KR102125695B1 - A method of purifying proteins - Google Patents

Abstract

The present invention relates generally to protein purification methods. More specifically, the present invention relates to a method for purifying haptoglobin and hemopexin from the same starting material, and uses thereof.

Description

Protein purification method {A METHOD OF PURIFYING PROTEINS}

The present invention relates generally to protein purification methods. More specifically, the present invention relates to a method for purifying haptoglobin and hemopexin from the same starting material, and uses thereof.

Hemolysis is characterized by the destruction of red blood cells, and red blood cell abnormalities, such as enzyme deficiency, hemoglobinopathy, hereditary globular hemoglobinemia, paroxysmal nocturnal hemoglobinuria and acupuncture cell anemia, and external factors, such as Spleen hypertrophy, autoimmune disorders ( e.g., hemolytic disease in newborns), genetic disorders ( e.g. sickle cell disease or G6PD deficiency), microangiopathic hemolysis, Gram-negative bacterial infections ( e.g. strepto Lactococcus (Streptococcus), Enterococcus (Enterococcus) and Staphylococcus (Staphylococcus)), parasitic infections (e. g., plastic Modified Titanium (Plasmodium)), toxins and trauma (e.g., characteristics of anemic disorders associated with the image) to be. In addition, hemolysis is a common disorder in patients with transfusions, especially mass transfusions and extracorporeal cardio-pulmonary support.

The side effects seen in patients with hemolysis-related conditions are largely due to the release of iron and iron-containing compounds from red blood cells, such as hemoglobin (Hb) and heme. Under physiological conditions, released hemoglobin is bound by soluble proteins, such as haptoglobin, and transported to macrophages and hepatocytes. However, when the incidence of hemolysis is accelerated and becomes pathological in nature, the buffering capacity of haptoglobin is subdued. As a result, hemoglobin rapidly oxidizes to ferri-haemoglobin, which eventually releases free heme (including protoporphyrin IX and iron). Heme plays an important role in some biological processes ( eg, as part of essential proteins, such as hemoglobin and myoglobin), but free heme is very toxic. Free heme is a source of redox-active iron, which produces highly toxic reactive oxygen species (ROS) that damage lipid membranes, proteins and nucleic acids. Heme toxicity is further exacerbated by its ability to intercalate with lipid membranes, where it causes oxidation of membrane components and promotes cell lysis and death.

The gradual pressurization of sustained low-level extracellular Hb/hem exposure resulted in a compensatory mechanism to control the side effects of free Hb/hem under physiological steady-state conditions and during moderate hemolysis. These systems include the release of a group of plasma proteins that bind Hb or heme, including Hb scavenger haptoglobin (Hp) and heme scavenger proteins hemopexin (Hx) and α1-microglobulin. However, endogenous Hp and Hx control the side effects of free Hb/hem under physiological steady-state conditions, but they maintain steady-state Hb/hem levels under pathological conditions, such as pathological conditions associated with hemolysis. As it has little effect.

The present invention provides a method for purifying Hp and Hx from the same starting material. The purified protein can be used in compositions for treating conditions associated with hemolysis and abnormal Hb/hem levels.

Summary of the invention

In one aspect of the invention, a method for purifying haptoglobin and hemopexin from a solution containing both haptoglobin and hemopexin protein, the method comprising:

(i) providing a solution containing both haptoglobin and hemopexin;

(ii) precipitating haptoglobin from the solution by adding ammonium sulfate to the solution;

(iii) separating the precipitated haptoglobin from the solution containing hemopexin; And

(iv) separately purifying haptoglobin and/or hemopexin in one or more steps

A method for purifying haptoglobin and hemopexin from a solution containing both haptoglobin and hemopexin protein is provided.

In another aspect of the invention, a composition is provided comprising haptoglobin recovered by a method disclosed herein.

In another aspect of the invention, a composition is provided, comprising hemopexin recovered by a method disclosed herein.

In another aspect of the invention, a composition is provided, comprising transferrin recovered by a method disclosed herein.

In another aspect of the invention, a composition is provided comprising haptoglobin recovered by a method disclosed herein and hemopexin recovered by a method disclosed herein.

In another aspect of the invention, a composition is provided comprising a haptoglobin content of at least 95% of the total protein. In another aspect of the invention, a composition is provided comprising a hemopexin content of at least 80% of the total protein. In another aspect of the invention, a composition is provided comprising a combined hemopexin and haptoglobin content of at least 80% of the total protein.

