the starting material while also providing a matrix compatible with, for example, clarification and further downstream purification of the hemopexin.

[0077] In embodiments, the extraction buffer comprises 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, 240mM, 250mM, 260mM, 270mM, 280mM, 290mM, 300mM, 310mM, 320mM, 330mM, 340mM, 350mM, 360mM, 37 0mM, 380mM, 390mM, 400mM, 410mM, 420mM, 430mM, 440mM, 450mM, 460mM, 470mM, 480mM, 490mM or 500mM NaCl).

[0078] Thus, in embodiments, the extraction buffer comprises 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, preferably about 260 mM 480mM, preferably about 490mM or preferably about 500mM NaCl.

[0079] In embodiments, the extraction buffer comprises about 400 mM NaCl.

[0080] In embodiments, the extraction buffer comprises a buffer of about 20mM to about 60mM. Suitable buffers are familiar to those skilled in the art, and illustrative examples include sodium phosphate. In embodiments, the extraction buffer comprises a sodium phosphate of about 20mM to about 60mM (e.g., about 20mM, 30mM, 40mM, 50mM, 60mM). In embodiments, the extraction buffer comprises a sodium phosphate of about 30mM to about 50mM. In embodiments, the extraction buffer comprises about 20mM sodium phosphate. In embodiments, the extraction buffer comprises about 30mM sodium phosphate. In embodiments, the extraction buffer comprises about 40mM sodium phosphate. In embodiments, the extraction buffer comprises about 50mM sodium phosphate. In embodiments, the extraction buffer comprises about 60mM sodium phosphate.

[0081] In one embodiment, the extraction buffer comprises about 40 mM sodium phosphate (Na2HPO4 / NaH2PO4) and about 400 mM NaCl.

[0082] In embodiments, 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).

[0083] Thus, in embodiments, 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.

[0084] In embodiments, the extraction buffer has a pH of about 6.2 to about 7.5.

[0085] In another embodiment, the extraction buffer has a pH of about 7.5.

[0086] The conductivity of the extraction buffer can 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, about 45 mS / cm). Thus, in embodiments, the conductivity of the extraction buffer is about 30 mS / cm to about 45 mS / cm. In embodiments, the conductivity of the extraction buffer is about 31 mS / cm to about 44 mS / cm. In embodiments, the conductivity of the extraction buffer is about 32 mS / cm to about 43 mS / cm. In embodiments, the conductivity of the extraction buffer is about 33 mS / cm to about 42 mS / cm. In embodiments, the conductivity of the extraction buffer is about 35 mS / cm to about 41 mS / cm. In embodiments, the conductivity of the extraction buffer is about 35 mS / cm to about 40 mS / cm. In embodiments, the conductivity of the extraction buffer is about 35 mS / cm to about 39 mS / cm. In embodiments, the extraction buffer has a conductivity of about 42 mS / cm. Suitable methods for determining (measuring) solution conductivity, including those described herein, will be familiar to those skilled in the art, an illustrative example of which includes using a Thermo Fisher Orion Star A212 conductivity meter. The conductivity of the extraction buffer can be measured at any suitable temperature, preferably at ambient temperature, for example, at 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 embodiments, the conductivity is measured at ambient temperature. In embodiments, the conductivity is measured at a temperature of about 18°C to about 25°C.

[0087] Resuspension of the hemopexin in the extraction buffer can be suitably achieved by mixing the material comprising the hemopexin and the extraction buffer for a period of time and under conditions suitable for achieving resuspension (i.e., solubilization) of the hemopexin. In embodiments, the material comprising the hemopexin and the extraction buffer are mixed for a period of time from 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 material comprising hemopexin and the extraction buffer are mixed for a period of ≥ 120 minutes, e.g., 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).

[0088] In embodiments, the material comprising hemopexin and the extraction buffer are mixed for at least about 20 minutes. In preferred embodiments, the material comprising hemopexin and the extraction buffer are mixed (ie, stirred) for at least about 60 minutes.

[0089] The mixing of the material comprising hemopexin and the extraction buffer can be achieved using any method known to those skilled in the art, illustrative examples of which include stirring, vortexing, shaking, rotating, oscillating, and any other suitable form of agitation. In an embodiment, the material comprising hemopexin and the extraction buffer are mixed by vortexing. According to this embodiment, the material comprising hemopexin and the extraction buffer are vortexed to a liquid depth of about 5% to about 20% (e.g., about 5%, 10%, 15%, or 20% of the liquid depth). In a preferred embodiment, the material comprising hemopexin and the extraction buffer are vortexed to a liquid depth of about 10%.

[0090] In embodiments disclosed herein, a solution comprising hemopexin is prepared by a process comprising mixing the hemopexin in a ratio of 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 1:12. The hemopexin-containing material is resuspended in extraction buffer at a ratio of hemopexin-containing material to extraction buffer of 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.

[0091] In embodiments, a solution comprising hemopexin is prepared by a process comprising resuspending the material comprising hemopexin in extraction buffer at a ratio of about 1:2.5 of hemopexin-containing material to extraction buffer. In embodiments, the starting material comprising hemopexin is Cohn Fraction IV. In embodiments, Cohn Fraction IV is Cohn Fraction IV4. In embodiments, Cohn Fraction IV is Cohn Fraction IV4 precipitate.

[0092] In embodiments, the resuspended hemopexin solution is passed through a depth filter.

[0093] In embodiments, the depth filter is a cellulose depth filter (e.g., 3M TM 90SP Zeta Plus TM 、Pall TM EK1P TM 、ErtelAlsop TM 953P TM 、Cytiva TM Stax TM Depth filter, 3M TM 70CA Zeta Plus TM Lens-shaped depth filter).

[0094] Optimization of filter flux can be achieved by adjusting any one or more of, for example, filter area, frame depth, and flow-through pressure. One skilled in the art will appreciate that any adjustment to filtration parameters may alter the clarity, flux, and viscosity of the clarified solution containing hemopexin.

[0095] In embodiments, the filter throughput is about 50 L / m 2 About 200L / m 2 In embodiments, the filter throughput is about 100 L / m 2 .

[0096] In embodiments, the filter area is about 0.5 m 2 / 9kg to about 2m 2 / 9 kg of Cohn fraction IV-4 precipitate. In embodiments, the filter area is about 1 m 2 / 9 kg of Cohn fraction IV-4 precipitate.

[0097] In another embodiment, the filter area is less than about 1 m 2 / 9 kg Cohn fraction IV-4 precipitate (i.e., about 0.0312 m 2 / L Cohn Fraction IV extract). Those skilled in the art will appreciate that when the filter area is reduced to less than about 1 m 2 When the precipitate is 1 kg / 9 kg Cohn Fraction IV-4, other filtration parameters may also need to be adjusted accordingly to maintain energy, for example, increasing the frame depth / volume due to the amount of solids present.

[0098] In an embodiment, the frame depth is from about 1 cm to about 10 cm, preferably from about 2 cm to about 6 cm, more preferably from about 3 cm to about 5 cm. In an embodiment, the frame depth is 4 cm.

[0099] Further increases in filter throughput can be achieved by pre-coating (i.e., pre-rinsing) the depth filter with a filter aid (e.g., Celpure C1000) under conditions and in an amount sufficient to uniformly coat the filter. If necessary, the depth filter can be washed with a suitable buffer prior to pre-coating. In embodiments, the depth filter comprises a filter aid. In some embodiments, the depth filter does not comprise a filter aid. In embodiments, the buffer is an extraction buffer described elsewhere herein (e.g., 40 mM sodium phosphate, 400 mM NaCl, pH 7.2).

[0100] In embodiments, the amount of filter aid is sufficient to provide a precoat of about 2 mm, e.g., about 0.625 kg / m 2In an embodiment, the flush volume pre-coated with the filter aid is about 1 times the press volume. The flow rate of the filter aid is set to provide uniform coating of the filter and prevent the filter aid from pooling at the bottom of the frame. In an embodiment, the flow rate is about 6.25 L / m 2 In embodiments, the filter aid is applied to the filter under pressure (eg, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 bar).

[0101] In embodiments, step (b) comprises passing the resuspended hemopexin solution of step (a) through the filter at a flow rate and pressure sufficient to prevent clogging of the filter and maintain flux and clarity. In embodiments, the flow rate is 6.25 L / m 2 / min. In an 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.

[0102] After filtration, the depth filter can be washed to optimize the recovery of hemopexin, i.e., post-wash. Suitable post-wash solutions and conditions are known to those skilled in the art. In embodiments, the post-wash solution is an equilibrium buffer (e.g., 40 mM sodium phosphate, 225 mM NaCl, pH 6.4) described elsewhere herein. In embodiments, the flush volume of the post-wash solution sufficient to maximize recovery of hemopexin from the filter is 2.5 times the ballast volume. In embodiments, the flush volume is approximately 3.0 times the ballast volume.

[0103] In embodiments, the pH of the solution comprising 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).

[0104] Thus, in embodiments, the pH of the solution comprising hemopexin is adjusted to 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.

[0105] In embodiments, the pH of the solution comprising hemopexin is adjusted to about 6.4 (ie, 6.4 ± 0.1).

[0106] The pH of the solution containing hemopexin can be adjusted with any suitable acidic solution known to those skilled in the art, illustrative examples of which include hydrochloric acid (HCl).

[0107] The conductivity of the solution containing hemopexin can be suitably 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 solution containing hemopexin can also be suitably 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).

[0108] Thus, in embodiments, the conductivity of the solution comprising hemopexin 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 embodiments, the conductivity of the solution comprising hemopexin 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 embodiments, the conductivity of the solution comprising hemopexin is about 27 mS / cm. In another embodiment, the conductivity of the solution comprising hemopexin 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.

[0109] In another embodiment, the conductivity of the solution comprising hemopexin is about 20 mS / cm to about 28 mS / cm. In another embodiment, the conductivity of the solution comprising hemopexin is about 20 mS / cm to about 26 mS / cm. In embodiments, the conductivity of the solution comprising hemopexin is about 21 mS / cm to about 26 mS / cm. In embodiments, the conductivity of the solution comprising hemopexin is about 22 mS / cm to about 26 mS / cm. In embodiments, the conductivity of the solution comprising hemopexin is about 23 mS / cm to about 26 mS / cm. In embodiments, the conductivity of the solution comprising hemopexin is about 22 mS / cm to about 25 mS / cm. In embodiments, the conductivity of the solution comprising hemopexin is about 23 mS / cm. In embodiments, the conductivity of the solution comprising hemopexin is about 24 mS / cm. In embodiments, the conductivity of the solution comprising hemopexin is about 25 mS / cm. The conductivity of the solution can be measured at any suitable temperature, preferably at ambient temperature, for example, at 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 embodiments, the conductivity is measured at ambient temperature. In embodiments, the conductivity is measured at a temperature of about 18°C to about 25°C.

[0110] The solution containing hemopexin can be stored for future use.

[0111] In an embodiment, a solution comprising hemopexin is prepared by further filtering a Cohn fraction IV extract. For example, a fraction IV-4 (FIV-4) paste can be used as a starting material and resuspended in a solution comprising 40 mM sodium phosphate, 400 mM NaCl, pH 7.5 + / - 0.1 at a ratio of 2.5 kg of buffer per kg of paste. The solution is then filtered in a filter press using, for example, 1 m 2 The resuspended FIV-4 paste was filtered through 3M90SP zeta plus filter media with a filter area of 32 L of extraction paste solution and a frame depth of 4 cm. The filter may optionally be pre-coated with a filter aid such as Celpure C1000.

[0112] In embodiments, the solution comprising hemopexin is passed through a fine filter having a pore size of about 0.5 μm or less to obtain a clear solution comprising hemopexin.

[0113] In an embodiment, the fine filter has an area of about 10 cm 2 / L to about 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 In another embodiment, the filter area of the fine filter is about 33 cm 2 / L deep filtrate.

[0114] In an 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 <1.5 bar.

[0115] The clear solution comprising hemopexin may be stored for future use. In embodiments, the clear solution comprising hemopexin may be stored at <23°C for up to about 24 hours.

[0116] In another embodiment, the clear solution comprising hemopexin can be stored at about 2°C to about 8°C for up to 48 hours.

[0117] Chromatographic purification of hemopexin

[0118] Protein purification by chromatography can be performed using axial flow chromatography columns (such as those available from Cytiva, Sartorius and Bio-Rad) or using radial flow chromatography columns (such as those available from Proxcys). Chromatography can also be performed using expanded bed techniques known to those skilled in the art.

[0119] Most chromatographic procedures utilize solid supports, also referred to herein interchangeably as resins or matrices. Suitable solid supports are familiar to those skilled in the art, and selection depends on the type of product to be purified. Examples of suitable solid supports include inorganic supports such as glass and silica gel, organic, synthetic or naturally occurring supports such as agarose, cellulose, dextran, polyamides, polyacrylamides, vinyl copolymers of difunctional acrylates and various hydroxylated monomers, and the like. Commercially available supports are sold under the following names: Eshmuno TM 、Nuvia TM , Sephadex TM 、Sepharose TM 、Hypercel TM 、Capto TM 、Fractogel TM 、MacroPrep TM 、UnosphereTM 、GigaCap TM 、Trisacryl TM , Ultrogel TM , Dynospheres TM 、Macrosorb TM and XAD TM Resin.

[0120] As described elsewhere herein, the present inventors have unexpectedly discovered that mixed-mode cation exchange chromatography resins can be used to selectively retain hemopexin.

[0121] Mixed-mode chromatography

[0122] Mixed-mode chromatography (MMC) is a chromatographic technique for separating proteins that is based on two or more forms of interaction (e.g., hydrophobic, hydrophilic, ionic) between the stationary phase and the proteins to be separated. For a given mixed-mode chromatography column, the dominant separation interaction will depend on the nature of the solution and the mobile phase conditions.

[0123] Those skilled in the art will appreciate that any mixed-mode cation exchange chromatography resin can be used to purify hemopexin from solution, as long as the hemopexin is able to bind to the chromatography resin while allowing some impurities in the solution to pass through the resin. Suitable resins are known to those skilled in the art. Examples of suitable mixed-mode cation exchange chromatography resins are resins comprising a ligand having the structure of formula (I) or (II):

[0124]

[0125] In embodiments, a mixed mode cation exchange chromatography resin comprises a ligand having the structure of Formula I (e.g., Capto MMC TM ).

[0126] In embodiments, a mixed-mode cation exchange chromatography resin comprises a ligand having the structure of Formula II (e.g., Nuvia cPrime TM ).

[0127] The chromatography step is typically performed under non-denaturing conditions and at a convenient temperature in the range of about 5°C to +30°C, more typically at about ambient temperature. The chromatography step can be performed batchwise or continuously, as convenient.

[0128] Optimization of the chromatographic performance of mixed-mode cation exchange chromatography resins can be achieved by adjusting variables such as pressure, temperature, column length, column bed height, height equivalent to a theoretical plate (HETP), and linear flow rate. Those skilled in the art will understand that any adjustment of these variables can alter the selective binding of hemopexin and other proteins to the chromatography column.

[0129] The height of the chromatographic column bed will vary according to the pressure and performance range of the specified product load and chromatographic column. In embodiments, the column bed height is approximately 15 cm (i.e., 15 ± 2 cm). For large-scale processes, the column bed height can be approximately 10 cm to approximately 25 cm.

[0130] 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 an embodiment, the column linear flow rate is about 120 cm / hr.

[0131] Before loading the solution comprising hemopexin, an equilibration buffer can be applied to the mixed mode chromatography column to ensure that the pH and conductivity are comparable to the solution comprising hemopexin (e.g., a clarified Cohn fraction IV extract). Suitable equilibration buffers are known to those skilled in the art, and illustrative examples thereof include wash buffers (e.g., 40 mM sodium phosphate, 225 mM NaCl, pH 6.4) described elsewhere herein. In embodiments, the volume of the equilibration buffer required for pre-equilibration is ≥ 1 column volume (CV). In embodiments, the pH of the mixed mode chromatography column after equilibrium is from 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 from 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). Thus, in embodiments, the conductivity of the equilibration buffer is from about 20 mS / cm to about 30 mS / cm, preferably from about 21 mS / cm to about 29 mS / cm, preferably from about 22 mS / cm to about 28 mS / cm, preferably from about 23 mS / cm to about 28 mS / cm, preferably from about 23 mS / cm to about 27 mS / cm, preferably from about 23 mS / cm to about 26 mS / cm, or more preferably from about 23 mS / cm to about 25 mS / cm. In embodiments, the conductivity of the equilibration buffer is about 20 mS / cm to about 30 mS / cm. In embodiments, the conductivity of the equilibration buffer is about 20 mS / cm to about 30 mS / cm. In embodiments, the conductivity of the equilibration buffer is about 20 mS / cm to about 30 mS / cm. In embodiments, the conductivity of the equilibration buffer is about 20 mS / cm to about 30 mS / cm. In embodiments, the conductivity of the equilibration buffer is about 23 mS / cm to about 28 mS / cm. In embodiments, the conductivity of the equilibration buffer is about 23 mS / cm to about 25 mS / cm. The conductivity of the equilibration buffer can be measured at any suitable temperature, preferably at ambient temperature, such as at 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 embodiments, the conductivity is measured at ambient temperature. In embodiments, the conductivity is measured at a temperature of about 18°C to about 25°C.

[0132] The present inventors have surprisingly discovered that solutions comprising 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 mixed-mode cation exchange chromatography resins to promote selective binding of hemopexin to the resin.

[0133] Thus, in embodiments, the solution comprising hemopexin comprises 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.

[0134] In an embodiment, the solution comprising hemopexin comprises about 160 mM to about 250 mM NaCl. In another embodiment, the solution comprising hemopexin comprises about 200 mM to about 250 mM NaCl. In a preferred embodiment, the solution comprising hemopexin comprises about 250 mM NaCl. In another embodiment, the solution comprising hemopexin comprises about 220 mM to about 230 mM NaCl. In a preferred embodiment, the solution comprising hemopexin comprises about 225 mM NaCl.

[0135] The inventors have also shown that solutions containing hemopexin and other proteins maintained at a pH below about 8 (e.g., 7, 6, 5, 4, and values therebetween) are optimal for loading mixed-mode cation exchange chromatography resins to promote selective binding of hemopexin to the resin.

[0136] Thus, in embodiments, the pH of the solution comprising hemopexin is less than about 7, preferably less than about 6.5, preferably less than about 6, preferably less than about 5.5, preferably less than about 5, preferably less than about 4.5 or preferably less than about 4.

[0137] In an embodiment, the solution comprising hemopexin has a pH of about 6.2 to about 6.6. In another embodiment, the solution of step (i) has a pH of about 6.4.

[0138] In an embodiment, the solution comprising hemopexin comprises:

[0139] (a) a pH of about 6.2 to about 6.6;

[0140] (b) about 20 mM to about 60 mM phosphate buffer; and

[0141] (c) about 160 mM to about 250 mM NaCl.

[0142] In another embodiment, the solution comprising hemopexin comprises:

[0143] (a) a pH of about 6.4;

[0144] (b) about 40 mM phosphate buffer; and

[0145] (c) About 225 mM NaCl.

[0146] In embodiments, the amount of hemopexin passed through the resin in step (ii) is from 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, 40 mg per mL of resin).

[0147] Thus, in embodiments, 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, 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 3 Preferably, the amount of hemopexin passed through the resin in step (ii) 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 embodiments, the amount of hemopexin passed through the resin in step (ii) is about 10 mg / mL resin to about 20 mg / mL resin. In embodiments, the amount of hemopexin loaded onto the resin in step (ii) is from 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, about 40 mg per mL of resin). In embodiments, the amount of hemopexin loaded onto the resin in step (ii) is from about 10 mg / mL resin to about 20 mg / mL resin.

[0148] Once hemopexin is bound to the mixed mode cation exchange chromatography resin, the resin can be washed to remove any residual impurities while maintaining hemopexin bound to the resin. Suitable washing solutions and conditions are well known to those skilled in the art. In embodiments, the washing solution comprises 40mM sodium phosphate, 225mM NaCl, and a pH of 6.4. In embodiments, the volume of the washing solution applied to the mixed mode cation exchange chromatography resin is about 3CV. If desired, the flow-through wash fractions can also be collected and stored for future use. The bound hemopexin can be eluted from the mixed mode cation exchange chromatography resin by methods well known to those skilled in the art.