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

In another aspect of the invention, there is provided a method of treating a condition related to hemolysis, comprising administering a composition or formulation of the invention as disclosed herein to a subject in need thereof. .

In another aspect of the invention, the use of a composition or formulation of the invention as disclosed herein in the manufacture of a medicament for treating a condition associated with hemolysis is provided.

1 is a flow diagram of a cone fractionation process (Cohn Fractionation Process). Those skilled in the art will appreciate that variations on the process parameters described in FIG. 1 (eg, pH, ethanol concentration, temperature, etc.) can also be used to generate the cone fraction.
FIG. 2 shows transferrin (TRF), albumin (Alb), hemopexin (HPX) and haptoglobin in the residual filtrate following precipitation in the presence of 2.0M, 2.5M, 3.0M, 3.5M and 4.0M ammonium sulfate. HAP)d.
3 shows a preferred degree plot of the experimental (DOE) design, which shows the preferred conditions in which haptoglobin can precipitate from the plasma fraction while maintaining hemopexin in solution. DOE is a set of controlled experiments used to evaluate the effect of pH and ammonium sulfate concentration on the ability to separate hemopexin from haptoglobin. Designed using industrial mathematical design, data was analyzed. The preferred degree plot shown in FIG. 3 uses this mathematical design to determine the most desirable conditions that would result in the best separation and recovery of both hemopexin and haptoglobin.
4 shows SDS-PAGE electrophoresis of hemopexin recovered according to various steps in the purification process disclosed herein. SDS-PAGE analysis was performed using a pre-cast 10% Tris-glycine gel (Novex, EC6075). All samples were diluted in Tris-glycine SDS sample buffer (LC2676) at a concentration of 0.1 mg/mL, and 20 μL of each sample was loaded into the sample well of the gel. Run times and voltages were set according to the gel manufacturer's recommendations (125V constant voltage). Each gel was stained with an easy-to-use type of Coomassie Brilliant Blue staining solution (Novex, Simply Blue Safestain, LC6065). Lane 1 (LMWS) contains Novex Sharp Unstained Protein Standards, LC5801.
5 shows SDS-PAGE electrophoresis (using the method described above in FIG. 4) of haptoglobin intermediates recovered according to various steps in the purification process disclosed herein. The haptoglobin standard in Lane 2 is from Benesis (T043HPX).
FIG. 6 shows a Western blot of the Capto Q eluate on a 10% Tris-glycine non-reducing SDS-PAGE electrophoresis gel (using the method described in FIG. 4). The isolated proteins are then transferred to the nitrocellulose membrane and the membrane is blocked to prevent any non-specific binding of the antibody. The nitrocellulose membrane is then incubated with a solution containing antibodies to human haptoglobin (rabbit anti-human haptoglobin, Sigma H8636). The secondary antibody linked to horseradish peroxidase (HRP) is then incubated with nitrocellulose membrane (goat anti-rabbit HRP, Sigma A6154). Subsequently, nitrocellulose is developed into a solution containing peroxide to visualize only protein bands specifically containing human haptoglobin. Lanes used Capto Q ImpRes eluate (T0209001) at different concentrations (lane 2-1:10 dilution, 0.1 mg/mL, 20 μL loaded; lanes 3-1:20; lanes 4-1:40; lanes 5-1: 80; Lane 6-1:160; Lane 7-1:320; Lane 8-1:640; Lane 9-1:1280; and Lane 10-1:2560). Western blots indicate that most of the bands shown in the Capto Q eluate are haptoglobin.
FIG. 7 shows SDS-PAGE electrophoresis (using the method described in FIG. 4) of transferrin recovered from ion-exchange chromatography (Capto DEAE) according to various steps in the purification process disclosed herein. Peak 1 (lane 4) is heavily loaded, but is found to be pure transferrin (compared to lane 2, human transferrin, Sigma T8158). This indicates that it is possible to purify transferrin from the octyl sepharose wash fraction, and also it is possible to purify hemopexin, haptoglobin, and transferrin from the same starting material.
8 shows a chromatogram of transferrin, a linear concentration gradient from an ion-exchange chromatography column during the purification process disclosed herein. Peaks labeled 1-4 were analyzed by SDS-PAGE in FIG. 7.
9 is a flow diagram of a hemopexin purification process according to one embodiment disclosed herein.
10 is a flow diagram of a haptoglobin purification process according to one embodiment disclosed herein.
11 is a flow diagram of a combined haptoglobin/hemopexin/transferrin purification process according to embodiments disclosed herein.

Throughout this specification, unless the context requires otherwise, variations such as the words “comprises” or “comprises” or “comprising” refer to the inclusion of the stated element or integer or elements or group of elements. It will be understood that it does not imply the exclusion of any other element or integer or group of elements or integers.

References to any existing publications (or information derived therefrom) or to any known material in this specification include references to existing publications (or information derived therefrom) or known materials. It is not considered and should not be considered as a knowledge or permit or any form of proposal that forms part of the common general knowledge in the field of attempts.

It should be noted that the singular forms “one”, “one” and “above” as used in the subject specification include multiple aspects unless the context clearly indicates otherwise. Thus, for example, reference to “one resin” includes a single resin, and two or more resins; Reference to “the above composition” includes a single composition, and two or more compositions.

In the absence of any contrasting indication, references made to “%” content throughout this specification should be considered to mean% w/w (weight/weight). For example, a solution comprising a haptoglobin content of at least 95% of the total protein is considered to mean a solution comprising a haptoglobin content of at least 95% w/w of the total protein.

The present invention is predicted for the discovery that haptoglobin and hemopexin can be purified, at least in part, from the same starting material. Thus, in one aspect of the invention, a method for purifying haptoglobin and hemopexin from a solution containing both haptoglobin and hemopexin protein, the method comprising:

(i) providing a solution containing both haptoglobin and hemopexin;

(ii) precipitating haptoglobin from the solution by adding ammonium sulfate to the solution;

(iii) separating the precipitated haptoglobin from the solution containing hemopexin; And

(iv) separately purifying haptoglobin and/or hemopexin in one or more steps

A method for purifying haptoglobin and hemopexin from a solution containing both haptoglobin and hemopexin protein is provided.

Haptoglobin (Hp) is a 4-chain (α ₂ β ₂ ) glycoprotein that is synthesized by the adult liver and secreted into the plasma. The propeptide form of Hp is proteolytically cleaved into α- and β-chains. The two α-subunits and two β-subunits of the Hp protein are then joined by interchain disulfide bonds to form a mature peptide, which can be (αβ)-dimer or (αβ)-multimer. Hemopexin (Hx) is a 60-kD plasma β-1B-glycoprotein comprising a single 439 amino acid long peptide chain that forms two domains linked by an interdomain linker. It has the best known affinity for heme of any characterized heme-binding protein (Kd<1pM) and is in equimolar ratio between the two domains of Hx in the pocket formed by the interdomain linker. Bind to heme.

We separate both proteins from the same starting material with an ammonium sulfate concentration in the range of about 2.0M to about 2.5M, preferably in the range of about 2.2M to about 2.5M, more preferably about 2.4M. It was found to be optimal. Thus, in one embodiment, the method includes precipitating haptoglobin from the solution by adding about 2.0M to about 2.5M ammonium sulfate to the solution. In another embodiment, the method comprises precipitating haptoglobin from the solution by adding about 2.2M to about 2.5M ammonium sulfate to the solution. In another embodiment, the method includes precipitating haptoglobin from the solution by adding about 2.4M ammonium sulfate to the solution. In certain embodiments, the ammonium sulfate concentration is 2.0M or 2.1M or 2.2M or 2.3M or 2.4M or 2.5M.

In addition, the inventors have found that a pH maintained in the range of about 8 or less, preferably about 6 to about 8, more preferably about 7, is optimal for separating both proteins from the starting material. Thus, in one embodiment, the method comprises precipitating haptoglobin from the solution at a pH of 8 or less. In another embodiment, the method includes precipitating haptoglobin from the solution at a pH in the range of about 6 to about 8. In another embodiment, the method comprises precipitating haptoglobin from the solution at a pH of about 7. In certain embodiments, the method sums from the solution at a pH within the range of 6.0 to 8.0, or 6.25 to 7.75, or 6.5 to 8.0, or 6.5 to 7.75, or 6.5 to 7.5, or 6.75 to 7.25, or 6.75 to 7.5. And precipitating toglobin. The pH is typically measured in haptoglobin and hemopexin solutions, and then during the addition of ammonium sulfate and precipitation of haptoglobin. The pH can be adjusted as needed (typically the concentration of acid (eg HCl) or base (eg NaOH) used to adjust the pH is in the range of 0.05M to 0.6M).