[0149] Buffers suitable for eluting hemopexin from the resin are also known to those skilled in the art, illustrative examples of which include phosphates. In an embodiment, the elution buffer comprises 40 mM sodium phosphate at a pH of about 7.5.

[0150] In embodiments, the elution buffer further comprises about 100 mM to about 200 mM NaCl. This corresponds to an elution buffer having a conductivity range of about 10 mS / cm (100 mM NaCl) to about 18 mS / cm (200 mM NaCl). In specific embodiments, the elution buffer comprises about 140 to 160 mM NaCl. In embodiments, the elution buffer comprises about 150 mM NaCl. However, those skilled in the art will appreciate that the NaCl concentration of the elution buffer may depend on the composition of the protein applied to the column and may require adjustment beyond the stated limits to achieve the necessary recovery and purity of the hemopexin eluted from the resin.

[0151] In embodiments, the elution buffer comprises about 20mM to about 60mM sodium phosphate (e.g., about 20mM, 30mM, 40mM, 50mM, 60mM sodium phosphate). In embodiments, the extraction buffer comprises about 30mM to about 50mM sodium phosphate. In embodiments, the elution buffer comprises about 20mM sodium phosphate. In embodiments, the elution buffer comprises about 30mM sodium phosphate. In embodiments, the elution buffer comprises about 40mM sodium phosphate. In embodiments, the elution buffer comprises about 50mM sodium phosphate. In embodiments, the elution buffer comprises about 60mM sodium phosphate.

[0152] The conductivity of the elution buffer may suitably be from 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 embodiments, the conductivity of the elution buffer is from about 16 mS / cm to about 24 mS / cm, preferably from about 17 mS / cm to about 23 mS / cm, preferably from about 17 mS / cm to about 22 mS / cm, preferably from about 17 mS / cm to about 21 mS / cm, preferably from about 17 mS / cm to about 20 mS / cm, or more preferably from about 17 mS / cm to about 19 mS / cm. In embodiments, the conductivity of the elution buffer is from about 17 mS / cm to about 21 mS / cm. In embodiments, the conductivity of the elution buffer is about 17 mS / cm to about 20 mS / cm. In embodiments, the conductivity of the elution buffer is about 17 mS / cm to about 19 mS / cm. The conductivity of the elution buffer can be measured at any suitable temperature, preferably at ambient temperature, such as at 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 embodiments, the conductivity is measured at ambient temperature. In embodiments, the conductivity is measured at a temperature of about 18°C to about 25°C.

[0153] In some embodiments, it is advantageous that the volume of elution buffer applied to the mixed-mode cation exchange chromatography resin is sufficient to completely elute the bound hemopexin from the resin. However, in some cases, it may be sufficient to elute only a portion of the bound hemopexin from the resin. In embodiments, the volume of elution buffer applied to the mixed-mode cation exchange chromatography resin is about 3 CV.

[0154] In embodiments, collection of the hemopexin eluate begins after applying about 0.5 column volumes (CV) of elution buffer to the mixed mode cation exchange chromatography resin and continues until the elution peak is below A 280nm ≤50mAU (2mm path length).

[0155] In embodiments, the recovered hemopexin eluate will suitably have a purity (e.g., substantially pure) 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%).

[0156] Thus, in embodiments, the purity of the recovered hemopexin eluate is preferably 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 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%.

[0157] In embodiments, the recovered hemopexin eluate has a purity of about 70% to about 99%.

[0158] In embodiments, 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 embodiments, the conductivity of the recovered hemopexin eluate is about 17 mS / cm to about 21 mS / cm. In embodiments, the conductivity of the recovered hemopexin eluate is about 17 mS / cm to about 20 mS / cm. In embodiments, the conductivity of the recovered hemopexin eluate is about 17 mS / cm to about 19 mS / cm. In embodiments, the conductivity of the recovered hemopexin eluate is about 17 mS / cm. In embodiments, the conductivity of the recovered hemopexin eluate is about 18 mS / cm. In embodiments, the conductivity of the recovered hemopexin eluate is about 19 mS / cm. In embodiments, the conductivity of the recovered hemopexin eluate is about 20 mS / cm. In embodiments, the conductivity of the recovered hemopexin eluate is about 18 to about 19 mS / cm. The conductivity of the recovered hemopexin eluate can be measured at any suitable temperature, preferably at ambient temperature, such as at 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 embodiments, the conductivity of the recovered hemopexin eluate is measured at ambient temperature. In embodiments, the conductivity of the recovered hemopexin eluate is measured at a temperature of about 18°C to about 25°C.

[0159] In an embodiment, the recovered hemopexin eluate can be stored for future use. In an embodiment, the recovered hemopexin eluate can be stored at a temperature below about 23° C. for up to 48 hours. In another embodiment, the recovered hemopexin eluate can be stored at a temperature of about 2° C. to about 8° C. for at least 7 days.

[0160] In embodiments, 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 by further chromatographic purification. In embodiments, the recovered hemopexin eluate is further purified by ultrafiltration. In embodiments, 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.

[0161] Mixed-mode anion exchange chromatography

[0162] In embodiments, the methods described herein further comprise:

[0163] (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; and

[0164] (vii) recovering the unbound fraction containing hemopexin.

[0165] One skilled in the art will appreciate that any mixed-mode anion exchange chromatography resin can be used to further purify hemopexin from the recovered hemopexin eluate, so long as other proteins and impurities in the recovered hemopexin eluate are capable of binding to the chromatography resin while suitably allowing hemopexin from the recovered hemopexin eluate to pass through the resin. By evaluating a number of different mixed-mode anion exchange chromatography resins, the present inventors unexpectedly discovered that a mixed-mode anion exchange chromatography resin comprising an N-benzylmethylethanolamine ligand (e.g., Capto Adhere) TM ) are particularly suitable for further purification of hemopexin protein, including for commercial or industrial scale production. Thus, in embodiments, the mixed mode anion exchange chromatography resin comprises N-benzylmethylethanolamine ligand.

[0166] Optimization of the chromatographic performance of mixed-mode anion exchange chromatography resins can be achieved by adjusting variables such as temperature, column length, column bed height, height equal to a theoretical plate (HETP), and linear flow rate. Those skilled in the art will appreciate that any adjustment of such variables can alter the binding of impurities to the chromatography column or the elution of hemopexin in the unbound fraction.

[0167] In embodiments, the column bed height is about 15 cm (ie, 15 ± 2 cm).

[0168] In embodiments, the column linear flow rate is about 120 cm / hr.

[0169] The solution (also referred to herein as equilibrium buffer) suitable for mixed mode anion exchange resin equilibrium can comprise a concentration of about 10mM to about 200mM, preferably about 10mM to about 60mM, or more preferably a buffer of about 40mM. The pH of the equilibrium buffer can be in the range of 5 to about 9, and the conductivity of the equilibrium buffer can be suitably less than about 18mS / cm. In embodiments, the conductivity of the equilibrium buffer is about 16mS / cm to about 22mS / cm (e.g., about 16mS / cm, about 17mS / cm, about 18mS / cm, about 19mS / cm, about 20mS / cm, about 21mS / cm or about 22mS / cm). In embodiments, the conductivity of the equilibrium buffer is about 17mS / cm. In embodiments, the conductivity of the equilibrium buffer is about 18mS / cm. In embodiments, the conductivity of the equilibrium buffer is about 19mS / cm. In embodiments, the conductivity of the equilibrium buffer is about 20mS / cm. The conductivity of the equilibration buffer may be measured at any suitable temperature, preferably at ambient temperature, such as at 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 embodiments, the conductivity of the equilibration buffer is measured at ambient temperature. In embodiments, the conductivity of the equilibration buffer is measured at a temperature of about 18°C to about 25°C.

[0170] In embodiments, 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.

[0171] In embodiments, the pH of the equilibration buffer is about 7.5.

[0172] In embodiments, 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).

[0173] Thus, in embodiments, 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.

[0174] In another embodiment, the equilibration buffer comprises about 150 mM NaCl.

[0175] In embodiments, the volume of equilibration buffer required for pre-equilibration is ≥ 1 column volume (CV). In embodiments, the volume of equilibration buffer required for pre-equilibration is ≥ 3 CV. In embodiments, the volume of equilibration buffer required for pre-equilibration is about 3 CV. In embodiments, the pH of the effluent 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 embodiments, the conductivity of the effluent from the mixed-mode anion exchange chromatography resin after pre-equilibration is about 20 mS / cm.

[0176] Prior to step (vi), the concentration of hemopexin in the recovered hemopexin eluate may be suitably concentrated, for example, by passing the recovered hemopexin eluate through an ultrafiltration membrane. Thus, in embodiments, the concentration of hemopexin in the recovered hemopexin eluate is concentrated prior to step (vi). In embodiments, the concentration of hemopexin in the recovered hemopexin eluate is concentrated by passing the recovered hemopexin eluate through an ultrafiltration membrane. In embodiments, the concentration of hemopexin in the concentrated hemopexin eluate is from 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 from about 10 mg / mL to about 30 mg / mL, preferably from about 15 mg / mL to about 25 mg / mL, or more preferably about 20 mg / mL.

[0177] In embodiments, the amount of hemopexin passed through the resin in step (vi) is from 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 / L of resin.

[0178] The unbound fraction comprising 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 embodiments, the conductivity of the unbound fraction recovered is about 17 mS / cm to about 21 mS / cm. In embodiments, the conductivity of the unbound fraction recovered is about 17 mS / cm to about 20 mS / cm. In embodiments, the conductivity of the unbound fraction recovered is about 17 mS / cm. In embodiments, the conductivity of the unbound fraction recovered is about 18 mS / cm. In embodiments, the conductivity of the unbound fraction recovered is about 19 mS / cm. In embodiments, the conductivity of the unbound fraction recovered is about 20 mS / cm. The conductivity of the recovered unbound fraction can be measured at any suitable temperature, preferably at ambient temperature, for example, at 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 embodiments, the conductivity of the recovered unbound fraction is measured at ambient temperature. In embodiments, the conductivity of the recovered unbound fraction is measured at a temperature of about 18°C to about 25°C.

[0179] After the hemopexin eluate of the recovery of step (v) has passed through the resin in step (vi), the resin can be washed to ensure that all hemopexins are collected in the unbound hemopexin fraction. Suitable washing solutions and conditions are well known to those skilled in the art. In embodiments, the washing solution comprises 40mM sodium phosphate, 150mM NaCl, and pH is 7.5. In embodiments, the volume of the washing solution applied to the mixed mode anion exchange chromatography resin is about 3CV. In embodiments, the conductivity of the washing solution is about 16mS / cm to about 22mS / cm (e.g., about 16mS / cm, about 17mS / cm, about 18mS / cm, about 19mS / cm, about 20mS / cm, about 21mS / cm or about 22mS / cm). In embodiments, the conductivity of the washing solution is about 17mS / cm. In embodiments, the conductivity of the washing solution is about 18mS / cm. In embodiments, the conductivity of the washing solution is about 19mS / cm. In embodiments, the conductivity of the wash solution is about 20 mS / cm. The conductivity of the wash solution may be measured at any suitable temperature, preferably at ambient temperature, for example, at 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 embodiments, the conductivity of the wash solution is measured at ambient temperature. In embodiments, the conductivity of the wash solution is measured at a temperature of about 18°C to about 25°C.

[0180] In an embodiment, the collection of the unbound fraction comprising hemopexin begins at A 280nm ≥50 mAU (when measured at the column outlet; 2 mm path length) and ends at A 280nm ≤50 mAU (when measured at the column outlet; 2 mm path length).

[0181] In an embodiment, the unbound fraction comprising hemopexin can be stored for future use. In an embodiment, the unbound fraction comprising hemopexin can be stored at <23°C for up to 48 hours. In another embodiment, the unbound fraction comprising hemopexin can be stored at about 2°C to about 8°C for up to 7 days.

[0182] Virus inactivation

[0183] When hemopexin is used in clinical or veterinary applications (e.g., for administration to subjects with hemolysis-related disorders), it may be necessary to reduce the level of active viral content (i.e., viral titer) and other potential infectious agents (e.g., prions). This may be desirable when, for example, the raw materials (i.e., starting materials / solutions) comprising hemopexin and other proteins are derived from plasma. Methods for reducing viral titers in solutions are 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, virus filtration (e.g., passing the solution through a nanofilter, e.g., a cutoff of 20nm) and / or treating the solution with a suitable organic solvent and surfactant under conditions and for a period of time that inactivates the virus in the solution. Solvent detergents (SD) have been used to inactivate enveloped viruses, particularly enveloped viruses in plasma-derived products, for more than 20 years. Therefore, it can be carried out using various reagents and methods known in the art (see, for example, US 4540573 and US 4764369, which are hereby incorporated by reference). Suitable solvents include tri-n-butyl phosphate (TnBP) and ethers, preferably TnBP (usually about 0.3%). Suitable detergents include polysorbate (Tween) 80, polysorbate (Tween) 20 and Triton X-100 (usually about 0.3%). The selection of treatment conditions, including solvent and detergent concentration, depends in part on the characteristics of the raw material. Raw materials with lower purity generally require higher concentrations of reagents and more extreme reaction conditions. The preferred detergent is polysorbate 80, and a particularly preferred combination is polysorbate 80 and tri-n-butyl phosphate (TnBP). The raw material can be stirred with the solvent and detergent reagent at a temperature and time sufficient to inactivate any enveloped viruses that may be present. For example, the solvent detergent treatment can be carried out at 23±2°C for about 2 to 24 hours. The solvent detergent chemicals are subsequently removed by, for example, adsorption onto a chromatographic medium such as a C-18 hydrophobic resin or eluting them in the flow-through fraction of an ion exchange resin under conditions in which the protein of interest is adsorbed.

[0184] The virus inactivation step can be performed at any suitable stage of the method disclosed herein. In embodiments, after step (vii), the virus inactivation step is performed to the unbound fraction comprising hemopexin. Before the virus inactivation step, the unbound fraction comprising hemopexin recovered in step (vii) can be appropriately concentrated, for example, to minimize the product volume processed in subsequent processing steps (including virus inactivation). Therefore, in embodiments, the method described herein further comprises concentrating the unbound fraction comprising hemopexin recovered in step (vii). In embodiments, before the virus inactivation step, the unbound fraction comprising hemopexin recovered in step (vii) is concentrated. Suitable methods for concentrating the unbound fraction containing hemopexin recovered in step (vii) are familiar to those skilled in the art, illustrative examples of which include ultrafiltration / diafiltration using, for example, ultrafiltration / diafiltration membranes such as Millipore Pellicon 3 cassettes (PES), Pellicon 2 cassettes (Millipore) or polyethersulfone or Hydrosart cassettes (Sartorius) with Biomax. In an embodiment, the unbound fraction containing hemopexin recovered in step (vii) is concentrated by ultrafiltration.

[0185] The use of a viral inactivation step, such as a solvent detergent treatment after passing the partially purified hemopexin (i.e., the unbound fraction containing hemopexin) through a mixed-mode anion exchange chromatography resin, advantageously avoids interference with the purification by solvents and detergents and limits the number of purification steps that need to be performed. This is particularly advantageous for industrial-scale manufacturing.

[0186] In embodiments disclosed herein, the viral inactivation step comprises exposing the unbound fraction comprising hemopexin of step (vii) to a solution comprising a surfactant and a solvent.

[0187] In embodiments, the temperature of the unbound fraction comprising hemopexin is about 21°C to about 25°C (eg, 21°C, 22°C, 23°C, 24°C, or 25°C) prior to adding the solution comprising a surfactant and a solvent.

[0188] In embodiments, the solvent is tri-n-butyl phosphate (TnBP).

[0189] In an embodiment, the surfactant is polysorbate 80 (PS80).

[0190] In embodiments, 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).

[0191] The unbound fraction comprising hemopexin and the solution comprising a surfactant and a solvent can be incubated under a time and conditions suitable for achieving viral clearance. For example, any method known to those skilled in the art can be used to mix (i.e., stir) the unbound fraction comprising hemopexin and the solution comprising a surfactant and a solvent, illustrative examples of which include stirring, shaking, rotating, and oscillating. In an embodiment, the unbound fraction comprising hemopexin and the solution comprising a surfactant and a solvent are mixed by stirring. According to this embodiment, the unbound fraction comprising hemopexin and the solution comprising a surfactant and a solvent are stirred so that a vortex occurs at about 5% to about 20% of the liquid depth (e.g., about 5%, 10%, 15%, or 20% of the liquid depth). In an embodiment, the unbound fraction comprising hemopexin and the solution comprising a surfactant and a solvent are stirred with a vortex of about 5-10% of the liquid depth.

[0192] In an embodiment, the unbound fraction comprising hemopexin and the solution comprising a surfactant and a solvent are incubated at about 23° C. (i.e., 23° C. ± 2° C.). In another embodiment, the unbound fraction comprising hemopexin and the solution comprising a surfactant and a solvent are 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).

[0193] Virus inactivation, including virus inactivation as 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 embodiments, the low pH virus inactivation is performed in the presence of caprylate.

[0194] In another example, viral inactivation can be achieved by contacting the hemopexin-containing fraction with n-octyl-β-D-glucopyranoside (OG), thereby forming an OG-IgG mixture.

[0195] In another example, low pH viral inactivation is performed in the presence of N,N-dimethyltetradecylamine N-oxide (TDAO).

[0196] In another example, viral inactivation can be achieved by exposing the fraction containing hemopexin to a solvent-detergent inactivation step. Suitable solvent-detergent treatments are known to those skilled in the art, and illustrative examples include detergents, including biodegradable and / or environmentally friendly detergents. Exemplary biodegradable and / or environmentally friendly detergents suitable for use in viral inactivation steps, particularly for inactivating lipid enveloped viruses, include N,N-dimethyltetradecylamine N-oxide (TDAO), polysorbate 80 (PS80), polyoxyethylene (10) isooctylcyclohexyl ether ( X-100-reduced) and nonionic surfactants prepared from glucose and alcohol (e.g., Simulsol™ formulations). In one embodiment, the detergent is N,N-dimethylmyristamine N-oxide (TDAO). In another embodiment, the detergent is polysorbate 80. In another embodiment, the detergent is polyoxyethylene (10) isooctylcyclohexyl ether ( X-100-reduced). In yet another embodiment, the detergent is a nonionic surfactant prepared from glucose and alcohol.

[0197] The methods disclosed herein may also include a virus filtration step. For example, a virus filtration membrane having a pore size of about 15 nm to about 20 nm can be used to remove microorganisms and viruses from the solution. Illustrative examples of suitable nanofilters include Planova S20N (Asahi), Virosart HC (Sartorius), and Planova 20N (Asahi). In embodiments, the recovered hemopexin is subjected to virus filtration. In embodiments, the virus filtration comprises passing the recovered hemopexin through a virus filter having a pore size of about 15 nm to about 20 nm.

[0198] In embodiments, the methods described herein further comprise ultrafiltration / diafiltration of any of the solutions described herein comprising hemopexin. Illustrative examples of suitable ultrafiltration / diafiltration membranes include Pellicon 2 cassettes (Millipore) or polyethersulfone or Hydrosart cassettes (Sartorius).

[0199] Ion exchange chromatography

[0200] In embodiments, the methods described herein further comprise:

[0201] (ix) passing the virus-inactivated hemopexin solution through an ion exchange chromatography resin under conditions that allow hemopexin to bind to the resin;

[0202] (x) optionally washing the resin after step (ix); and

[0203] (xi) eluting the hemopexin bound to the resin in step (ix); and

[0204] (xii) Recovering the eluted hemopexin from step (xi).

[0205] Ion exchange chromatography is based on the attachment of amino acids (e.g., histidine) to positively or negatively charged functional groups, which allows proteins with a net negative charge (i.e., captured by positively charged anion exchange resins) or a net positive charge (i.e., captured by negatively charged cation exchange resins) to be retained on immobilized functional groups such as -N+(C2H5)2, -N+(CH3)3, -COO - or -SO3 - in the column.

[0206] In embodiments, 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 TM In an embodiment, the ion exchange chromatography resin is a cation exchange chromatography resin. In an embodiment, the ion exchange chromatography resin is an anion exchange chromatography resin.

[0207] Optimization of the chromatographic performance of ion exchange chromatography resins can be achieved by adjusting variables such as pressure, temperature, column length, column bed height, height equal to the theoretical plate (HETP), and linear flow rate. Those skilled in the art will understand that any adjustment of such variables can alter the selective binding of hemopexin to the resin.

[0208] In embodiments, the column bed height is from 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 preferably at least about 25 cm. In embodiments, the column bed height is about 15 cm (i.e., 15 ± 2 cm).

[0209] In embodiments, the column linear flow rate is from about 100 cm / hr to about 200 cm / hr, preferably from about 100 cm / hr to about 150 cm / hr, preferably from about 110 cm / hr to about 130 cm / hr, or more preferably from about 115 cm / hr to about 125 cm / hr. In embodiments, the column linear flow rate is about 120 cm / hr.

[0210] The ion exchange chromatography resin can be suitably balanced to prepare for the passage of the virally inactivated hemopexin solution in step (ix) as described herein. This can be suitably achieved by passing a suitable equilibration buffer through the ion exchange chromatography resin before step (ix). The pH of the equilibration buffer can suitably be, preferably, about 5 to 7, more preferably about 6. In embodiments, the equilibration buffer comprises a buffer of about 20mM to about 60mM. Suitable buffers are familiar to those skilled in the art, and illustrative examples thereof include sodium phosphate. In embodiments, the equilibration buffer comprises sodium phosphate of about 20mM to about 60mM (e.g., about 20mM, 30mM, 40mM, 50mM, 60mM). In embodiments, the equilibration buffer comprises sodium phosphate of about 30mM to about 50mM. In embodiments, the equilibration buffer comprises about 20mM sodium phosphate. In embodiments, the equilibration buffer comprises about 30mM sodium phosphate. In embodiments, the equilibration buffer comprises about 40mM sodium phosphate. The solution (i.e., pH balancing buffer) suitable for the pH value of balancing ion exchange chromatography resin is generally in the range of 5 to about 9. In embodiments, the pH balancing buffer comprises 40nM sodium phosphate, and the pH is 6.0. Other solutions (i.e., balancing buffer) for balancing the conductivity of ion exchange chromatography resin are also contemplated herein, and it is generally comprised that the buffer has a concentration of about 20mM to about 100mM, to obtain a conductivity of about 10mS / cm. In embodiments, the balancing buffer comprises 25mM sodium phosphate, 25mM sodium acetate, 38mM NaCl, and the pH is 6.0. In embodiments, the conductivity of the balancing buffer of ion exchange chromatography resin is about 8mS / cm to about 12mS / cm, preferably about 8mS / cm, preferably about 9mS / cm, preferably about 10mS / cm, preferably about 11mS / cm or preferably about 12mS / cm. In embodiments, 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 embodiments, the conductivity of the equilibration buffer for the ion exchange chromatography resin is about 8 mS / cm to about 10 mS / cm. In embodiments, 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 the equilibration buffer for the ion exchange chromatography resin can be measured at any suitable temperature, preferably at ambient temperature, such as at 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 embodiments, the conductivity of the equilibration buffer for the ion exchange chromatography resin is measured at ambient temperature. In embodiments, the conductivity of the equilibration buffer for the ion exchange chromatography resin is measured at a temperature of about 18°C to about 25°C.

[0211] In embodiments, the volume of equilibration buffer required for pre-equilibration is ≥ 1 column volume (CV). In embodiments, the pH of the effluent 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 embodiments, the conductivity of the effluent from the mixed-mode anion exchange chromatography resin after pre-equilibration is about 10 mS / cm.

[0212] In embodiments, prior to step (ix), the pH of the virally inactivated hemopexin solution is adjusted to about pH 5.9 to about 6.2 (eg, pH 5.9, 6.0, 6.1 or 6.2).

[0213] Thus, in embodiments, prior to step (ix), the pH of the virally inactivated hemopexin solution is adjusted to about 5.9 to about 6.2, preferably about 5.9, preferably about 6.0, preferably about 6.1 or preferably about 6.2.

[0214] In embodiments, prior to step (ix), the pH of the virally inactivated hemopexin solution is adjusted to about 6.0.

[0215] In embodiments, 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 embodiments, 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 embodiments, the conductivity of the virally inactivated hemopexin solution is about 8 mS / cm to about 10 mS / cm. In embodiments, 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 can be measured at any suitable temperature, preferably at ambient temperature, such as at 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 embodiments, the conductivity of the virus-inactivated hemopexin solution is measured at ambient temperature. In embodiments, the conductivity of the virus-inactivated hemopexin solution is measured at a temperature of about 18°C to about 25°C.

[0216] In embodiments, prior to step (ix), the conductivity of the virus-inactivated hemopexin solution is adjusted to about 10 mS / cm (ie 10 ± 2 mS / cm).

[0217] In embodiments, the amount of hemopexin passed through the resin in step (ix) is from 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 / L of resin.

[0218] Once the virally inactivated hemopexin solution has passed through the ion exchange chromatography resin, the flow-through fraction can be collected and stored for future use.

[0219] Can be by any method known to those skilled in the art from ion exchange chromatography resin eluting bound hemopexin.Before eluting hemopexin from resin, can optionally under the condition that keeps hemopexin and resin-bound, use suitable washing solution or buffer solution washing resin.Suitable washing buffer solution and condition are well known to those skilled in the art.The washing buffer solution concentration depends on the load of post to a certain extent, yet typical washing solution has buffering effect under the pH of about 6 to about 8.In one embodiment, washing buffer solution comprises 25mM sodium phosphate, 25mM sodium acetate and 38mM sodium chloride, and pH is 6.0.In embodiments, the volume of washing solution applied to the mixed mode anion exchange chromatography resin is about 3CV.If desired, can also collect and store the flow-through washing fraction for future use.

[0220] In embodiments, the bound hemopexin can be eluted from the ion exchange chromatography resin using an elution buffer comprising 20 mM sodium phosphate and 0.6 M sodium chloride, pH 7.2. In embodiments, the volume of the elution buffer applied to the ion exchange chromatography resin is about 3 CV. The elution buffer can 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). Therefore, in embodiments, the conductivity of the elution buffer is about 45 mS / cm to about 60 mS / cm. In embodiments, the conductivity of the elution buffer is about 47 mS / cm to about 56 mS / cm. In embodiments, the conductivity of the elution buffer is about 47 mS / cm. In embodiments, the conductivity of the elution buffer is about 48 mS / cm. In embodiments, the conductivity of the elution buffer is about 49 mS / cm. In embodiments, the conductivity of the elution buffer is about 50 mS / cm. In embodiments, the conductivity of the elution buffer is about 51 mS / cm. In embodiments, the conductivity of the elution buffer is about 52 mS / cm. In embodiments, the conductivity of the elution buffer is about 53 mS / cm. In embodiments, the conductivity of the elution buffer is about 54 mS / cm. In embodiments, the conductivity of the elution buffer is about 55 mS / cm. In embodiments, the conductivity of the elution buffer is about 56 mS / cm. The conductivity of the elution buffer can be measured at any suitable temperature, preferably at ambient temperature, such as at 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 embodiments, the conductivity is measured at ambient temperature. In embodiments, the conductivity of the elution buffer is measured at a temperature of about 18°C to about 25°C.

[0221] In an embodiment, the collection of the eluted hemopexin begins with A 280nm ≥50mAU (2mm path length), ending at A 280nm ≤50 mAU (when measured at the column outlet; 2 mm path length).

[0222] The eluted hemopexin recovered from the ion exchange chromatography resin may suitably 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 embodiments, the eluted hemopexin has a conductivity of about 35 mS / cm to about 50 mS / cm. In embodiments, the eluted hemopexin has a conductivity of about 35 mS / cm to about 45 mS / cm. In embodiments, the eluted hemopexin has a conductivity of about 40 mS / cm to about 45 mS / cm. In embodiments, the conductivity of the eluted hemopexin is about 40 mS / cm. In embodiments, the conductivity of the eluted hemopexin is about 41 mS / cm. In embodiments, the conductivity of the eluted hemopexin is about 42 mS / cm. In embodiments, the conductivity of the eluted hemopexin is about 43 mS / cm. In embodiments, the conductivity of the eluted hemopexin is about 44 mS / cm. In embodiments, the conductivity of the eluted hemopexin is about 45 mS / cm. The conductivity of the eluted hemopexin can be measured at any suitable temperature, preferably at ambient temperature, such as at 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 embodiments, the conductivity of the eluted hemopexin is measured at ambient temperature. In embodiments, the conductivity of the eluted hemopexin is measured at a temperature of about 18°C to about 25°C.

[0223] 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 <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.

[0224] The eluted hemopexin can be further purified as needed, for example, by concentrating and diafiltering the hemopexin through an ultrafiltration membrane and / or by sterile filtering the concentrated and / or diafiltered hemopexin. In some embodiments, the eluted hemopexin is subjected to virus removal.

[0225] Virus removal techniques based on size differences are known to those skilled in the art, illustrative examples of which include filtration and nanofiltration.

[0226] In embodiments, virus removal is performed by nanofiltration.

[0227] In embodiments, virus removal is performed by prefiltration in series with a virus filter. Suitable prefilters are known to those skilled in the art, illustrative examples of which include nanofilters or other suitable filters having a pore size of about 0.1 μm or about 0.2 μm (e.g., Sartopore 2XLM 0.1 μm). Other suitable filters are also known to those skilled in the art, illustrative examples of which include Planova BioEX and Virosart HF. In embodiments, the pore size of the virus filter is less than about 0.2 μm, or preferably less than about 0.1 μm.

[0228] In embodiments, the prefilter area is sufficient to provide adequate prefiltration without product loss, e.g., >0.6 m 2 / 100L eluted hemoglobin.

[0229] In embodiments, the virus filter area is sufficient to maintain flux and achieve viral clearance, e.g., 1 m 2 / 100L eluted hemoglobin.

[0230] In embodiments, the pre-filter area is at least 0.6 times the area of the virus filter.

[0231] In embodiments, before the eluted hemopexin is subjected to virus filtration, the pre-filter and the virus filter are contacted with a washing solution. Suitable virus filtration washing solutions are well known to those skilled in the art. In embodiments, the virus filtration washing solution comprises NaCl and sodium phosphate. In embodiments, the volume of the virus filtration washing solution applied to the pre-filter and the virus filter is at least about 6 capsule volumes. In embodiments, the virus filtration washing solution is applied to the pre-filter and the 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). It will be appreciated by those skilled in the art that different pressures may be applied at different stages of filter preparation (i.e., pre-washing).

[0232] In embodiments, the eluted hemopexin is applied to a pre-filter in series with the virus filter at a pressure of about 3.0 bar (ie, 3.0 ± 0.1).

[0233] Those skilled in the art will appreciate that the protein load applied to the virus filter will depend on the filter used and the filter area. In embodiments, the filter protein load is <1400 g / m 2 .

[0234] In embodiments, the pre-filter and virus filter are washed (i.e., post-washed) after eluting the hemopexin. In embodiments, the post-wash solution is a virus filtration solution described elsewhere herein. In embodiments, the volume of the virus filtration wash solution applied to the pre-filter and virus filter is at least about 3 capsule volumes. In embodiments, the post-wash solution is applied to the pre-filter in series with the virus filter at a pressure of about 3.0 bar (i.e., 3.0 ± 0.1).

[0235] The filtrate containing hemopexin recovered from the pre-filter and the virus filter can be stored for future use. In an embodiment, the filtrate containing hemopexin can be stored at <23°C for up to 24 hours. In another embodiment, the filtrate containing hemopexin can be stored at about 2°C to about 8°C for up to 7 days.

[0236] Concentration and diafiltration

[0237] In embodiments, the methods described herein further comprise exposing the eluted hemopexin recovered in step (xii) to ultrafiltration and / or diafiltration. In embodiments, the eluted hemopexin recovered in step (xii) is exposed to ultrafiltration and / or diafiltration to adjust 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). , 61mg / mL, 62mg / mL, 63mg / mL, 64mg / mL, 65mg / mL, 66mg / mL, 67mg / mL, 68mg / mL, 69mg / mL, 70mg / mL, 71m g / 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, 92m g / mL, 93mg / mL, 94mg / mL, 95mg / mL, 96mg / mL, 97mg / mL, 98mg / mL, 99mg / mL, 100mg / mL, 101mg / mL, 102mg / mL, 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) value.

[0238] Thus, in embodiments, the concentration of the eluted hemopexin is adjusted to 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, 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 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, preferably about 100 mg / mL, preferably about 101 mg / mL, preferably about 102 mg / mL, preferably about 10 Preferably, the value of the present invention is about 100 mg / mL, preferably about 101 mg / mL, preferably about 102 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 about 117 mg / mL, preferably about 118 mg / mL, preferably about 119 mg / mL or preferably about 120 mg / mL.

[0239] In embodiments, the concentration of the eluted hemopexin is adjusted to about 100 mg / mL.

[0240] 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 protein in the filtrate will be familiar to those skilled in the art, illustrative examples of which include diafiltration.

[0241] In embodiments, the concentration of hemopexin in the filtrate is adjusted to 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, 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 g / 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, preferably about 100 mg / mL, preferably about 101 mg / mL, preferably about 102 mg / mL, preferably about 10 Preferably, the value of the present invention is about 100 mg / mL, preferably about 101 mg / mL, preferably about 102 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 about 117 mg / mL, preferably about 118 mg / mL, preferably about 119 mg / mL or preferably 120 mg / mL.

[0242] In embodiments, the concentration of hemopexin in the filtrate is adjusted to about 100 mg / mL.

[0243] In another aspect disclosed herein, there is provided a method for purifying hemopexin from a solution containing hemopexin and other proteins, the method comprising:

[0244] (i) providing a solution comprising 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;

[0245] (ii) passing the solution of step (i) through a mixed-mode cation exchange chromatography resin under conditions that promote selective binding of hemopexin to the resin over binding of the other proteins to the resin;

[0246] (iii) washing the resin after step (ii) to remove unbound protein;

[0247] (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;

[0248] (v) recovering the hemopexin eluted in step (iv);

[0249] (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 the hemopexin to pass through the resin as an unbound fraction;

[0250] (vii) recovering an unbound fraction comprising hemopexin; and

[0251] (viii) Optionally, exposing the recovered unbound fraction of step (vii) to a virus inactivation step to obtain a virus-inactivated hemopexin solution.

[0252] In another aspect disclosed herein, there is provided a method for purifying hemopexin from a solution containing hemopexin and other proteins, the method comprising:

[0253] (i) providing a solution comprising 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 23 to about 25 mS / cm;

[0254] (ii) passing the solution of step (i) through a mixed-mode cation exchange chromatography resin under conditions that promote selective binding of hemopexin to the resin over binding of the other proteins to the resin;

[0255] (iii) washing the resin after step (ii) to remove unbound protein;

[0256] (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;

[0257] (v) recovering the hemopexin eluted in step (iv);

[0258] (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 the hemopexin to pass through the resin as an unbound fraction;

[0259] (vii) recovering an unbound fraction comprising hemopexin; and

[0260] (viii) concentrating the recovered unbound fraction from step (vii); and

[0261] (ix) Optionally, exposing the concentrated unbound fraction of step (viii) to a viral inactivation step to obtain a virally inactivated hemopexin solution.

[0262] The processes described herein may be suitably performed batchwise or continuously, depending on convenience.

[0263] Composition

[0264] In one aspect of the invention, a composition comprising a hemopexin recovered by the methods disclosed herein is provided. In embodiments, the composition comprises a hemopexin content that is at least 80% (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 total protein.

[0265] 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%.

[0266] Compositions comprising hemopexins recovered by the methods of the invention disclosed herein will be substantially free of other components typically associated with them (e.g., other plasma-derived proteins). Thus, in embodiments, compositions comprising hemopexins will contain less than 20% of total protein, preferably less than 10% of total protein, and more preferably less than 5% of total protein of other components typically associated with them (i.e., impurities). It will be understood by those skilled in the art that the level of impurities present in the compositions of the invention may depend on the intended use of the composition. For example, when the composition is to be administered to a human subject in need (i.e., for clinical use), it is desirable that the composition contain less than 5% impurities (based on total protein). In contrast, where the protein is to be used in vitro, it is acceptable if the composition contains more than 5% impurities (based on total protein).

[0267] In another aspect disclosed herein, a formulation is provided comprising a composition comprising a hemopexin as described herein and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers, diluents, and / or excipients are known to those skilled in the art, illustrative examples of which include solvents, dispersion media, antifungal and antibacterial agents, surfactants, isotonic agents, and absorbents.

[0268] The preparation can also be prepared by adding a suitable stabilizer (e.g., amino acid, carbohydrate, salt and detergent) (or a combination thereof). In a specific embodiment, the stabilizer comprises a mixture of sugar alcohol and amino acid. The stabilizer can comprise a mixture of sugar (e.g., sucrose or trehalose), sugar alcohol (e.g., mannitol or sorbitol) and amino acid (e.g., proline, glycine and arginine). In an embodiment, the preparation comprises amino acid such as arginine. In other embodiments, the preparation comprises a divalent metal ion at a concentration of up to 100mM and a complexing agent as described in US 7045601. In an embodiment wherein pH is preferably about 6.5 to 7.5 and osmotic pressure concentration is at least 240mosmol / kg.

[0269] The preparation can also be sterilized by filtration before distribution and long-term storage. Preferably, the preparation will maintain its original stability characteristics for at least 2, 4, 6, 8, 10, 12, 18, 24, 36 months or longer. For example, when stored for 6 months or longer, the preparation stored at 2-8°C or 25°C can generally maintain the same molecular size distribution as measured by HPLC-SEC. When stored at 2-8°C and / or room temperature, the specific embodiments of the pharmaceutical preparation can be stable and suitable for commercial pharmaceutical use for at least 6 months, 12 months, 18 months, 24 months, 36 months or even longer.

[0270] Compositions as herein described or preparations can be formulated into any of many possible dosage forms (such as injection preparations). Preparations and subsequent use (administration) thereof are within the skill of those skilled in the art. Administration depends on the responsiveness of the subject to treatment, but as long as it is desired to achieve the desired effect (e.g., the reduction in the level of free Hb / heme), administration will always continue. Those of ordinary skill can easily determine optimal dose, administration method, and repetition rate.

[0271] In embodiments disclosed herein, the formulation has a volume of at least 5 mL and comprises 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) of hemopexin. In another embodiment, the pharmaceutical formulation has a volume of at least 5 mL and comprises at least 20 mg / mL of 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. In another aspect, a container comprising at least 5 mL of a formulation comprising hemopexin is provided, wherein the concentration of hemopexin in the formulation is at least 20 mg / mL.

[0272] In a preferred embodiment, the formulation comprises about 15 mM citrate-phosphate buffer, about 150 mM NaCl, wherein the pH is preferably about 7.2, and a hemopexin concentration of about 100 mg / mL.

[0273] In an embodiment, the composition or formulation comprises a hemopexin content of about 95 mg / mL to about 110 mg / mL. In an embodiment, the composition or formulation comprises a hemopexin content of about 100 mg / mL to about 110 mg / mL. In an embodiment, the composition or formulation comprises a hemopexin content of about 103 mg / mL to about 106 mg / mL. In an embodiment, the composition or formulation comprises a hemopexin content of about 103 mg / mL to about 104 mg / mL. In an embodiment, the composition or formulation comprises a hemopexin content of about 106 mg / mL to about 107 mg / mL. In an embodiment, the composition or formulation comprises a hemopexin content of about 100 mg / mL. In an embodiment, the composition or formulation comprises a hemopexin content of about 103 mg / mL. In an embodiment, the composition or formulation comprises a hemopexin content of about 106 mg / mL.

[0274] In embodiments, the composition or formulation comprises about 1000 μM to about 2000 μM heme binding activity. In embodiments, the composition or formulation comprises about 1300 μM to about 1900 μM heme binding activity. In embodiments, the composition or formulation comprises about 1500 μM to about 1800 μM heme binding activity. In embodiments, the composition or formulation comprises about 1600 μM to about 1800 μM heme binding activity. In embodiments, the composition or formulation comprises about 1700 μM heme binding activity.