In the methods disclosed herein, the majority of haptoglobin from the starting material will be found in the ammonium sulfate precipitate, and the majority of hemopexin from the starting material will be found in the residual solution (also referred to as suspension). However, a person skilled in the art, the precipitate may contain some hemopexin ( e.g. , trace Hx), and the residual solution (or suspension) may contain some haptoglobin ( e.g. trace Hp). It will be understood that it may include. When trace amounts of Hp and Hx are present in the suspension and precipitate respectively, it may be desirable to remove them by separately purifying haptoglobin and/or hemopexin in one or more steps according to the methods disclosed herein. However, those skilled in the art can understand that, for example, when both proteins are eventually present in the same composition, trace amounts of Hp and Hx, respectively, which may be present in the suspension and sediment are acceptable.

Any solution comprising both haptoglobin and hemopexin can be used as starting material in the methods of the invention disclosed herein. In certain embodiments, the solution containing both haptoglobin and hemopexin further comprises transferrin. Suitable starting materials are known to those skilled in the art, examples of which include various supernatants and precipitates derived from plasma fractionation, eg, ethanol fractionation processes. Examples of such ethanol fractionation processes include Cohn fractionation and Keithler-Nitschmann fractionation. Examples of suitable plasma fractions are derived from the cone fractions I, II, III, II+III, I+II+III, IV and V (see Figure 1) or the Kisler-Nisschmann fraction, e.g. sediment A or B. Included. In one embodiment, the solution is a human plasma fraction. In another embodiment, the solution is Cone Fraction IV. In another embodiment, the solution is cone fraction IV ₄ . In certain embodiments, cone fraction IV ₄ is derived from cone fraction II+III or cone fraction I+II+III. In a particularly preferred embodiment, the solution is derived from fraction IV ₄ precipitate.

It will be appreciated that if the starting material is provided as a precipitate ( eg , Fraction IV ₄ precipitate), the precipitate will need to be initially resolubilized to provide a starting solution suitable for the method of the present invention. The buffer used to resolubilize the precipitate can be any agent or combination of agents that has a buffering capacity at approximately pH 7 (eg, ADA, PIPES, ACES, MOPSO, MOPS, BES, TRIS). Can). Typically, the buffer will be present at a concentration of about 5mM to about 100mM. In one embodiment, the resolubilization buffer is 50 mM Tris, pH 7. In some embodiments, resolubilization buffer may be added at about 5 to about 30 grams per gram of starting material. In certain embodiments, resolubilization buffer is added at 5-10 grams per gram of starting material, added at 10-15 grams per gram of starting material, added at 15-20 grams per gram of starting material, or starting material It is added at 20 to 25 grams per gram of, or at 25 to 30 grams per gram of starting material.

The method of the invention is suitable for commercial/industrial scale purification of hemopexin, haptoglobin, and optionally transferrin. For example, only using a plasma fraction as a starting material, using the method of the invention on a commercial/industrial scale, may include the use of a plasma fraction derived from at least about 500 kg of plasma. More preferably, the plasma fraction will be derived from plasma of at least about 5,000 kg, 7,500 kg, 10,000 kg and/or 15,000 kg per batch.

A person skilled in the art is aware that the plasma for fractionation is a liquid portion of blood remaining after separating cellular elements from blood collected in a receptacle containing an anticoagulant, or any known to those skilled in the art. It will be appreciated that by other suitable means, for example, it is a liquid portion of blood separated by continuous filtration or centrifugation of the anticoagulated blood in apheresis procedures.

In one embodiment, a solution containing precipitated haptoglobin, and hemopexin is recovered and stored separately prior to separately purifying haptoglobin and/or hemopexin in one or more steps according to the present invention. In another embodiment, a solution containing precipitated haptoglobin and/or hemopexin is recovered and immediately subjected to further purification steps according to the method of the present invention; That is, haptoglobin and/or hemopexin are separately purified in one or more steps.

Thus, in one embodiment, the method comprises:

(i) dissolving the precipitated haptoglobin in a buffer solution to obtain a haptoglobin solution;

(ii) passing the haptoglobin solution through an anion exchange chromatography resin under conditions such that the haptoglobin binds to the anion exchange chromatography resin;

(iii) eluting the haptoglobin from the resin; And

(iv) recovering the eluted haptoglobin

It further includes.

In certain embodiments, the anion exchange chromatography resin of step (ii) above is a strong anion exchange chromatography resin. In another embodiment, the anion exchange chromatography resin of step (ii) is a weak anion exchange chromatography resin.

Purification of the protein by chromatography uses an axial flow column, such as those available from GE Healthcare, Pall Corporation, Millipore and Bio-Rad, or a radial flow column, for example For example, it can be performed using those available from Proxcys or Sepragen. In addition, chromatography can be performed using an expanded bed technique known to those skilled in the art.

Most chromatographic processes use solid supports, which are also referred to interchangeably as resins or matrices herein. Suitable solid supports will be familiar to those skilled in the art, and the choice will depend on the type of product being purified. Examples of suitable solid supports include inorganic carriers such as glass and silica gels, organic, synthetic or naturally occurring carriers such as agarose, cellulose, dextran, polyamides, polyacrylamides, bifunctional acrylates Vinyl copolymers, and various hydroxylated monomers, and the like. Commercially available carriers are solids under the trade names Sephadex™, Sepharose™, Hypercel™, Capto™, Fractogel™, MacroPrep™, Unosphere™, GigaCap™, Trisacryl™, Ultrogel™, Dynospheres™, Macrosorb™ and XAD™ resins.

The chromatography step will generally be performed under non-denaturing conditions, and at a convenient temperature of about -10°C to +30°C, more typically at about ambient temperature. The chromatography step can be performed batch-wise or continuously as desired. Any convenient separation method can be used, such as column, centrifugation, filtration, or decanting.

Suitable buffers for dissolving haptoglobin precipitates will be familiar to those skilled in the art and may depend on the conditions required to perform the chromatography purification step. Typically, the buffer will have a buffer (ie Tris) concentration from about 5 mM to about 100 mM. In certain embodiments, the buffer is from about 10 mM to about 60 mM. In addition, those skilled in the art will recognize that a buffer may contain more than one buffer. Examples of suitable buffers are sodium acetate and Tris with a pH range of 5.5 to 9.0. In some embodiments, the pH of the buffer used to dissolve the haptoglobin precipitate is pH 7.5 to 9.0, or pH 7.75 to 9.0, or pH 8.0 to 9.0, or pH 8.25 to 9.0, or pH 8.4 to 8.6. In a preferred embodiment, the buffer is Tris at about 50 mM with a pH of about pH 8.4 to about pH 8.6.

In embodiments, the lipid content of the extracted precipitate comprising haptoglobin (i.e., haptoglobin solution) is obtained by exposing the haptoglobin solution to a lipid remover under conditions that allow the lipid to bind to the lipid remover. Is reduced. Examples of such lipid removers include fumed silica, for example Aerosil. In one embodiment, the lipid remover is fumed silica. In one embodiment, the fumed silica is Aerosil (eg Aerosil 380). In embodiments, a lipid scavenger such as Aerosil may be added at about 0.5 g to about 4 g per liter of plasma equivalent to the extracted precipitate comprising haptoglobin. In certain embodiments, a lipid removal agent, such as Aerosil, is added at 1 to 2 g per liter of plasma equivalent to the extracted precipitate comprising haptoglobin. In another embodiment, a lipid remover, such as fumed silica (e.g. Aerosil), is added to the extracted precipitate containing haptoglobin at 1.6 to 1.8 g per liter of plasma of sugar. In another embodiment, a lipid removal agent, such as Aerosil, is added at an equivalent of 1.8 to 2.0 grams per liter of plasma to the extracted precipitate comprising haptoglobin. In another embodiment, a lipid removal agent, such as Aerosil, is added at 1.6 g per liter of plasma equivalent to the extracted precipitate comprising haptoglobin. Lipid removal was determined to be the most effective within a certain pH range. A pH range of 5.5 to 9.0 was found to be effective with Aerosil. In some embodiments, the pH range is 6.5 to 8.6. In other embodiments, the pH during the lipid removal step is pH 5.5 to 9.0, or pH 5.75 to 9.0, or pH 6.0 to 9.0, or pH 6.25 to 9.0, or pH 6.5 to 9.0, or pH 6.75 to 9.0, or pH 7.0 To 9.0, or pH 7.25 to 9.0, or pH 7.5 to 9.0, or pH 7.75 to 9.0, or pH 8.0 to 9.0, or pH 8.25 to 9.0, or pH 8.4 to 9.0 or pH 8.6 to 9.0. Preferred embodiments use a pH range of 8.4 to 8.6.