[0275] In embodiments, the composition or formulation comprises at least about 80% of the heme-specific binding activity of the total protein. In embodiments, the composition or formulation comprises at least about 85% of the heme-specific binding activity of the total protein. In embodiments, the composition or formulation comprises at least about 90% of the heme-specific binding activity of the total protein. In embodiments, the composition or formulation comprises at least about 91% of the heme-specific binding activity of the total protein. In embodiments, the composition or formulation comprises at least about 92% of the heme-specific binding activity of the total protein. In embodiments, the composition or formulation comprises at least about 93% of the heme-specific binding activity of the total protein. In embodiments, the composition or formulation comprises at least about 94% of the heme-specific binding activity of the total protein. In embodiments, the composition or formulation comprises at least about 95% of the heme-specific binding activity of the total protein. In embodiments, the composition or formulation comprises at least about 96% of the heme-specific binding activity of the total protein. In embodiments, the composition or formulation comprises at least about 97% of the heme-specific binding activity of the total protein. In embodiments, the composition or formulation comprises at least about 98% heme-specific binding activity of total protein. In embodiments, the composition or formulation comprises at least about 99% heme-specific binding activity of total protein. In embodiments, the composition or formulation comprises about 100% heme-specific binding activity of total protein.

[0276] In embodiments, compositions or preparations include the CD91 dissociation constant (KD) of about 0.50 μM to about 2.0 μM. In embodiments, compositions or preparations include the CD91 dissociation constant (KD) of about 1.00 μM to about 1.5 μM. In embodiments, compositions or preparations include the CD91 dissociation constant (KD) of about 1.05 μM to about 1.15 μM. In embodiments, compositions or preparations include the CD91 dissociation constant (KD) of about 1.10 μM to about 1.20 μM. In embodiments, compositions or preparations include the CD91 dissociation constant (KD) of about 1.13 ± 0.06 μM. In embodiments, compositions or preparations include the CD91 dissociation constant (KD) of about 1.06 ± 0.05 μM.

[0277] In embodiments, the composition or preparation comprises a transferrin content lower than about 0.50mg / mL. In embodiments, the composition or preparation comprises a transferrin content lower than about 0.40mg / mL. In embodiments, the composition or preparation comprises a transferrin content lower than about 0.30mg / mL. In embodiments, the composition or preparation comprises a transferrin content lower than about 0.25mg / mL. In embodiments, the composition or preparation comprises a transferrin content lower than about 0.20mg / mL. In embodiments, the composition or preparation comprises a transferrin content lower than about 0.15mg / mL. In embodiments, the composition or preparation comprises a transferrin content lower than about 0.12mg / mL. In embodiments, the composition or preparation comprises a transferrin content lower than about 0.11mg / mL.

[0278] In an embodiment, the composition or formulation comprises an albumin content of less than about 0.05 mg / mL. In an embodiment, the composition or formulation comprises an albumin content of less than about 0.025 mg / mL. In an embodiment, the composition or formulation comprises an albumin content of less than about 0.020 mg / mL. In an embodiment, the composition or formulation comprises an albumin content of less than about 0.015 mg / mL. In an embodiment, the composition or formulation comprises an albumin content of less than about 0.010 mg / mL. In an embodiment, the composition or formulation comprises an albumin content of less than about 0.009 mg / mL. In an embodiment, the composition or formulation comprises an albumin content of less than about 0.0085 mg / mL. In an embodiment, the composition or formulation comprises an albumin content of less than about 0.008 mg / mL.

[0279] In embodiments, the composition or formulation comprises a haptoglobin content of less than about 0.05 mg / mL. In embodiments, the composition or formulation comprises a haptoglobin content of less than about 0.04 mg / mL. In embodiments, the composition or formulation comprises a haptoglobin content of less than about 0.03 mg / mL. In embodiments, the composition or formulation comprises a haptoglobin content of less than about 0.025 mg / mL. In embodiments, the composition or formulation comprises a haptoglobin content of less than about 0.02 mg / mL. In embodiments, the composition or formulation comprises a haptoglobin content of less than about 0.015 mg / mL. In embodiments, the composition or formulation comprises a haptoglobin content of less than about 0.010 mg / mL. In embodiments, the composition or formulation comprises a haptoglobin content of less than about 0.005 mg / mL.

[0280] In an embodiment, the composition or formulation comprises an Apo-A1 content of less than about 0.10 mg / mL. In an embodiment, the composition or formulation comprises an Apo-A1 content of less than about 0.09 mg / mL. In an embodiment, the composition or formulation comprises an Apo-A1 content of less than about 0.08 mg / mL. In an embodiment, the composition or formulation comprises an Apo-A1 content of less than about 0.07 mg / mL. In an embodiment, the composition or formulation comprises an Apo-A1 content of less than about 0.06 mg / mL. In an embodiment, the composition or formulation comprises an Apo-A1 content of less than about 0.05 mg / mL.

[0281] In embodiments, the composition or formulation comprises a high molecular weight (HMW) hemopexin aggregate content of less than about 1.0% of total protein. In embodiments, the composition or formulation comprises a HMW hemopexin aggregate content of less than about 0.9% of total protein. In embodiments, the composition or formulation comprises a HMW hemopexin aggregate content of less than about 0.8% of total protein. In embodiments, the composition or formulation comprises a HMW hemopexin aggregate content of less than about 0.7% of total protein. In embodiments, the composition or formulation comprises a HMW hemopexin aggregate content of less than about 0.6% of total protein. In embodiments, the composition or formulation comprises a HMW hemopexin aggregate content of less than about 0.5% of total protein. In embodiments, the composition or formulation comprises a HMW hemopexin aggregate content of less than about 0.4% of total protein. In embodiments, the composition or formulation comprises a HMW hemopexin aggregate content of about 0.4% to about 0.5% of total protein.

[0282] In embodiments, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 1.0% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.9% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.8% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.7% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises an HMW hemopexin aggregate content of less than about 0.6% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a HMW hemopexin aggregate content of less than about 0.5% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a HMW hemopexin aggregate content of less than about 0.4% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a HMW hemopexin aggregate content of about 0.4% to about 0.5% of total protein as determined by size exclusion-HPLC.

[0283] In an embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 90% of the total protein. In an embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 91% of the total protein. In an embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 92% of the total protein. In an embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 93% of the total protein. In an embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 94% of the total protein. In an embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 95% of the total protein. In an embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 96% of the total protein. In an embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 97% of the total protein. In an embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 98% of the total protein. In an embodiment, the composition or formulation comprises a hemopexin monomer content of at least about 99% of the total protein.

[0284] In embodiments, the composition or formulation comprises a hemopexin monomer content of at least about 90% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a hemopexin monomer content of at least about 91% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a hemopexin monomer content of at least about 92% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a hemopexin monomer content of at least about 93% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a hemopexin monomer content of at least about 94% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a hemopexin monomer content of at least about 95% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a hemopexin monomer content of at least about 96% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a hemopexin monomer content of at least about 97% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a hemopexin monomer content of at least about 98% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a hemopexin monomer content of at least about 99% of total protein as determined by size exclusion-HPLC.

[0285] In embodiments, the composition or formulation comprises a low molecular weight (LMW) impurity content of less than about 1.0% of the total protein. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.9% of the total protein. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.8% of the total protein. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.7% of the total protein. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.6% of the total protein. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.5% of the total protein. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.4% of the total protein. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.3% of the total protein.

[0286] In embodiments, 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-HPLC. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.9% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.8% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.7% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.6% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.5% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.4% of total protein as determined by size exclusion-HPLC. In embodiments, the composition or formulation comprises a LMW impurity content of less than about 0.3% of total protein as determined by size exclusion-HPLC.

[0287] In an embodiment, the composition or formulation comprises a hemopexin content that is at least about 80% pure of the total protein. In an embodiment, the composition or formulation comprises a hemopexin content that is at least about 82% pure of the total protein. In an embodiment, the composition or formulation comprises a hemopexin content that is at least about 84% pure of the total protein. In an embodiment, the composition or formulation comprises a hemopexin content that is at least about 86% pure of the total protein. In an embodiment, the composition or formulation comprises a hemopexin content that is at least about 88% pure of the total protein. In an embodiment, the composition or formulation comprises a hemopexin content that is at least about 90% pure of the total protein. In an embodiment, the composition or formulation comprises a hemopexin content that is at least about 92% pure of the total protein. In an embodiment, the composition or formulation comprises a hemopexin content that is at least about 94% pure of the total protein. In an embodiment, the composition or formulation comprises a hemopexin content that is at least about 96% pure of the total protein. In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 98% of total protein. In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 99% of total protein.

[0288] In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 80% of the total protein as determined by reducing SDS-PAGE. In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 82% of the total protein as determined by reducing SDS-PAGE. In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 84% of the total protein as determined by reducing SDS-PAGE. In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 86% of the total protein as determined by reducing SDS-PAGE. In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 88% of the total protein as determined by reducing SDS-PAGE. In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 90% of the total protein as determined by reducing SDS-PAGE. In an embodiment, the composition or formulation comprises a hemopexin purity content of at least about 92% of the total protein as determined by reducing SDS-PAGE. In an embodiment, the composition or formulation comprises a hemopexin purity content of at least about 94% of the total protein as determined by reducing SDS-PAGE. In an embodiment, the composition or formulation comprises a hemopexin purity content of at least about 96% of the total protein as determined by reducing SDS-PAGE. In an embodiment, the composition or formulation comprises a hemopexin purity content of at least about 98% of the total protein as determined by reducing SDS-PAGE. In an embodiment, the composition or formulation comprises a hemopexin purity content of at least about 99% of the total protein as determined by reducing SDS-PAGE.

[0289] In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 80% of the total protein as determined by non-reducing SDS-PAGE. In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 82% of the total protein as determined by non-reducing SDS-PAGE. In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 84% of the total protein as determined by non-reducing SDS-PAGE. In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 86% of the total protein as determined by non-reducing SDS-PAGE. In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 88% of the total protein as determined by non-reducing SDS-PAGE. In embodiments, the composition or formulation comprises a hemopexin purity content of at least about 90% of the total protein as determined by non-reducing SDS-PAGE. In an embodiment, the composition or formulation comprises a hemopexin purity content of at least about 92% of the total protein as determined by non-reducing SDS-PAGE. In an embodiment, the composition or formulation comprises a hemopexin purity content of at least about 94% of the total protein as determined by non-reducing SDS-PAGE. In an embodiment, the composition or formulation comprises a hemopexin purity content of at least about 96% of the total protein as determined by non-reducing SDS-PAGE. In an embodiment, the composition or formulation comprises a hemopexin purity content of at least about 98% of the total protein as determined by non-reducing SDS-PAGE. In an embodiment, the composition or formulation comprises a hemopexin purity content of at least about 99% of the total protein as determined by non-reducing SDS-PAGE.

[0290] In embodiments, the protein content of a composition or preparation comprises an isoelectric point (pi) of about 5.0 to about 6.5. In embodiments, the protein content of a composition or preparation comprises an isoelectric point (pi) of about 5.2 to about 6.4. In embodiments, the protein content of a composition or preparation comprises an isoelectric point (pi) of about 5.4 to about 6.3. In embodiments, the protein content of a composition or preparation comprises an isoelectric point (pi) of about 5.5 to about 6.2. In embodiments, the protein content of a composition or preparation comprises an isoelectric point (pi) of about 5.6 to about 6.1. In embodiments, the protein content of a composition or preparation comprises an isoelectric point (pi) of about 5.7 to about 6.0. In embodiments, the protein content of a composition or preparation comprises a minimum isoelectric point (pi) of about 5.4 to about 5.6. In embodiments, the protein content of a composition or preparation comprises a minimum isoelectric point (pi) of about 5.5. In embodiments, the protein content of a composition or preparation comprises a major isoelectric point (pi) of about 5.8 to about 5.9. In embodiments, the protein content of the composition or formulation comprises a principal isoelectric point (pi) of about 5.8 to about 5.85. In embodiments, the protein content of the composition or formulation comprises a maximum isoelectric point (pi) of about 6.00 to about 6.10. In embodiments, the protein content of the composition or formulation comprises a maximum isoelectric point (pi) of about 6.00 to about 6.05. In embodiments, the protein content of the composition or formulation comprises a maximum isoelectric point (pi) of about 6.03. In embodiments, the protein content of the composition or formulation comprises a maximum isoelectric point (pi) of about 6.04.

[0291] In embodiments, the protein content of a composition or formulation comprises an isoelectric point (pi) of about 5.0 to about 6.5, as determined by capillary isoelectric focusing (cIEF). In embodiments, the protein content of a composition or formulation comprises an isoelectric point (pi) of about 5.2 to about 6.4, as determined by cIEF. In embodiments, the protein content of a composition or formulation comprises an isoelectric point (pi) of about 5.4 to about 6.3, as determined by cIEF. In embodiments, the protein content of a composition or formulation comprises an isoelectric point (pi) of about 5.5 to about 6.2, as determined by cIEF. In embodiments, the protein content of a composition or formulation comprises an isoelectric point (pi) of about 5.6 to about 6.1, as determined by cIEF. In embodiments, the protein content of a composition or formulation comprises an isoelectric point (pi) of about 5.7 to about 6.0, as determined by cIEF. In embodiments, the protein content of a composition or formulation comprises a minimum isoelectric point (pi) of about 5.4 to about 5.6 as determined by cIEF. In embodiments, the protein content of a composition or formulation comprises a minimum isoelectric point (pi) of about 5.5 as determined by cIEF. In embodiments, the protein content of a composition or formulation comprises a main isoelectric point (pi) of about 5.8 to about 5.9 as determined by cIEF. In embodiments, the protein content of a composition or formulation comprises a main isoelectric point (pi) of about 5.8 to about 5.85 as determined by cIEF. In embodiments, the protein content of a composition or formulation comprises a maximum isoelectric point (pi) of about 6.00 to about 6.10 as determined by cIEF. In embodiments, the protein content of a composition or formulation comprises a maximum isoelectric point (pi) of about 6.00 to about 6.05 as determined by cIEF. In embodiments, the protein content of a composition or formulation comprises a maximum isoelectric point (pi) of about 6.03 as determined by cIEF. In embodiments, the protein content of the composition or formulation comprises an isoelectric point (pi) maximum of about 6.04 as determined by cIEF.

[0292] In embodiments, the composition or formulation comprises a protease activity level of less than about 5 nKat / L. In embodiments, the composition or formulation comprises a protease activity level of less than about 4 nKat / L. In embodiments, the composition or formulation comprises a protease activity level of less than about 3 nKat / L.

[0293] In embodiments, the composition or formulation comprises a level of prokallikrein activity of less than about 30 IU / mL. In embodiments, the composition or formulation comprises a level of prokallikrein activity of less than about 25 IU / mL. In embodiments, the composition or formulation comprises a level of prokallikrein activity of less than about 20 IU / mL.

[0294] In an embodiment, the composition or preparation comprises a tri(n-butyl) phosphate (TnBP) content of less than about 10 μg / mL. In an embodiment, the composition or preparation comprises a TnBP content of less than about 8 μg / mL. In an embodiment, the composition or preparation comprises a TnBP content of less than about 7 μg / mL. In an embodiment, the composition or preparation comprises a TnBP content of less than about 6 μg / mL. In an embodiment, the composition or preparation comprises a TnBP content of less than about 5 μg / mL. In an embodiment, the composition or preparation comprises a TnBP content of less than about 4 μg / mL. In an embodiment, the composition or preparation comprises a TnBP content of less than about 3 μg / mL. In an embodiment, the composition or preparation comprises a TnBP content of less than about 2 μg / mL.

[0295] In embodiments, the composition or preparation comprises a PS80 content of less than about 20 mg / mL. In embodiments, the composition or preparation comprises a PS80 content of less than about 18 mg / mL. In embodiments, the composition or preparation comprises a PS80 content of less than about 16 mg / mL. In embodiments, the composition or preparation comprises a PS80 content of less than about 14 mg / mL. In embodiments, the composition or preparation comprises a PS80 content of less than about 12 mg / mL. In embodiments, the composition or preparation comprises a PS80 content of less than about 10 mg / mL. In embodiments, the composition or preparation comprises a PS80 content of less than about 8 mg / mL.

[0296] Compositions or formulations comprising a combination of any two or more of the features described herein are also contemplated herein. As non-limiting examples, the compositions or formulations described herein may comprise a combination of any two or more of the following features:

[0297] (a) a hemopexin content of about 95 mg / mL to about 110 mg / mL;

[0298] (b) a heme binding activity of about 1000 μM to about 2000 μM;

[0299] (c) Heme-specific binding activity accounting for at least about 80% of the total protein

[0300] (d) a CD91 dissociation constant (KD) of about 0.50 μM to about 2.0 μM;

[0301] (e) a transferrin content of less than about 0.50 mg / mL;

[0302] (f) an albumin content of less than about 0.05 mg / mL;

[0303] (g) a haptoglobin content of less than about 0.05 mg / mL;

[0304] (h) an Apo-A1 content of less than about 0.10 mg / mL;

[0305] (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;

[0306] (j) a hemopexin monomer content of at least about 90% of total protein as determined by size exclusion-high performance liquid chromatography;

[0307] (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;

[0308] (1) 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;

[0309] (m) an isoelectric point (pi) of about 5.0 to about 6.5 as determined by capillary isoelectric focusing (cIEF);

[0310] (n) a protease activity level of less than about 5 nKat / L;

[0311] (o) a prokallikrein activity level of less than about 30 IU / mL;

[0312] (p) a tri(n-butyl) phosphate (TnBP) content of less than about 10 μg / mL; and

[0313] (q) a PS80 content of less than about 20 mg / mL.

[0314] In embodiments, a composition or formulation described herein comprises a combination of any two or more of the following features:

[0315] (a) a hemopexin content of about 95 mg / mL to about 110 mg / mL;

[0316] (b) a heme binding activity of about 1600 μM to about 1800 μM;

[0317] (c) a heme-specific binding activity of at least about 97% of the total protein;

[0318] (d) a CD91 dissociation constant (KD) of about 1.10 μM to about 1.20 μM;

[0319] (e) a transferrin content of less than about 0.25 mg / mL;

[0320] (f) an albumin content of less than about 0.009 mg / mL;

[0321] (g) a haptoglobin content of less than about 0.03 mg / mL;

[0322] (h) an Apo-A1 content of less than about 0.06 mg / mL;

[0323] (i) a high molecular weight (HMW) hemopexin aggregate content of less than about 0.6% of the total protein;

[0324] (j) a hemopexin monomer content of at least about 99% of the total protein;

[0325] (k) a low molecular weight (LMW) impurity content of less than about 0.4% of the total protein;

[0326] (1) a hemopexin purity content of at least about 88% of total protein;

[0327] (m) an isoelectric point (pi) of about 5.4 to about 6.3;

[0328] (n) a protease activity level of less than about 3 nKat / L;

[0329] (o) a prokallikrein activity level of less than about 20 IU / mL;

[0330] (p) a tri(n-butyl) phosphate (TnBP) content of less than about 5 μg / mL; and

[0331] (q) a PS80 content of less than about 18 mg / mL.

[0332] In embodiments, the compositions or formulations described herein comprise (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. In another embodiment, the compositions or formulations further comprise (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 haptoglobin.

[0333] In embodiments, the compositions or formulations described herein further comprise no detectable apolipoprotein A1 and / or albumin content. In embodiments, 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.

[0334] In embodiments, the compositions or formulations described herein further do not comprise detectable protease activity. Suitable methods for measuring protease activity are familiar to those skilled in the art, and illustrative examples thereof are described in, for example, Zhang et al. (ed. 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).

[0335] In embodiments, a composition or formulation described herein is suitable for pharmaceutical administration after storage for 12 months at 2°C to 8°C and / or ambient temperature (eg, room temperature).

[0336] Treatment

[0337] In another aspect of the present invention, there is provided a method of treating a disorder associated with hemolysis, the method comprising administering to a subject in need thereof a composition or formulation of the invention disclosed herein.

[0338] As used herein, the term "subject" refers to an animal, including primates (lower or higher primates). Higher primates include humans. Although the present invention is particularly suitable for targeting human diseases, it will be understood by those skilled in the art that non-human animals can also benefit from the compositions and methods disclosed herein. Therefore, it will be understood by those skilled in the art that the present invention has both human and veterinary applications. For convenience, "animal" includes livestock and companion animals such as cattle, horses, sheep, pigs, camels, goats, donkeys, dogs and cats. In the case of horses, these horses include horses used in the racing industry as well as horses used for entertainment or animal husbandry.

[0339] As described herein, the compositions or formulations comprising hemopexin can be administered to a subject by any suitable route. Illustrative examples of suitable routes of administration include intravenous, subcutaneous, intraarterial, or by infusion. In embodiments, the compositions or formulations described herein are administered intravenously.

[0340] If necessary, the methods of treatment described herein may further include administering a second therapeutic agent. A second therapeutic compound may be co-administered to the subject sequentially (before or after administering a composition or formulation disclosed herein) or simultaneously. In embodiments, the second therapeutic agent is an iron chelator (e.g., deferoxamine or deferiprone).

[0341] In another aspect disclosed herein, there is provided use of a composition or formulation comprising a hemopexin as described herein in the preparation of a medicament for treating a disorder associated with hemolysis. In an embodiment, the composition or formulation described herein is formulated for use in humans.

[0342] The compositions and formulations described herein are particularly suitable for treating subjects suffering from conditions associated with hemolysis, including those conditions associated with hemoglobin / heme-mediated toxicity risks. Conditions associated with hemolysis, including conditions associated with hemoglobin / heme-mediated toxicity risks, are known in the art. In embodiments, the condition is selected from acute hemolysis and / or chronic hemolysis. In embodiments, the condition is selected from the group consisting of: hemolytic anemia, transfusion hemolysis, hemolytic uremic syndrome, autoimmune diseases, malarial infection, trauma, blood transfusion, open heart surgery using cardiopulmonary bypass, and burns, including in the treatment of hemoglobinemia or hemoglobinuria with hemolysis after burns. In embodiments, the disease is selected from sickle cell anemia, hereditary spherocytosis, hereditary elliptocytosis, thalassemia, congenital dyserythropoietic anemia and paroxysmal nocturnal hemoglobinuria, systemic lupus erythematosus, and chronic lymphocytic leukemia.

[0343] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications that come within the spirit and scope. The invention also includes all steps, features, compositions and compounds referred to or indicated in this specification, whether singly or collectively, and any and all combinations of any two or more of said steps or features.

[0344] Certain embodiments of the present invention will now be described with reference to the following examples, which are provided for illustrative purposes only and are not intended to limit the general scope described above. Example

[0345] Example 1 - Screening of mixed-mode chromatography resins for hemopexin purification

[0346] Initial studies found that the isoelectric points of hemopexin and the major contaminating proteins in FIV-4 were very similar, ranging from approximately pI 4.8 to 6.3. This suggested that ion-exchange chromatography would not provide a robust and effective 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 by charge. In an initial resin screening study, several mixed-mode cation- and anion-exchange resins were screened in a high-throughput format and evaluated for their utility in the purification of hemopexin.

[0347] Mixed-mode cation exchange

[0348] The mixed-mode cation exchange resins Capto MMC (Cytiva), Eshmuno HCX (Merck), Nuvia cPrime (Bio-Rad), and Toyopearl MX-TRP 650M (Tosoh) were screened in this study.

[0349] The FIV-4 paste was resuspended in acetate buffer at pH 4.5 and loaded onto a mixed-mode cation exchange column equilibrated in the same buffer. Unbound material was washed with the same buffer and bound protein was eluted with a stepwise NaCl gradient.

[0350] For Eshmuno HCX or Toyopearl MX-TRP 650M resins, no reasonable selectivity was observed for hemopexin, with the majority of all proteins eluting in the unbound fraction. In contrast, no protein eluted from Capto MMC or Nuvia cPrime, indicating that all proteins were strongly retained. Considering that it is expected (based on pI) that hemopexin should be one of the more strongly retained proteins, these resins are identified as having the potential for a suitable hemopexin capture step. These resins were further developed to determine whether sufficient selectivity could be obtained for effective purification (Example 4).

[0351] Mixed-mode anion exchange

[0352] Mixed mode anion exchange resins; Capto Adhere (Cytiva), MEP Hypercel (Sartorius), HEA Hypercel (Sartorius), and PPA Hypercel (Sartorius) were screened in this study.

[0353] The FIV-4 paste was resuspended in phosphate buffer at pH 7.5 and loaded onto a mixed-mode anion exchange column equilibrated in the same buffer. Unbound material was washed away with the same buffer, and bound protein was eluted using 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 an anion exchange column, and eluted using a stepwise decreasing pH gradient.

[0354] The mixed mode resins MEP Hypercel, HEA Hypercel and PPA Hypercel showed poor selectivity, with hemopexin eluting with contaminating proteins in several fractions ( Figure 1 ).

[0355] When loaded onto Capto Adhere resin in the presence of salt, hemopexin was found in a fairly high purity in the unbound fraction. While this clearly indicates a useful purification step, when loading a paste extract, the binding of all other proteins will result in a low hemopexin capacity, where the column quickly becomes saturated with contaminants. This suggests that Capto Adhere chromatography may be best used as a second purification step once most contaminating proteins have been removed.

[0356] Further studies to optimize conditions for downstream purification using Capto Adhere are discussed in Example 5.

[0357] Additional cation / anion exchange resins

[0358] Initial studies focused on the purification of hemopexin from extracted FIV-4 paste and involved the cation exchange resins Eshmuno CPX, Eshmuno S, Eshmuno COO, and Fractogel EMD COO. - Screening of (M). Both EshmunoCPX and EshmunoCOO resins exhibited selective binding for hemopexin, but recoveries were poor, with 40% and 20% of hemopexin present in the flowthrough fraction, respectively, and low binding capacities were observed for both resins. Therefore, these cation exchange resins were deemed unsuitable for the first purification step.

[0359] The feasibility of the anion exchange chromatography resins Eshmuno Q, FractogelTMAE Hicap(M), and FractogelDMAE(M) was investigated for potential use as a polishing step. This study was conducted using hemopexin, which contained only small amounts of contaminating proteins, as expected after partial purification. While Eshmuno Q produced favorable results, further development of this resin was discontinued in favor of cation exchange resins.

[0360] When 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 at the final stage of the hemopexin purification process, hemopexin may be essentially pure, with some contaminating transferrin remaining. Therefore, further development work utilized an alternative 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.

[0361] This chromatography step is expected to serve as the final polishing step in the hemopexin purification process. In the overall process design, this may result in the feed to the 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, it has been shown that solvent / detergent reagents have no effect on the separation characteristics of Eshmuno CPS. During the loading and washing steps, the solvent / detergent reagents elute from the flowthrough, resulting in low concentrations of PS80 and TnBP in the hemopexin product. This demonstrates that Eshmuno CPS resin is a viable option for purity polishing and removal of solvents / detergents from the product.

[0362] Because Eshmuno CPS chromatography resin was not tested in the initial screening of cation exchange resins using paste extracts, and because it showed high binding selectivity for hemopexin, its potential use as a preliminary purification step was re-evaluated. When the extract paste was used as the loading material, a constant breakthrough of hemopexin was observed during loading, with a loss of approximately 40% of the hemopexin, regardless of the conductivity of the load (5 and 10 mS / cm). This may indicate competition for binding sites with another component in the crude extract and / or the presence of a different form of hemopexin, such as a heme complex. Regardless, due to the low recovery, further development of Eshmuno CPS resin as an initial purification step was not pursued.

[0363] Eshmuno CPS chromatography resin was further optimized for use as a post-virus inactivation polishing step. These studies are described in Example 7.

[0364] Example 2—Evaluation of Extraction Conditions of Fraction IV-4 Paste for Hemopexin Purification

[0365] The first step in the process of purifying hemopexin from FIV-4 paste is resuspension in an aqueous buffer. Because several different proteins are present in FIV-4 paste, certain extraction conditions can preferentially solubilize hemopexin while leaving some or all of the contaminating proteins in the insoluble fraction. It is desirable to maximize the amount of hemopexin extracted from the FIV-4 paste and provide conditions for solubilizing 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, a number of extraction buffers and conditions were investigated to optimize the extraction process steps. These studies are briefly described below.

[0366] Preliminary FIV-4 paste extraction development for bench-scale batches

[0367] Preliminary extraction studies performed over a pH range of pH 4 to pH 8 indicated that only small amounts of hemopexin and haptoglobin were soluble at pH < 5.5, whereas significant amounts of albumin and transferrin were present in the extracts at all pH levels ( Figure 2 ). Within the pH range investigated, no conditions were identified that favored the preferential extraction of hemopexin.

[0368] At a neutral or near-neutral pH, extraction of hemopexin was shown to be most efficient and productive. Under these conditions, hemopexin was not preferentially extracted, nor were most contaminating proteins extracted under these conditions. Highly efficient resuspension of the FIV-4 paste was demonstrated at a paste:buffer ratio of 1:5 to 1:20 and a mixing time of 1 to 4 hours, with comparable hemopexin intermediates being produced under these combinations of conditions.

[0369] Following initial extraction process development studies, bench-scale batch production using the second generation hemopexin process (Example 13) was performed using extraction conditions of pH 6.5 and 250 mM NaCl, a 1:10 paste:buffer ratio, and a 2 hour mixing time.

[0370] FIV-4 paste batch consistency

[0371] In early experiments, the hemopexin concentration of extracted FIV-4 paste solutions was observed to vary by more than 50%, and it was hypothesized that this variation was due to batch variation of the FIV-4 paste.

[0372] To test this, a consistency study of FIV-4 paste extraction and depth filtration was conducted on 10 batches of FIV-4 paste. The experiment was conducted for 2 hours at room temperature using 0.2 kg of paste from each batch, dissolved in 40 mM phosphate, 180 mM NaCl, pH 6.2 at a paste:buffer ratio of 1:10. The extract was clarified by filtration through a 3M 90LP depth filter with a 0.04 m 2 filter area, pre-coated with 625g / m 2 Filter area of Celpure C1000. Hemopexin concentration of extracts was measured by reverse phase HPLC before and after depth filtration.

[0373] When analyzed by reverse phase HPLC, the hemopexin concentration of the extract paste varied between 0.6 and 0.76 g / L ( Figure 3 The performance of the depth filtration process step remained consistent across the batches tested, with recoveries of approximately 80% to 95% for hemopexin, regardless of the quantification method.

[0374] Optimization of FIV-4 paste extraction for pilot-scale production

[0375] Although earlier studies showed that extraction of hemopexin was maximal at pH 7.5, extractions in laboratory studies were performed at pH 6.2 to adequately match the equilibrium conditions of the first chromatographic step. To improve pilot-scale yields, studies were conducted to investigate whether extraction at pH 7.5 extracted sufficiently more hemopexin to justify the additional steps involved in post-clarification pH adjustment.

[0376] 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 at the given pH in 40 mM phosphate, 180 mM NaCl with a 1:10 paste:buffer ratio. The results showed that increasing the extraction pH to 7.5 resulted in approximately 20% more hemopexin solubility, regardless of the paste batch. Figure 4 ). This is considered a significant increase in yield.

[0377] As part of the Capto MMC chromatography process development, robustness studies were performed around the loading conditions and a concentration of 225 mM NaCl was identified as the target loading condition (Example 4).

[0378] Based on these studies, the extraction buffer conditions were changed to 225 mM NaCl (to match the conductivity to the Capto MMC equilibrium) and pH 7.5 to maximize yield. These conditions were used for the remaining laboratory-scale and pilot-scale batches (Examples 13 and 14).

[0379] Prior to pilot-scale batch production, solubilization of FIV-4 paste in a small amount of extraction buffer was investigated to minimize tank size in a production environment. Hemopexin extractions performed at paste:buffer ratios of 1:2.5 and 1:5 demonstrated extraction of substantial amounts of hemopexin, ranging from 5.5 to 6.6 g / kg of paste. Initially, there was concern that residual ethanol in the paste might result in lower recoveries in the first chromatographic purification step (Capto MMC resin) when solubilization ratios were lower. Capto MMC chromatography of clarified extract pastes from 1:2.5, 1:5, and 1:10 extracts demonstrated no effect on purity or recovery at lower ratios, indicating that residual ethanol had no significant impact and that extracts with ratios as low as 1:2.5 were suitable. Studies using an extraction paste:buffer ratio of 1:1 showed lower recoveries of hemopexin and protein during the Capto MMC chromatography step.

[0380] At lower extraction rates, larger post-press wash volumes were required to maximize protein recovery, with up to three times the press volume required at an extraction rate of 1:2.5.

[0381] Optimization of FIV-4 paste extraction for production scale

[0382] On a production scale, the paste may be frozen before use, in which case solubilization directly from the frozen paste would provide simpler paste handling. To evaluate this, the efficiency of solubilization of hemopexin was assessed after extraction of the frozen paste and thawing of the paste at 2-8°C and resuspension in buffer at room temperature (approximately 22°C) and 4°C. The results showed that extraction efficiencies using the frozen paste were slightly higher than those obtained using the thawed paste, regardless of the temperature of the extraction buffer. As the paste was stored at 4°C for several days before extraction, this suggests that some degradation of the hemopexin in the FIV-4 paste may occur under these storage conditions. Recovery rates by depth filtration were consistent regardless of the temperature of the paste and extraction buffer.

[0383] As part of further optimization of the process for production scale, the NaCl concentration of the extraction buffer was modified to achieve a conductivity of the clarified extract paste comparable to that of the equilibration buffer used for the Capto MMC column. Extraction of FIV-4 paste in a buffer containing 400 mM NaCl yielded a clarified extract paste process intermediate with a conductivity of 27 mS / cm (comparable to the Capto MMC equilibration buffer). This eliminated the need for conductivity adjustment after depth filtration and clarification.

[0384] Final process extraction method

[0385] The FIV-4 paste containing hemopexin was resuspended in 40 mM sodium phosphate, 400 mM sodium chloride, pH 7.5 ± 0.1 extraction buffer at a 1:2.5 w / w paste to buffer ratio. The paste was broken into small pieces and slowly added to the extraction buffer. The extract was stirred at room temperature for at least 120 minutes before clarification, and after all visible clumps had dissociated, stirred for at least 60 minutes.

[0386] Example 3 - Evaluation of Fraction IV-4 Paste Clarification Conditions for Hemopexin Purification

[0387] Prior to downstream chromatographic purification steps, it was essential to develop an efficient filtration step to remove suspended particulate matter and prevent column fouling. After developing initial conditions for solubilizing hemopexin from FIV-4 paste, clarification process development was conducted in conjunction with further extraction studies to ensure compatibility of the extraction and clarification conditions. These depth filtration and clarification studies were conducted under the assumption that the filter press would be used for production-scale depth filtration. These studies are briefly described below.

[0388] Initial development of deep filtration and clarification

[0389] Several different depth filter media from manufacturers 3M and Pall were screened to obtain the best throughput and clarification of the extracted FIV-4 paste. Fraction IV-4 paste was resuspended in phosphate buffer (250 mM NaCl) at pH 6.5 at a paste:buffer ratio of 1:10. The extract was clarified using a 47 mm disc filter, a 60 mm disc filter, or a cartridge at a constant pressure of 2 bar.

[0390] Acceptance criteria for filter performance were initially set at at least 200 L / m 2 The filter area has a throughput of 100 nm and a turbidity of less than 80 NTU. The NTU quality standard is based on initial studies that found that at this turbidity level after 0.22 μm filtration of the extract, the material did not significantly foul the column.

[0391] Double-layer cartridge filters and sandwich filters provide some of the highest levels of clarification, but have very low flux characteristics due to clogging of the cartridges with residual filter aid from the original FIV-4 paste. A two-step filtration process (which includes a first coarse filter such as a 3M 30SP filter plate, followed by a second fine-grade depth filter plate) has been shown to produce a highly clarified product intermediate at high throughput. However, in a production environment, a two-step or sandwich filter filtration process is not ideal.

[0392] 3M zeta plus 90SP filter media was selected to produce product intermediates with reasonable clarity and throughput when used as a single layer filter.

[0393] The best flux and clarification are achieved when the depth filter is pre-coated with filter aid. Adding filter aid to the extract paste feed is less effective than pre-coating the filter with filter aid (pre-mixed filter aid), with reduced clarification and lower flux observed. Using two different filter aids, Celpure C1000 and Celpure C300, little difference in depth filtration performance was observed.

[0394] The filter press capacity is determined to be 0.11m 2 A filter area of 1000 μg / kg paste and a frame depth of 4 cm provided sufficient capacity for deep filtration of the extract paste solution.

[0395] In order to produce a uniform filter aid coating and filter cake in the filter press, a relatively high flow rate is required. At low flow rates, filter aid has been observed to pool at the bottom of the filter frame, reducing filter efficiency and causing fouling in the inlet line. Studies conducted have shown that 6.25 L / m 2 A flow rate of filter area / minute produces a uniform coating of filter aid on the filter plates.

[0396] Following initial development of the depth filtration step, further studies were conducted using 3M 90LP depth filters with a 0.11 m 2 / kg paste filter area, pre-coated with 625g / m 2 Filter area of Celpure C1000.

[0397] Optimization of depth filtration and clarification

[0398] Variation in the recovery of the depth filtration step was observed in laboratory-scale batches, with one particular batch recovering only 50% to 60% of the hemopexin in this step (Example 13). Investigation into the cause indicated that the recovery was significantly worse when filtration was performed without a post-wash step. Lower extract protein concentrations, alternative extraction buffers, or post-washes with high salt had little or no effect on product recovery. Taken together, this suggests that losses in the depth filtration step are unlikely to be due to protein binding to the filter, but most likely due to insufficient post-wash volume.

[0399] The filter area used is 0.012m 2 The optimal volume for post-depth filter washes for 1:10 paste:buffer extractions was determined using a 20 x 20 cm filter press with 1 L of extract, and the results showed that almost two filter press volumes were required to recover all the protein.

[0400] To better suit large-scale production, the FIV-4 paste extraction conditions were optimized to reduce the volume and were performed at a paste:buffer ratio of 1:2.5. When performing the 1:2.5 extraction, the filter area used was adjusted based on the amount of paste used, resulting in the same solid loading per unit area as with the 1:10 extraction. This is equivalent to 9 kg paste / m 2 Filter area or 0.031m 2 The filtration efficiency of the 1:2.5 extraction ratio was not significantly different from that of the 1:10 extraction, however, a larger post-wash volume of 3 times the filter press volume was required to recover the majority of the protein.

[0401] Estimation of hemopexin concentration in clarified extracts

[0402] To calculate the appropriate loading on the Capto MMC chromatography resin, the hemopexin concentration in the clarified extract paste solution needs to be measured. At the extract paste stage, the hemopexin content is low. Therefore, immunoturbidimetry or reversed-phase HPLC are used to provide hemopexin concentrations in early development. In the production process, it is not feasible to perform complex or time-consuming methods to provide the concentration to be loaded on the Capto MMC resin. Therefore, a method using the OD value of the extract solution was developed. 280nm A simple in-process testing method.

[0403] The OD values from the FIV-4 paste batch consistency study (Example 4) were 280nm The data were compared with the hemopexin concentration of each batch measured by reverse phase HPLC. It was found that on average, the OD 280nm Divide by 13 to estimate the hemopexin concentration (Table 1).

[0404] Final clarification method

[0405] The filter area is 1m 2 The FIV-4 extract paste solution was clarified by passing it through 3M 90SP zetaplus depth filter media in a filter press with a 4 cm frame depth. 1 filter press volume of 3M zetaplus filter media containing 625 g Celpure 1000 filter aid was used. 2 The extraction buffer of the filter area was 6.25 L / m 2 / min flow rate to pre-wash the filter press. Then 2 The FIV-4 extract paste solution was filtered at a flow rate of 100 / min, which typically resulted in a pressure of <1.5 bar (maximum 2 bar) during filtration. The filter press was then washed with 3 times the filter press volume of Capto MMC equilibration buffer. The pH of the resulting deep filtered extract was adjusted to pH 6.4 ± 0.1 with 0.5 M HCl and a conductivity of 26 to 28 mS / cm was confirmed. A 33 cm 2 The depth filtered extract is further clarified using a Millipak 200 0.2 μm filter cartridge or similar device at 400 nm to produce a clarified extract paste.

[0406] Example 4 - Mixed Mode Chromatography Using Capto MMC Resin

[0407] Two mixed-mode resins, Capto MMC and Nuvia cPrime, were optimized and compared for use as the first purification step in the hemopexin protein production process described herein. Preliminary studies demonstrated little difference in the yield and purity of hemopexin purified using Capto MMC and Nuvia cPrime resins. However, after several studies to optimize the performance of both resins, Capto MMC resin demonstrated slightly better purity than Nuvia cPrime resin. The following is an overview of the studies conducted during the development and optimization of Capto MMC resin.

[0408] Development and optimization of Capto MMC chromatography

[0409] Earlier studies have shown that Capto MMC resin binds all proteins very strongly at low pH (Example 1). Presumably, if hemopexin is among the more strongly bound proteins, conditions can be established under which Capto MMC can constitute an efficient capture step.

[0410] Initial evaluation of binding conditions was performed over a pH range of pH 5.0 to 7.0 with the goal of maximizing hemopexin binding while minimizing contaminant binding. At each pH, elution conditions were evaluated over a range of NaCl concentrations (50 mM, 150 mM, 300 mM, 500 mM, and 1 M). For loading at pH 7.0, the highest contaminant levels were observed in the unbound fraction. Although some hemopexin did not bind to the Capto MMC resin and was observed in the unbound fraction, in the combination of higher NaCl concentrations, approximately 50% of the loaded hemopexin was eluted at a purity level of approximately 30% ( Figure 5 ).

[0411] Based on the results of the preliminary studies, determining the optimal binding conditions required further manipulation of pH, NaCl concentration, or both. Therefore, a study was conducted to investigate binding conditions between pH 6.0 and pH 7.5 over a range of NaCl concentrations, with elution conditions between pH 7.0 and pH 7.5.

[0412] Hemopexin starting material was loaded in 200 mM NaCl at either pH 6.0 or pH 6.5, and elution at pH 7.5 resulted in high recoveries of ≥90% hemopexin. However, a higher purity (approximately 90%) was observed when loading at pH 6.5, compared to 58% purity at pH 6.0. Under the pH 6.5, 200 mM NaCl loading conditions, hemopexin appeared to be almost the only protein bound, with most other proteins eluting in the unbound fraction ( Figure 6). Clearly, these loading and elution conditions are well suited for the purification of hemopexin using Capto MMC resin.

[0413] Binding and elution conditions for hemopexin purification were established, and the hemopexin binding capacity and appropriate loading capacity of Capto MMC resin were determined. The clarified extract paste was loaded onto a 5 mL Capto MMC column to a total hemopexin loading of 250 mg / mL resin with a contact time of 7.5 minutes. Fractions were collected during the loading step and analyzed for hemopexin concentration by immunoturbidimetry.

[0414] After loading approximately 4 mg of hemopexin / mL of resin, a small amount of hemopexin was observed in the unbound fraction. This amount remained fairly stable until approximately 20 mg of hemopexin was loaded per mL of resin, after which the amount of hemopexin breakthrough increased significantly ( Figure 7 This suggests that under the current conditions, there is a certain proportion of hemopexin that does not bind to Capto MMC, and the remaining hemopexin can be captured to a capacity of approximately 20 mg / mL of resin with some moderate losses.

[0415] This is consistent with the existence of two hemopexin populations with distinct physicochemical characteristics and suggests the existence of a heme-hemopexin complex (Example 10).

[0416] The optimal hemopexin loading for the Capto MMC chromatography step was further investigated by loading varying amounts of clarified extract paste and quantifying the eluted hemopexin. The elution amount generally mirrored that seen in the unbound fraction. Recovery was fairly consistent, ranging from 70% to 75%, until the hemopexin loading exceeded approximately 15 mg / mL resin, decreasing to approximately 65% hemopexin recovery at a 20 mg / mL resin loading. Based on this, the optimal loading was determined to be 14 mg / mL resin, with 20 mg / mL resin considered the maximum acceptable loading.

[0417] Likewise, the initial constant recovery of 70% to 75% suggests the presence of a second population of hemopexins with weaker binding characteristics (Example 10).

[0418] In other brief studies, the effect of different loading contact times on column capacity was evaluated. These studies showed that a 3 minute contact time produced very similar results to the 7.5 minute contact time described above. While this would allow for more rapid loading of the feed, it was decided that a 7.5 minute contact time with a 15 cm bed height would allow the entire method to be run at a linear velocity of 120 cm / hr, which is easily achievable for most instruments and avoids potential overpressure issues at higher flow rates. A 15 cm bed height also falls within the range recommended for ease of industrial-scale column packing.

[0419] Loading conditions

[0420] A robustness study of the Capto MMC loading conditions was conducted to more specifically define the optimal loading conditions and determine the acceptable operating range for these conditions.

[0421] Capto MMC loading conditions were investigated over a pH range of 6.0 to 6.6 and NaCl concentrations from 160 to 250 mM, with FIV-4 extraction performed in Capto MMC equilibration buffer.

[0422] In the Capto MMC chromatography step, hemopexin purity and recovery were unaffected when NaCl concentrations ranged from 160 mM to 250 mM over a pH range of 6.2 to 6.6. However, material processed at pH 6.0 exhibited poorer purity and recovery, with 160 mM NaCl being inferior to 200 mM NaCl.

[0423] To determine the impact of Capto MMC loading parameters on final product quality, the material was processed using different Capto MMC loading conditions throughout a putative purification method.

[0424] After treatment with the Capto MMC chromatography step under each loading condition (except pH 6.6, 250mM NaCl), the hemopexin product (Examples 5-7) was treated by Capto Adhere chromatography, solvent detergent treatment, and Eshmuno CPS chromatography under the optimal conditions for these steps. After treatment with the Capto Adhere and Eshmuno CPS chromatography steps, an increase in hemopexin protein purity was observed, wherein all conditions demonstrated comparable final purity, with hemopexin being approximately 96% as determined by RP-HPLC (Figure 8). All loading conditions showed equivalent protein contaminant characteristics at the Eshmuno CPS eluate process intermediate, wherein only a faint transferrin band was visible by non-reducing SDS-PAGE. This indicates that the downstream processing steps of Capto Adhere and Eshmuno CPS chromatography are robust and capable of producing equivalent final products, despite the suboptimal loading conditions of Capto MMC.

[0425] From the loading condition data, the target Capto MMC chromatography loading conditions were defined as pH 6.4 and 225 mM NaCl, with an acceptable range of pH 6.2 to 6.6 and 200 to 250 mM NaCl.

[0426] Stability of Capto MMC eluate

[0427] During development, Capto MMC eluate is required as raw material for the evaluation of other chromatography resins. This can be accelerated by purifying large quantities of material that can be stored and used for these development studies. During commercial production, there may also be situations where product intermediates need to be stored. Therefore, understanding the stability of stored product intermediates is important.

[0428] To ensure that the stored material was of adequate quality, a short stability study was performed, which is summarized below.

[0429] Fraction 4-IV paste was extracted in 40 mM phosphate, 250 mM NaCl, pH 6.50 at a ratio of 1:10 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.

[0430] Partially purified hemopexin was stored at 4°C and room temperature for up to 7 days. Samples were taken at the beginning of the study (T=0), 2 days later, and 7 days later and analyzed for protein concentration, purity, monomer content, heme binding activity, and charge heterogeneity. To ensure that samples could be frozen without affecting the results, a single freeze-thaw event was performed on the samples and analyzed as described above. This enabled the use of a frozen T=0 control at each time point.

[0431] The results showed no significant changes in hemopexin under any storage conditions (see Table 2). This indicates that Capto MMC eluates can be stored at room temperature or lower for up to 7 days without compromising product quality. Alternatively, the product can be frozen at least once without detrimental effects.

[0432] Capto MMC chromatography process steps

[0433] The clarified extract paste solution (Example 3) was loaded onto a mixed mode Capto MMC column and equilibrated with 5CV of equilibration buffer (40mM sodium phosphate, 225mM NaCl, pH 6.4). The product was loaded to a target load of 14g hemopexin / L resin. After loading the product, the column was further washed with 3CV of equilibration buffer to remove unbound protein. The hemopexin was eluted with 3CV of elution buffer (40mM sodium phosphate, 150M NaCl, pH 7.5). The collection of the Capto MMC eluate began 5 minutes after entering the elution buffer application phase and continued until the UV absorbance returned to baseline. Figure 9 An exemplary chromatogram is shown. The eluted hemopexin concentration is typically between 3 mg / mL and 6 mg / mL with a purity of 70% to 85%. After product collection, the Capto MMC column is regenerated with 40 mM phosphate, 1 M NaCl pH 7.5 buffer.

[0434] Example 5 - Development and Optimization of Capto Adhere Chromatography

[0435] Initial chromatographic screening studies have identified Capto Adhere as a potential resin for the purification of hemopexin (embodiment 3). In these studies, when loaded at pH 7.5 with NaCl and KCl, hemopexin eluted from the unbound fraction of this resin with a reasonable purity. Owing to finding hemopexin in the unbound fraction, and most of other proteins being bound to the post, this resin seems best suited for the low situation of contaminating protein load. Therefore, further research was conducted to optimize the conditions of Capto Adhere as the second step, followed by partial purification by Capto MMC. The following is a general overview of the research carried out in the development and optimization of Capto Adhere resin.

[0436] Initial chromatographic screening studies showed that the flow-through fraction of Capto Adhere contained primarily hemopexin and a small amount of transferrin when operated under loading conditions of 140 mM NaCl, 30 mM KCl, pH 7.5. To optimize the binding of contaminating proteins, the effects of varying loading salt concentrations and pH conditions were investigated.

[0437] Before obtaining partially purified hemopexin (i.e., Capto MMC eluate), a first study was performed using the clarified extract paste as starting material. Combinations of loading conditions pH 6.5, 7.0, and 7.5 and NaCl concentrations of 100 mM, 150 mM, and 250 mM were investigated, and the concentrations of the main protein species in the unbound fraction were measured by immunoturbidimetry. The results showed that the highest hemopexin purity and recovery were observed under pH 7.5 mM NaCl with 100 mM NaCl loading conditions. Although most conditions were very effective in removing albumin, many other substances were still present ( Figure 10 ). Presumably, these other species remained due to the large contaminating protein load and potential overloading of the chromatography column. No further studies were performed using the clarified extract as starting material.

[0438] Because the Capto Adhere chromatography step appeared most suitable as a second purification step, hemopexin was used to further develop loading conditions after partial purification using Capto MMC resin. In this study, the Capto MMC eluate was adjusted to pH 7.5 over a range of NaCl concentrations and applied to a small Capto Adhere column equilibrated with the same buffer. Since the feed used for this experiment was 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 loading contact time of 1 minute in most cases. The 150 mM NaCl condition was then repeated at pH 7.5 and 8.0, with a contact time of 8 minutes.

[0439] At loading NaCl concentrations between 100 mM and 200 mM NaCl, very high hemopexin recoveries (92% to 97%) were observed, while the highest removal rates of contaminating proteins (primarily transferrin) were achieved at 150 mM and 8 minutes of contact time and 200 mM and 1 minute of contact time ( FIG. 11 ). These results were confirmed by SDS-PAGE analysis, which showed very high hemopexin purity between 100 mM NaCl and 200 mM NaCl ( Figure 12 ).

[0440] The elution step in the previous process step (Capto MMC chromatography) used a buffer containing 150 mM NaCl and pH 7.5, which fully matches the optimal conditions for Capto Adhere described above. Therefore, the process can be effectively simplified if the same conditions are used for Capto Adhere loading. For this reason, 150 mM NaCl and pH 7.5 were selected as the most suitable loading conditions for Capto Adhere.

[0441] The binding capacity of Capto Adher resin was determined under the loading conditions described above using partially purified hemopexin (Capto MMC eluate) and a contact time of 7.5 minutes (120 cm / hr for a column with a 15 cm bed height). A hemopexin load of 250 mg / mL resin was applied to the resin, equivalent to approximately 60 mg of contaminating protein per mL of resin. The penetration of haptoglobin, albumin, and transferrin in the unbound fraction was measured by immunoturbidimetry, and purity was assessed by SDS-PAGE.

[0442] The analysis showed that haptoglobin began to appear in the unbound fraction after loading hemopexin at 36 mg hemopexin / mL resin. This was supported by SDS-PAGE analysis, which showed excellent hemopexin purity in the fractions up to this loading level ( Figure 13 For simplicity and to provide margin for error, it was decided to set the appropriate load limit for Capto Adhere chromatography to 30 g hemopexin / L resin. Transferrin breakthrough during Capto Adhere chromatography is almost immediate, meaning that any residual levels of transferrin in the product after Capto Adhere chromatography will require additional purification steps.

[0443] Loading conditions

[0444] To determine the robustness of the Capto Adhere loading conditions, the Capto MMC eluate was pH and conductivity adjusted and loaded onto Capto Adhere under a range of loading conditions (pH 7.0 to pH 8.0, NaCl concentrations from 100 mM to 200 mM).

[0445] The hemopexin recovery rate on the Capto Adhere post is suitable for all loading conditions, and observes albumin and haptoglobin by SDS-PAGE and removes (Figure 14) almost completely.When measuring with RP-HPLC, the recovery rate of Transferrins,iron complexes, between 9% and 25%, observes higher recovery rate under 200mM NaCl concentration level, shows that higher specific conductivity has reduced the combination (Figure 14) of pollutant.These data show, the optimal loading condition for the Capto Adhere chromatographic step is that pH is between 7.0 and 8.0 and NaCl concentration is 100mM to 150mM (about 15.8-21mS / cm), to guarantee to remove Transferrins,iron complexes to greatest extent.Yet, considering that the additional cation exchange step of having proved is removed the ability (embodiment 7) of Transferrins,iron complexes, it can be feasible that NaCl (specific conductivity) scope is expanded to 200mM (about 25.6mS / cm).

[0446] During production of laboratory and pilot-scale batches, the Capto MMC eluate was adjusted from pH 7.2-7.3 to pH 7.5 and from approximately 18.5 mS / cm to 20 mS / cm before loading onto the Capto Adhere resin. Capto Adhere robustness studies 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 adjusting the pH and conductivity to simplify the process in a production setting. Small-scale runs confirmed this, with no impact on the chromatographic profile, purity, or hemopexin recovery.

[0447] Further processing runs were performed to evaluate the feasibility of online operation of Capto MMC and Capto Adhere columns. Compared to the pooled Capto MMC eluate, the online processing runs demonstrated higher levels of contaminating proteins on non-reducing SDS-PAGE gels, with stronger transferrin and haptoglobin bands visible. Lower hemopexin recovery was observed for the online processing runs, with 33% recovered from the clarified extract paste compared to 47% when operated under current conditions. Online operation of Capto MMC and Capto Adhere columns was not considered suitable for production workflows under current conditions but may be feasible with further optimization.

[0448] Capto Adhere chromatography process steps

[0449] The Capto MMC eluate obtained from the previous step typically has a pH of 7.2 and a conductivity of approximately 18 to 19 mS / cm. Although this differs slightly from the equilibration conditions used for Capto Adhere, pH and conductivity adjustments of the Capto MMC eluate are not necessary. The product is loaded onto a Capto Adhere mixed-mode column and 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 of resin, and the unbound hemopexin fraction is collected and washed with an additional 3 CV of equilibration buffer. Figure 15 An exemplary chromatogram is shown. The eluted hemopexin is typically >90% pure with a concentration between 1 mg / mL and 3 mg / mL. After product collection, the Capto Adhere column is regenerated with 40 mM phosphate, 1 M NaCl pH 7.5 buffer.

[0450] Example 6 - Virus Inactivation by Solvent Detergent Treatment

[0451] Confirmation of Solvent-Detergent Virus Inactivation

[0452] Solvent detergent (SD) treatment with 1% polysorbate 80 (PS80) and 0.3% tri-n-butyl phosphate (TnBP) represents one of two specialized virus removal / inactivation steps for the hemopexin process. This treatment disrupts the membranes of enveloped viruses and is a well-established, robust method for viral inactivation in the plasma industry. The effectiveness of SD treatment of hemopexin was demonstrated in Capto MMC eluates using pseudorabies virus (PRV) as a model. In this study, approximately 5.7 log saturation was achieved after 60 minutes of incubation. 10 The viral titer was reduced without damaging the hemopexin intermediate ( Figure 16 ).

[0453] Stability studies of Capto MMC-purified hemopexin in SD solution at 25°C for 24 hours showed no change in product purity or aggregation, and negligible changes in heme-binding activity. Combined with clearance data, this suggests that SD treatment is a viable strategy for viral reduction in hemopexin processes.

[0454] However, if the Capto MMC eluate is subjected to SD treatment (e.g., in spike / scavenging studies), SD may interfere with the subsequent Capto Adhere purification step. Furthermore, this step is unlikely to remove SD. To avoid these issues, the material is subjected to SD treatment after Capto Adhere treatment. Because protein purity increases after Capto Adhere, viral inactivation is less likely to be adversely affected by this change. This will also limit the number of purification steps required in the post-Class VI area during commercial production.

[0455] Solvent Detergent Treatment Process Steps

[0456] The Capto Adhere eluate was filtered through a 0.22 μm filter into a jacketed container and heated to 21 to 25°C. A solvent stock solution containing 20% w / w polysorbate 80 (PS80) and 6% w / w tri-n-butyl phosphate (TnBP) and a detergent solution were slowly added to the Capto Adhere eluate over 5 to 10 minutes to achieve a target concentration of 1.0% w / w PS80 and 0.3% w / w TnBP. The solvent detergent-treated stock solution was stirred using a stirring rate and impeller size that caused vortexing at approximately 10% of the container depth without causing aeration. After at least 15 minutes, the stirring rate was reduced so that vortexing occurred at approximately 5% of the container depth without causing aeration. Once the solution temperature reached 23°C, incubation was started and carried out for 2 to 24 hours under stirring, with the temperature maintained at 23 ± 2°C.

[0457] Example 7 - Ion Exchange Chromatography Using Eshmuno CPS Resin

[0458] Four kinds of ion exchange chromatography resins Capto Q, Capto DEAE, Capto S and Eshmuno CPS have been studied for the removal of solvent detergent process reagents from hemopexin process intermediates. Capto Q chromatography resin is used for early stage hemopexin process (as previously described in WO2014 / 055552), and it is proven to bind hemopexin and contaminating proteins, such as transferrin, haptoglobin and albumin. Under the NaCl concentration of 50 to 100mM, the elution of hemopexin is observed, yet transferrin is proven to be eluted altogether with hemopexin. Further studies in the pH range of pH 7.0 to 8.0 show that transferrin pollution does not further reduce.

[0459] Capto DEAE chromatography resin was observed to bind hemopexin and contaminating proteins, however, a small loss of approximately 5% of hemopexin was observed in the flowthrough fraction. Transferrin was observed to coelute with hemopexin across a range of NaCl concentrations, haptoglobin eluted at ≥150 mM NaCl, and albumin eluted at ≥200 mM NaCl. Capto S chromatography resin proved unsuitable for binding hemopexin, which was present in the flowthrough fraction.

[0460] Chromatographic resin screening studies identified Eshmuno CPS ion exchange resin as a suitable resin for the chromatographic polishing step following the solvent detergent treatment process step (Example 1). Eshmuno CPS resin demonstrated high binding capacity for hemopexin, effectively removed transferrin, and demonstrated superior purification capabilities compared to Capto Q, Capto DEAE, and Capto S resins.

[0461] Development and Optimization of Eshmuno CPS Chromatography

[0462] The Eshmuno CPS resin was initially evaluated with SD-treated Capto Adhere eluate under equilibrium and loading conditions of pH 6.0 and a conductivity of 10 mS / cm (Example 1). Elution of bound hemopexin was performed using a step gradient with 100 mM, 200 mM, and 300 mM NaCl. Under these conditions, purification of hemopexin was demonstrated, with 99% of transferrin recovered in the flow-through fraction and 98% of hemopexin recovered with elution at 300 mM NaCl. The final purity of hemopexin after Eshmuno CPS chromatography was shown in SDS-PAGE analysis ( Figure 17 ).

[0463] To simplify the operation of the hemopexin process, eluting hemopexin from the Eshmuno CPS resin in the hemopexin formulation buffer will provide the advantages of reduced diafiltration time during UF / DF and reduced amount of buffer required. A small-scale process run resulted in 99.6% elution of the loaded hemopexin with formulation buffer (0.9mM citric acid, 14.1mM sodium phosphate, 150mM NaCl pH 7.2), demonstrating the feasibility of eluting directly into the hemopexin formulation buffer. In a later study on virus filtration, it was found that a NaCl concentration of 0.6M was able to achieve a greater filter flux (Example 8). Assuming that all contaminating proteins were found in the unbound fraction in the Eshmuno CPS chromatogram, it is likely that elution could be performed at this NaCl concentration, thereby enabling a simpler transition to the virus filtration step.

[0464] The binding capacity of the Eshmuno CPS resin was determined using the Capto Adhere eluate as feedstock and the column loaded to the breakthrough point. Binding conditions were pH 6.0, 10 mS / cm as described above. Breakthrough was observed after loading 46 g of hemopexin per liter of resin ( Figure 18 Therefore, the target loading of hemopexin on the Eshmuno CPS resin was set at approximately 90% breakthrough point, 40 g hemopexin / L resin, to minimize the risk of hemopexin overloading and loss during the process steps.

[0465] The ability of Eshmuno CPS resin to remove polysorbate 80 was demonstrated by loading the sample with 1% polysorbate 80. Analysis of the flowthrough, elution, and regeneration fractions showed that the recovery of the loaded polysorbate 80 in the flowthrough fraction was 100%, indicating that polysorbate 80 was not retained by the Eshmuno CPS resin.

[0466] Loading conditions

[0467] In order to determine the robustness of loading conditions, the Capto Adhere eluate of solvent detergent processing is mixed into Transferrins,iron complexes, and is loaded onto pH between 5.8 and 6.2 and specific conductivity is on the Eshmuno CPS post of 8 to 12mS / cm.Observe and adopt pH 6.0 to 6.2 and specific conductivity is the loading conditions of 10 to 12mS / cm, and it is best that the Transferrins,iron complexes on the Eshmuno CPS post removes, but has acceptable result under 8mS / cm.Under the loading conditions of pH 5.8, in the Eshmuno CPS eluate, Transferrins,iron complexes (Figure 19) is detected by RP-HPLC under the conductivity level of 8 and 10mS / cm.Therefore, the suggestion loading conditions of the EshmunoCPS chromatographic step is pH 6.0 to 6.2 and specific conductivity is 8.0 to 12mS / cm.

[0468] Eshmuno CPS Chromatography Process Steps

[0469] The pH of the solvent / detergent treated Capto Adhere eluate was adjusted to pH 6.0 ± 0.1 with 0.5 M acetic acid and diluted to a target conductivity of 10 mS / cm with PFW or WFI. The adjusted product was loaded onto an Eshmuno CPS ion exchange chromatography column and equilibrated with 5 CV of 25 mM sodium phosphate, 25 mM sodium acetate, 38 mM NaCl, pH 6.0. The adjusted product was loaded to a target load of 40 g hemopexin / L resin. After product loading, the column was further washed with 3 CV of equilibration buffer to remove SD and unbound proteins. Hemopexin was eluted with 3 CV of 20 mM sodium phosphate, 0.6 M NaCl, pH 7.2. When the UV absorbance was ≥ 50 mAU (2 mm path length), the Eshmuno CPS eluate was collected and continued to be collected until the UV absorbance returned to baseline (A at 2 mm path length). 280nm ≤50 mAU). The eluted hemopexin is typically >95% pure, with a concentration between 6 mg / mL and 10 mg / mL. After product collection, the Eshmuno CPS column is regenerated with 40 mM phosphate, 1 M NaCl, pH 7.5 buffer.

[0470] Example 8 - Optimization of Virus Filtration for Hemopexin Process

[0471] Virus filtration represents one of two specialized virus removal / inactivation steps in the hemopexin process. The virus filtration step removes viruses based on size differences and is a well-established, robust method for viral reduction in the plasma industry. As previously described in WO2014 / 055552, virus filtration methods from the first-generation hemopexin process using Planova BioEX virus filters laid the foundation for further development of the virus filtration step. Literature indicates that various factors influence nanofiltration, including pressure, protein concentration, solution conductivity, and pH, and the impact of each of these factors on virus filtration flux has been investigated. The following summarizes the development research conducted on the virus filtration step.

[0472] Development and optimization of virus filtration

[0473] Initial studies have shown that higher pressures result in greater filtration flux, with 113 L / m at 2 bar compared to 113 L / m at 2 bar. 2 In comparison, 152 L / m was observed at 3 bar for 1.5 hours. 2 However, a higher decay rate was also observed for filtration at 3 bar. When the data were fitted with an exponential decay model, it showed that the maximum flux was higher at the lower pressure of 2 bar, compared to 371 L / m at 3.0 bar. 2 Compared with 473L / m 2 , in contrast to the flux data at 1.5 hours. The lower flux 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 lower pressures, thus preferring higher pressures for viral clearance. Although the results of this study indicated that the flux at 3 bar was lower than that at 2 bar, the flux achieved was acceptable for commercial production of hemopexin. This, combined with the time saved at 3 bar and its preferred pressure for viral clearance, led to the decision to use 3 bar for all subsequent studies.

[0474] Development studies to optimize viral filtration flux were conducted around the pH and conductivity of the Eshmuno CPS eluate using a Sartopore 2XLM prefilter coupled to a BioEX filter.

[0475] No significant differences in filtration flux and flux decay were observed for the Eshmuno CPS eluate over 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 virus filtration would provide little or no flux advantage.

[0476] For the initial hemopexin process, as previously described in WO2014 / 055552, the conductivity of the pure hemopexin material used for virus filtration was equivalent to 600 mM NaCl. Early development studies of the second generation process also showed excellent flux at high conductivity. Confirmation studies showed that medium to high conductivity resulted in increased flux, with CPS eluates with conductivity values of 37 mS / cm (600 mM NaCl) and 54 mS / cm (1 M NaCl) showing equivalent flux, which was 13% higher than 18 mS / cm (150 mM NaCl). Figure 20 ). Despite the changes in flux, the rate of flux decay does not vary significantly with conductivity, suggesting that increased conductivity may not increase the permeability of the filter, or reduce the fouling rate, but rather allow the solution to enter a larger available area, or a greater number of pores.

[0477] During pilot scale production, low virus filtration fluxes were observed, ranging from only 45-70 L / m 2 When the ratio of pre-filter to virus filter surface area was investigated as a possible cause, the flux was shown to be significantly improved when a larger pre-filter area was used. In this experiment, a 0.75:1 pre-filter area:virus filter area ratio resulted in an approximately 30% increase in flux (compared to a 0.22:1 ratio). Figure 21A ). Flux decay also decreased with increasing prefilter area, suggesting that larger differences can become apparent with increasing load. To account for differences in flux decay and sample volume limitations, data from subsequent experiments were fitted to exponential curves and extrapolated to determine the theoretical maximum flux.

[0478] 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 predicted that 551 L / m 2 could be achieved using this prefilter at a filter area: virus filter area ratio of 0.25:1. 2 When the filter area: virus filter area ratio is 1.67:1, the maximum flux can rise to 1040L / m 2 This is significantly better than the maximum throughput achieved with the Sartopore XLM and allows the use of smaller Virosart Max prefilters.

[0479] Evaluation of alternative virus filters as an alternative for Asahi Kasei BioEX identified the Sartorius Stedim Virosart HF filter as a suitable replacement, with a maximum throughput of 1068 L / m 2, slightly higher than the 1044L / m of BioEX in the same experiment 2 Sartorius Stedim Virosart HC and Merck-Millipore Viresolve Pro filters are not suitable replacements because both filters have lower maximum flux than the BioEx filters. Since BioEX has performed early viral validation studies for the hemopexin process and was used to produce material for the hemopexin Phase 1 studies mentioned elsewhere herein (as described in WO2014 / 055552), it remains the preferred filter. However, the flux of the Virosart HF filter highlights its potential use as a replacement for the BioEX filter, if needed.

[0480] Virus spike experiments

[0481] The effectiveness of the Asahi BioEX filtration step was evaluated by its ability to remove the model virus MVM (parvovirus of mice) from the purified hemopexin. MVM (a worst-case model virus for filtration due to its small size) was spiked into the Eshmuno CPS eluate and filtered with a 0.0003 m 2 Filtration was performed on Asahi BioEx filters with a filter area of 100 μg / ml. Excellent viral clearance was achieved throughout the filtration process by evaluating relevant sample fractions under worst-case combined conditions for viral penetration. ≥7.2 log was also achieved for a representative pooled library. 10 This indicates that the BioEX filter provides sufficient virus clearance to be implemented in the hemopexin process.

[0482] Virus Filtration Process Steps

[0483] The Eshmuno CPS eluate was filtered through a 0.1 μm prefilter connected in series with a Planova BioEX filter at a pressure of 3.0 ± 0.1 bar and a filter area of 1 m 2 / 100LEshmuno CPS eluate. The pre-filter area should be at least 0.6 times the area of the virus filter. Wash the filter with three BioEX capsule volumes of virus filter wash buffer and combine with the reserve filtrate to form the BioEX filtrate.

[0484] Example 9 - Concentration and diafiltration of BioEX filtrate

[0485] The concentration and diafiltration process steps used in the original hemopexin process, as previously described in WO 2014 / 055552, were used in the current process without modification. The concentration and diafiltration process steps were successfully implemented into the hemopexin process, and no further development or optimization of the process steps was performed.

[0486] Example 10 - Identification and Quantification of Heme-Hemopexin Complexes in FIV-4 Paste

[0487] During the development of the Capto MMC chromatography process step, it was observed that the recovery of hemopexin was very low, with a significant hemopexin fraction observed in the unbound fraction. This appeared to vary depending on the batch of paste used. The fact that this occurred at low column load and under optimal binding conditions (Example 6) led to the hypothesis that a population of hemopexins with distinct physicochemical characteristics existed. Early studies demonstrating a lack of binding to heme agarose and literature review suggested that this population may consist of heme-hemopexin complexes.

[0488] In a key study, a 50% mixture of hemopexin and hemopexin complex was applied to Capto MMC under the above conditions, and the protein was monitored by absorbance at 280 nm and the hemopexin complex was monitored by absorbance at 411 nm. The results clearly showed that pure hemopexin eluted in the bound fraction and the heme-hemopexin complex eluted in the unbound fraction ( Figure 23 ).

[0489] While a lack of binding of the heme-hemopexin complex to Capto MMC has been demonstrated, the presence of the complex in the extract paste has not been confirmed. To address this issue, the heme-hemopexin complex was isolated from the unbound fraction of the Capto MMC-purified extract paste. Under the process operating conditions used for hemopexin purification, the heme-hemopexin complex was found to not bind to Capto Adhere, allowing this procedure to be used to purify the complex from the Capto MMC unbound fraction. In this experiment, with low-volume loading of Capto Adhere, the majority of contaminating proteins remained bound to the Capto Adhere resin, and the purified heme-hemopexin complex was collected in the flow-through fraction. The resulting purified heme-hemopexin complex contained a significant amount of transferrin by SDS-PAGE; however, absorption spectroscopy revealed a peak at 414 nm for the heme-hemopexin complex, while the absence of a peak at 475 nm for holo-transferrin was observed. This clearly demonstrates the presence of the heme-hemopexin complex in the Capto MMC unbound fraction.

[0490] This result indicates that the amount of heme-hemopexin complex can be estimated using Capto Adhere Impres chromatography using a small injection volume. Under these conditions, hemopexin and the heme-hemopexin complex elute as a single broad peak in the unbound fraction, while all other proteins are retained. Since the extinction coefficient of hemepexin at 280 nm and the extinction coefficient of the heme-hemopexin complex at 414 nm are almost identical, the proportion of the heme-hemopexin complex can be estimated by the ratio of the 414 nm peak area to the 280 nm peak area.

[0491] Preliminary analysis indicated that approximately 15% of the hemopexin in the clarified extract paste was present as a heme-hemopexin complex, although this was found to vary depending on the batch of paste.

[0492] A second assay was developed to measure the content of heme-hemopexin complex using a combination of RP-HPLC and size exclusion HPLC (SE-HPLC) with detection at 412 nm. Size exclusion HPLC of the extracted paste enabled identification of a fairly well-resolved peak for the heme-hemopexin complex, which could be integrated and quantified relative to a standard curve. The concentration of free hemopexin was determined by RP-HPLC, and from these two data sets, the fraction of hemopexin complexed with heme could be determined. This assay was used to quantify the proportion of heme-hemopexin complex in nine different batches of paste and showed that the amount of the complex varied widely, ranging from 3.7% to 17.2% of total hemopexin, depending on the paste batch.

[0493] Taken together, the data from the studies described above demonstrate that the amount of complex can vary significantly between different paste batches. Because the heme-hemopexin complex is not recovered during the hemopexin purification process, a clear understanding of the amount of complex is required to fully understand process recovery.

[0494] Example 11 - Process Description

[0495] The final process developed involves purifying hemopexin from FIV-4 paste using three chromatographic steps. After paste solubilization and clarification, hemopexin is purified by Capto MMC and Capto Adhere chromatography before viral inactivation via solvent / detergent treatment. The product is then polished using an Eshmuno CPS salt-tolerant cation exchange column and nanofiltered to remove viruses before diafiltration into formulation buffer.

[0496] While Examples 2-10 outline most of the progress in each of these steps, some further optimization and simplification were performed following extensive bench-scale and pilot-scale purification campaigns.

[0497] The final process including all optimizations is as follows Figure 24 shown.

[0498] Example 12 - Process Reproducibility

[0499] After initial development and optimization, the purification process was run three times to assess reproducibility. Although this study was conducted before all optimization and efficient streamlining of the process was complete, most process parameters were established at this stage with only minor changes made thereafter. The process was essentially developed according to Figure 24 Extraction was performed as described, but the extraction was performed in 180 mM NaCl at pH 6.2 with a paste:buffer ratio of 1:10, and the Capto MMC column was equilibrated in the same buffer.

[0500] Three batches were produced at a small laboratory scale from three batches of paste (Batch 1, 2, and 3). Batches 1 and 2 were derived from adsorption batches used for Berinert / Beriplex production. Process intermediates and the final purified product were analyzed for hemopexin concentration by immunoturbidimetry and RP-HPLC, and for purity by SDS-PAGE. The activity of the final purified product was further analyzed by heme binding.

[0501] The results showed that the process was highly reproducible regardless of the batch of paste used, and the purity and specific activity of the three batches of hemopexin were almost identical (Table 3 and Figure 25 In all cases, purity was very high, exceeding 99% as determined by SDS-PAGE and densitometry, with heme-binding activity ranging from 99.6% to 104.9% of total protein. While there was some variation in these activity values, they remained very high and likely reflected assay variability rather than process or batch inconsistencies.

[0502] One possible reason for variation in yield and recovery is the amount of heme-hemopexin complex present in the starting material. The hemopexin in this complex is included in the starting material quantification, but the complex is removed during the Capto MMC chromatography step of the process. Because the amount of complex varies between paste batches, this can lead to batch-to-batch variations in yield and recovery.

[0503] When comparing recoveries for each process step, it's clear that the majority of process losses occur during the Capto MMC chromatography step. This is somewhat expected, as the heme / hemopexin complex is removed during this step. Nevertheless, fairly consistent recoveries ranging between 61% and 71% were observed for this step. Recoveries for all other steps were high and consistent.

[0504] In summary, the data show that process yields and recoveries were fairly consistent between batches of paste. In all cases, product quality was very high and consistent, with no significant differences evident between the pastes that underwent Beriplex / Berbinert adsorption.

[0505] Example 13 - Large-Scale Laboratory Production

[0506] During the development of the hemopexin process, bench-scale batch production was conducted to provide baseline data and demonstrate process feasibility. Four bench-scale batches were produced at the 3 kg FIV-4 paste scale and processed into the hemopexin drug substance. During these batches, numerous changes to the hemopexin process were made, and process conditions were optimized.

[0507] Initial lab-scale batch

[0508] Initial bench-scale batches were run using a 1:10 FIV-4 paste to buffer extraction ratio. The extracts were clarified by depth filtration and purified by chromatography using Capto MMC and Capto Adhere resins. Following the initial two chromatography steps, the product was treated with a solvent detergent to inactivate the virus, and the solvent detergent was removed by chromatography using Capto Q resin. The resulting process intermediate was subjected to virus filtration using BioEX filters (bench-scale batch 2 only) before UF / DF into the formulation buffer to produce hemopexin DS.

[0509] These two bench-scale batches have been successfully produced to the drug substance stage.

[0510] Overall, the quality of hemopexin purified from these initial laboratory-scale batches was quite high, with heme specific activity >90%, hemopexin monomer content >95%, and contaminating proteins reduced to below detectable levels (Table 4). The quality of the final product was also quite consistent between the two batches.

[0511] Optimized lab-scale batches

[0512] After optimizing several process conditions, an additional batch was produced to evaluate the entire process at a bench-scale. Conditions for the FIV-4 extraction step (Example 4) were optimized, and the filter frame depth was increased to 4 cm to allow for higher solids loading. Loading conditions for the Capto MMC chromatography step (Example 4) were improved, the Capto Q chromatography step was replaced with Eshmuno CPS (Example 7), and various buffer conditions were optimized. The modified process conditions are listed in Table 5.

[0513] An optimized laboratory-scale batch was successfully produced to the drug substance stage, achieving a 73% hemopexin recovery throughout the entire process. Because a different batch of FIV-4 paste was used in this batch, the improved recovery compared to the previous batch is likely due to the varying amounts of hemopexin and heme / hemopexin complex present. Using a more appropriate frame size could also improve recovery through more efficient recovery from the clarification step.

[0514] The produced hemopexin drug substance had a heme-binding specific activity of 97%, with a monomer content of >99%, and the contaminating proteins albumin, transferrin, and haptoglobin reduced to undetectable levels (Table 6). Overall, the optimized bench-scale batches demonstrated that the modified conditions resulted in good process recovery and a highly pure hemopexin product.

[0515] Efficient and streamlined lab-scale batching

[0516] Following pilot-scale batch production (Example 14), further optimization was performed to streamline the process for efficient scale-up. Increasing the NaCl concentration in the extraction buffer eliminated the need to adjust the conductivity prior to Capto MMC chromatography (Example 2). It was also discovered that Capto Adhere chromatography (Example 5) could be performed without adjusting the pH and conductivity of the Capto MMC eluate.

[0517] A streamlined, bench-scale batch was produced to confirm that process changes did not affect the purity and yield of hemopexin. The bench-scale batch was produced up to the Eshmuno CPS eluate stage, where hemopexin was eluted from the column at lower NaCl concentrations to provide material for viral filtration conductivity studies. The modified process conditions are listed in Table 7.

[0518] The observed overall process recovery for the high-efficiency simplified batch was 55%, with a yield of 0.18 g / LPEQ. These recoveries and yields are low, but consistent with those of previous batches. Because different batches of paste were used in this study, the amounts of hemopexin and heme / hemopexin complex may have played a role. Overall, the high-efficiency simplified conditions are unlikely to have contributed to the reduced recovery.

[0519] By SDS-PAGE analysis, hemopexin was of considerable purity after Capto MMC chromatography, with no visible contaminating bands after subsequent chromatography steps (Figure 26). Very high purity was obtained after Eshmuno CPS chromatography, consistent with previous laboratory-scale batches.

[0520] Example 14 - Pilot scale batch production

[0521] Pilot-scale batches are conducted to demonstrate that the process is scalable and capable of producing consistent product intermediates and hemopexin drug substance. Additionally, these pilot-scale batches help identify potential process issues that may arise during large-scale production.

[0522] Two pilot-scale batches were produced at the 20 kg FIV-4 paste scale using the same batch of FIV-4 paste as starting material. The process followed the parameters listed in Figure 44 with the following exception: the paste was thawed at 4°C for 2.5 days prior to extraction with a buffer containing 225 mM NaCl at pH 6.4. Due to maximum pump speed limitations, the extract was split in half and clarified using a filter area appropriate for half the paste mass before recombining the filtrates. Due to limited column hardware availability, Capto Adhere and Eshmuno CPS columns were not large enough to accommodate the entire Capto MMC eluate. When the Capto MMC column was loaded to capacity, this meant that only approximately 65% of the Capto MMC eluate passed through the remainder of the process, with the remainder being discarded. While wasteful, this allowed for assessment of product quality with all chromatographic steps fully loaded.

[0523] In both pilot-scale batches, the virus filtration flux was surprisingly low, with a total flux of only 47 L / m for batches 1 and 2, respectively. 2 and 70L / m 2 This led to further development work around the filtration conditions, as discussed above in Example 10. Following this development work, two additional large-scale batches were run under conditions similar to the pilot scale, and this material was used to validate virus filter flux using the optimized conditions. Essentially, these conditions involved using a 0.1 m 2 BioEx Virus Filter 650cm2 Sartopore XLM prefilters and feed conditions with a conductivity of 37 mS / cm. Under these conditions, all available material was easily filtered with only moderate decay. For the first of these batches, the maximum throughput was calculated to be 521 L / m 2 , in the second batch it was 232L / m 2 While it is unclear why these two fluxes differ, they are clearly a substantial improvement over those seen in the first two pilot-scale batches.

[0524] Pilot-scale intermediate and process recovery

[0525] When comparing the two pilot-scale batches, recoveries were comparable for all steps, with the exception of the Capto Adhere chromatography step (Table 11). It is suspected that the low recovery observed in this step in batch B was due to insufficient product mixing before sampling, resulting in inaccurate concentration measurements. A similar error was also evident in the Eshmuno CPS recovery calculation, which returned a value of 130% in the same batch. The comparable overall process recoveries for the two batches are also incompatible with such variations in step recoveries.

[0526] In the deep filtration and clarification steps, approximately 80% recovery of hemopexin was observed for both batches, 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 the loss of product volume in this step, possibly due to insufficient post-filter press washing. In the second pilot-scale batch (Batch B), variable recoveries of contaminating proteins ranging from 60% to 90% were observed in the deep filtration and clarification steps. In this case, sampling variability is suspected.

[0527] The majority of contaminating proteins were removed during the Capto MMC step, with approximately 85% of the total hemopexin recovered (Tables 11 and 12). At least some of the loss of hemopexin can be attributed 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 extracts used in the pilot-scale batch indicated that up to 20% of the total hemopexin may be present in the form of heme complexes. This readily explains the loss of most of the hemopexin during this step.

[0528] Minimal protein or hemopexin loss was observed during the Eshmuno CPS chromatography, virus filtration, UF / DF, and sterile filtration process steps, with approximately 100% recovery observed.

[0529] The overall process recovery of hemopexin was approximately 50-60%. As described above in Example 10, FIV-4 paste contains a portion of hemopexin complexed with heme, which varies between paste batches. Since the heme-hemopexin complex is undesirable in the final product and is removed during the Capto MMC step of the process, it is understood that recovery of free hemopexin is more appropriate. When the concentration of the complex is taken into account, the recovery of free hemopexin for both pilot-scale batches was approximately 70%, indicating a high recovery of usable product.

[0530] In summary, the data outlined above show that fairly consistent recoveries are achieved at each step of the process, and throughout the process when performed at pilot manufacturing scale.

[0531] The process produced 50 and 51 grams of purified hemopexin from the two pilot-scale batches, derived from the equivalent of 13 kg of FIV-4 paste (corrected for previously discarded material from Capto Adhere). This equates to an input of 0.15 grams of purified hemopexin per liter of plasma.

[0532] Pilot-scale intermediate product quality

[0533] The purity of the process intermediates was consistent with expectations for both pilot batches, with the majority of contaminating proteins removed during the Capto MMC stage and the remaining transferrin almost completely removed in subsequent steps. The purity profile of each process intermediate was nearly identical across both batches.

[0534] Protease activity varied widely between the two batches of extract paste (Table 8). It is suspected that the high values seen in batch A were due to the presence of filter aid in the sample, leading to protease activation during the time between sampling and analysis. The sample in batch B was filtered immediately after sampling to prevent further activation by this mechanism. Regardless, in both batches, protease activity became very low after clarification and remained low throughout the remainder of the run.

[0535] Prokallikrein activator was found to be <20 IU / mL in the extract paste and remained at this level throughout the run, regardless of batch (Table 9).

[0536] Taken together, these data demonstrate that the process produces consistently pure hemopexin at every step. Protein contaminants, proteases, and PKA are consistently removed and present at very low levels in the final drug substance, even at a hemopexin concentration of 100 mg / mL.

[0537] Characterization of Hemopexin API

[0538] The pilot-scale process produced highly pure active hemopexin that met or exceeded all quality standards defined by the QTPP for Phase 1 production (Table 10). This was emphasized by very high values for specific activity, percentage of monomer, and purity as determined by non-reducing SDS-PAGE ( Figure 27 Specific protein contaminants albumin, haptoglobin, transferrin, and apolipoprotein A1 were reduced to very low levels, as were TnBP and polysorbate 80. Kallikrein activator activity was reduced to acceptable levels, and residual protease activity was very low.

[0539] The two pilot-scale batches produced highly comparable Hemopexin DS, with very similar results for all quality parameters. As the same batch of paste was used for both pilot-scale batches, this highlights the reproducibility of the process.

[0540] Example 15 - Production-scale purification method

[0541] Step 1: FIV-4 paste extraction

[0542] Prior to clarification, the hemopexin-containing Fraction IV-4 (FIV-4) paste was resuspended in 40 mM sodium phosphate, 400 mM NaCl pH 7.5 ± 0.1 (conductivity of about 36 to about 38 mS / cm) at a 1:2.5 w / w paste:buffer ratio by stirring at room temperature for about 20 to about 120 minutes.

[0543] Step 2 - Extract paste deep filtration and clarification

[0544] The FIV-4 extract paste solution was clarified by depth filtration to remove undissolved material and particles, and then further clarified by passing through a 0.22 μm filter. Briefly, the FIV-4 extract paste solution was depth filtered using a STAX disposable filter cartridge and EK1P filter media. The solution was then filtered at 6.25 L / m 2 The FIV-4 extract paste solution is filtered at a flow rate of 1000 ng / min, which typically results in a pressure of <1.5 bar during filtration. The filter press is then post-washed until the protein concentration is below 0.2 g / L, reaching a maximum of 2.0 filter volumes of Capto MMC equilibration buffer (40 mM sodium phosphate, 225 mM NaCl, pH 6.4; conductivity of approximately 23 to approximately 25 mS / cm). After depth filtration, the pH of the depth-filtered extract is adjusted to pH 6.4 ± 0.1 with 0.5 M HCl, and the conductivity is adjusted to 27 mS with 5 M NaCl or water for injection (WFI), as appropriate. Filters of 0.5 μm or smaller, such as Further clarify the depth-filtered extract using a 0.2 μm or similar filter. After filtration, store the clarified extract solution at <23°C overnight or at 2-8°C for up to 48 hours before processing by CaptoMMC chromatography.

[0545] Step 3 – Mixed-mode chromatography using Capto MMC resin

[0546] The clarified extract solution (27 ± 2 mS / cm) obtained from the previous step was loaded onto a pre-equilibrated mixed-mode Capto MMC column to a target load of 14 g hemopexin / L resin. 280nm The hemopexin concentration of the clarified extract was estimated by ÷13, and this value was used to calculate the loading volume. After loading the product, the Capto MMC column was washed with equilibration buffer (40 mM sodium phosphate, 225 mM NaCl pH 6.4; conductivity of about 23 to about 25 mS / cm). When the eluate collection was complete, the Capto MMC column was regenerated.

[0547] Hemopexin was eluted from the column with 40 mM sodium phosphate, 150 mM NaCl, pH 7.5 (conductivity of about 18 to about 19 mS / cm). The eluted hemopexin concentration was typically between 3 mg / mL and 6 mg / mL and 70% to 85% pure. The Capto MMC eluate was stored overnight at <23°C or at 2-8°C for up to 48 hours prior to further purification by Capto Adhere chromatography.

[0548] Step 4 – Mixed-mode chromatography using Capto Adhere resin

[0549] The Capto MMC eluate (typically pH 7.2, conductivity of approximately 18 to 19 mS / cm) obtained from the previous step is loaded onto a pre-equilibrated mixed-mode Capto Adhere chromatographic column (Cytiva) to a target load of 30 g hemopexin / L resin. Unbound hemopexin fractions are collected and further washed with equilibration buffer (40 mM sodium phosphate, 150 mM NaCl, pH 7.5, conductivity of approximately 18 to approximately 19 mS / cm) for 3 x CV to collect any remaining hemopexin. When the washing step is complete, the Capto Adhere column is regenerated. The hemopexin concentration of the eluted column is typically between 1 mg / mL and 3 mg / mL, and has a purity of >90%. The Capto Adhere eluate is directly transferred to a solvent detergent treatment. If desired, the eluate can be stored overnight at <23 ° C, or for 7 days at 2-8 ° C, before the solvent detergent treatment.

[0550] Step 5 - Pooling and concentration of Capto Adhere

[0551] The flow-through from the Capto Adhere column (Capto Adhere effluent; conductivity of approximately 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 of approximately 18 to approximately 19 mS / cm) to reduce the volume for subsequent S / D treatment and Eshmuno CPS column loading steps. The Capto Adhere concentrate, having a hemopexin content of approximately 20 mg / mL, was then stored at <23°C overnight or at 2-8°C for up to 7 days prior to solvent detergent treatment.

[0552] Step 6 - Solvent Detergent Viral Inactivation

[0553] A solvent / detergent (SD) treatment step is 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) is added to the concentrated CaptoAdhere flow-through / eluate to a target concentration of 0.3% w / w and 1.0% w / w, respectively, in the final product solution. The product solution containing TnBP and PS80 is incubated at 21-25°C for a period of 4-24 hours.

[0554] Step 7 – Ion exchange chromatography using Eshmuno CPS resin

[0555] The pH of the solvent / detergent-treated solution (conductivity of approximately 18-19 mS / cm) obtained from the previous step was 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 of resin. After product loading, the column was further washed with equilibration buffer (25 mM sodium phosphate, 25 mM sodium acetate, 38 mM NaCl, pH 6.0; conductivity of approximately 8.5-9.5 mS / cm) to remove SD and unbound protein. Hemopexin was eluted with 20 mM sodium phosphate, 0.6 M NaCl, pH 7.2 (conductivity of approximately 50 to approximately 52 mS / cm), and the column was regenerated by washing with 1 M NaCl.

[0556] Step 8 – Nanofiltration with Planova BioEX to remove viruses

[0557] This step constitutes the second viral reduction step in the hemopexin production process. Briefly, the EshmunoCPS eluate was passed through a filter with a filter area of 1 m 2 The eluate of Shmuno CPS was filtered through a Planova BioEX filter in series with a 0.1 μm prefilter.

[0558] The viral filtrate can be stored at <23°C overnight or at 2-8°C for up to 7 days before processing by ultrafiltration and diafiltration.

[0559] Step 9 - Concentration and diafiltration of BioEX filtrate

[0560] The BioEX filtrate was concentrated and buffer-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)) with a nominal molecular weight cut-off of no more than 10 kDa and a 0.01 mM 2 / L BioEX filtrate membrane area. The material was concentrated to a hemopexin concentration of 100 mg / mL.

[0561] Step 10 - Sterile Filtration

[0562] The hemopexin UF bulk stock solution is sterile filtered into an appropriate sterile container using a sterile 0.22 μm sterilizing grade filter at a pressure of <3 bar. The filtration is preferably performed aseptically in a laminar flow cabinet using sterile tubing and connectors. The sterile hemopexin solution can be stored at 2 to 8°C or frozen at -80°C.

[0563] Example 16 - Conductivity

[0564] In this study, the conductivity of the solutions 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 fraction IV-4 plasma fraction was used at a 1.5 kg scale. Figure 30The plasma fraction was filtered using a filter press before being loaded onto the Capto MMC resin, following the purification process described in

[15] . Conductivity measurements were taken twice at each step, using two different probes to allow simultaneous readings: probe PD (with a cell constant of 0.4750) and a recalibrated probe PP (with a cell constant of 0.4200). Multiple conductivity readings were taken for buffers and steps requiring conductivity adjustment.

[0565] Table 14 shows the buffer conductivity ranges, and Table 15 shows the in-process conductivity ranges.

[0566] The results show equivalent conductivity measurements using different cell constants. Through bench-scale runs, the conductivity measurements recommended for production were shown to be equivalent.

[0567] in conclusion

[0568] The strategy used to develop a hemopexin purification process involved screening a large number of candidate resins in both high-throughput and bench-scale configurations. Based on these screening studies, the top candidate resins were optimized for hemopexin purity, binding capacity, recovery, and operational simplicity. Virus clearance was performed by solvent / detergent treatment and nanofiltration, and nanofiltration conditions were optimized.

[0569] Following this strategy, a commercially viable method for the purification of hemopexin from Fraction IV-4 paste was developed. The method comprises three chromatographic purification steps: Capto MMC, Capto Adhere, and Eshmuno CPS, with two viral inactivation steps: solvent / detergent treatment and nanofiltration.

[0570] Because the step with the lowest binding capacity is Capto MMC chromatography, the process capacity is best expressed as a function of column size, allowing for scalability. For each liter of Capto MMC column volume, this process yields approximately 8-10 grams of pure hemopexin, making it a high-capacity process. With a 250-L column size, this would allow processing of approximately 450 kg of FIV-4 paste per run.

[0571] Because the heme-hemopexin complex is not purified in this process, an adequate estimate of process recovery requires knowledge of the amount of complex in the FIV-4 paste. This can be estimated by size exclusion chromatography or Capto Adhere chromatography monitored at 414 nm. When the concentration of the complex is omitted, the recovery of active (uncomplexed) hemopexin in the entire process is 70-72%. If the recovery is calculated based on the total amount of hemopexin present (including the complex), the recovery will be variable, depending on the concentration of the complex.

[0572] During development studies, the yield ranged from 0.15 g / L PEQ to 0.18 g / L PEQ. Similarly, the yield relative to plasma equivalent volume varied with the amount of complex in the starting material.

[0573] The quality of hemopexin purified using this procedure is very high, typically around 99% purity as measured by non-reducing SDS-PAGE, with approximately 99% monomer content. The protein activity exceeds 95%, and trace contaminants are very low.

[0574] The process was demonstrated to be fully reproducible and suitable for large-scale production through the successful production of two pilot-scale production batches.

[0575] Overall, this method is well suited for commercial production of high-quality hemopexin, as discussed in Example 16.

[0576] Table 1. OD280 of different batches of FIV-4 paste and concentrations measured by RP-HPLC

[0577]

[0578] Table 2. Amount of heme-binding protein after storage of Capto MMC eluate

[0579]

[0580] Table 3. Yield, recovery, and quality data of hemopexin purified from several batches at a small laboratory scale.

[0581] 2 10 1 Yield (g / LPEQ;neph) 0.12 0.15 0.15 Recovery rate (%; neph) 54 61 57 Purity (%; SDS-PAGE) 99.9 99.8 99.7 Specific activity (%)* 104.9 100.5 99.6

[0582] *Specific activity - the amount of hemopexin that can bind heme divided by the amount of total protein.

[0583] Table 4. Characterization results of the hemopexin drug substance from the initial bench-scale batch.

[0584]

[0585] Table 5. Modified process conditions for optimized bench-scale batches.

[0586]

[0587] Table 6. Characterization results of optimized laboratory-scale batches of hemopexin drug substance

[0588]

[0589] Table 7. Modified process conditions for efficient simplification of laboratory-scale batches.

[0590]

[0591] Table 8. Protease activity in intermediate fractions of pilot scale batches

[0592]

[0593] Table 9. Prokallikrein Activators in Intermediate Fractions from Pilot Scale Batches

[0594]

[0595] Table 10. Characterization results of sterile hemopexin drug substance

[0596]

[0597]

[0598] Table 11. Stepwise recovery of pilot scale batches of hemopexin as measured by RP-HPLC

[0599]

[0600] Table 12. Stepwise recoveries of transferrin, albumin, and haptoglobin from pilot-scale batches.

[0601]

[0602] T = transferrin; A = albumin; H = haptoglobin.

[0603] Table 13. Process recoveries for pilot-scale batches

[0604]

[0605] Table 14. Processed conductivity measurements

[0606]

[0607] *Indicates that the range is not provided, but the expected reading is provided

[0608] Table 15. Processed conductivity measurements

[0609]

[0610] *Indicates that this is not a conditioning step and provides expected readings.

Claims

1. A method for purifying hemopexin from a solution containing hemopexin and other proteins, the method comprising: (i) providing a solution comprising hemopexin and other proteins, wherein the solution comprises 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 selective binding of hemopexin to the resin over binding of the other proteins to the resin; (iii) washing the resin after step (ii) to remove unbound protein; (iv) eluting the hemopexin bound to the resin after step (iii); and (v) recovering the hemopexin eluted in step (iv).

2. The method of claim 1, wherein the mixed-mode cation exchange chromatography resin has a structure of formula (I) or (II):

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

4.

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

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

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

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

11. The method of claim 10, wherein the solution of step (i) comprises: (a) a pH of about 6.4; (b) about 40 mM phosphate buffer; and (c) About 225 mM NaCl.

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 / mL of resin.

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

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

17. The method of claim 16, wherein the solution of step (i) is derived from Cohn fraction IV4.

18. The method of claim 16 or claim 17, wherein the solution of step (i) is prepared by (a) resuspending the Cohn Fraction IV in 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).

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

20. The method of claim 19, wherein step (a) comprises resuspending Cohn Fraction IV in extraction buffer at a ratio of about 1:2.5 Cohn Fraction IV:extraction buffer.

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

5.

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

5.

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

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

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

4.

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 about 24 mS / cm to about 30 mS / cm.

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

33. The method of any one of claims 1 to 32, further comprising: (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 the hemopexin to pass through the resin as an unbound fraction; and (vii) recovering the unbound fraction comprising hemopexin.

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

0.

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

5.

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

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

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

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

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

42. The method of claim 40 or claim 41, wherein 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).

43. The method of any one of claims 38 to 42, further comprising: (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); as well as (xi) eluting the hemopexin bound to the resin in step (ix); and (xii) recovering the eluted hemopexin from step (xi).

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

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 about 8 mS / cm to about 12 mS / cm.

48. 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 about 10 mS / cm.

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

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

52. The method of claim 51, wherein the virus filtering comprises passing the recovered hemopexin through a virus 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. A composition comprising a combination of any two or more features selected from the group consisting of: (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 accounting for 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 the 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 the total protein as determined by size exclusion-high performance liquid chromatography; (1) 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) a protease activity level of less than about 5 nKat / L; (o) a prokallikrein 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.

55. A composition comprising a combination of any two or more features selected from the group consisting of: (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 the 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; (1) 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 prokallikrein 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.

56. 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 (iii) a heme-specific binding activity of at least about 97% of the total protein.

57. The composition of claim 56, further comprising (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 haptoglobin.

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

59. The composition of claim 56 or claim 57, further comprising (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.

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

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

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

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

65. The method of claim 64, wherein the condition is acute hemolysis or chronic hemolysis.

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

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

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

69. The use of claim 68, wherein the condition is selected from the group consisting of hemolytic anemia, transfusion hemolysis, hemolytic uremic syndrome, autoimmune disease, malarial infection, trauma, blood transfusion, open heart surgery with cardiopulmonary bypass, and burns, post-burn hemoglobinemia with hemolysis, or hemoglobinuria.

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

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

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

73. 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 dyserythropoietic anemia and paroxysmal nocturnal hemoglobinuria, systemic lupus erythematosus, and chronic lymphocytic leukemia.

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