Lipid removers can be removed using methods such as filtration or centrifugation. In certain embodiments, the lipid removal agent is removed by depth filtration. Examples of depth filters for use in this application are Cuno 70CA or similar or smaller particle size retention filters.

Those skilled in the art can use haptoglobin solution using any anion exchange chromatography resin, as long as haptoglobin can bind to the chromatography resin, while allowing certain impurities in the haptoglobin solution to pass through the resin. It will be understood that haptoglobin can be purified separately. In certain embodiments, the anion exchange chromatography resin is a strong anion exchange resin. In other embodiments, the anion exchange chromatography resin is a weak anion exchange resin. In addition, those skilled in the art, due to the ionic strength of the extraction buffer and subsequent pH adjustment of the loading solution; It can be determined that dilution, dialysis filtration, chromatographic desalting, or other methods of buffer exchange/reduction of ionic strength may be required to allow haptoglobin to bind to the resin. Suitable resins are known to those skilled in the art. Examples of suitable anion exchange resins are anion exchange resins comprising functional quaternary amine groups (Q) and/or diethylaminopropyl groups (ANX). In one embodiment, the strong anion exchange chromatography resin comprises functional quaternary amine groups ( eg , Capto Q ImpRes™).

Solutions suitable for equilibration of the chromatography medium (often also referred to as equilibration buffers) generally have a buffer concentration of about 5 mM to about 100 mM. The pH of the equilibration buffer will typically be in the range of about 5 to about 9, and the conductivity is typically less than about 9 mS/cm. These conditions generally enable the binding of haptoglobin to the anion exchange medium. In certain embodiments, the buffering agent will be in a concentration range from about 10 mM to about 60 mM. In certain embodiments, the pH of the equilibration buffer is within a pH range of about 5.0 to about 9.0. In some embodiments, the pH is in the range of about 5.0 to about 7.0, or about 5.0 to about 6.0, or about 5.3 to about 5.7. In certain embodiments, the pH of the equilibration buffer is pH 5.3±0.1, or pH 5.4±0.1, or pH 5.5±0.1, or pH 5.6±0.1, or pH 5.7±0.1, or pH 5.8±0.1. In some embodiments, the conductivity of the equilibration buffer is less than about 7.0 mS/cm. In certain embodiments, the conductivity of the equilibration buffer is less than 6.0 mS/cm, or less than 5.0 mS/cm. An example of a buffer for the equilibration buffer is sodium acetate. Certain embodiments use 50mM sodium acetate to a pH in the range of 5.0 to 6.0, and have a conductivity of less than 6.0mS/cm. Other embodiments use about 10 to about 40 mM sodium acetate at a pH of about 5 to about 6. In certain embodiments, the equilibration buffer comprises 50 mM sodium acetate with a pH in the range of pH 5.3 to pH 5.7, and a conductivity of less than 5.0 mS/cm.

The haptoglobin solution passed through the anion exchange chromatography resin may initially require buffer exchange, desalination or dilution to reduce ionic strength to enable binding to the resin. Solutions suitable for buffer exchange, desalting or dilution include solutions having a buffer at a concentration of about 5 mM to about 100 mM. The pH range of these solutions is typically in the range of about pH 5 to about 9. On the other hand, the conductivity is generally less than about 8.0 mS/cm. In certain embodiments, the solution for buffer exchange, desalination or dilution will include a buffer in the range of about 10 mM to about 60 mM, with a pH range of about 5 to about 9 and a conductivity of less than about 7.0 mS/cm. Examples of solutions for buffer efficacies, desalting or dilution are sodium acetate. Certain embodiments use 50 mM sodium acetate in a pH range of 5.0 to 6.0 with a conductivity of less than 6.0 mS/cm. Another embodiment uses about 10 to about 40 mM sodium acetate at a pH of about 5 to about 6.

Other embodiments include dilution of the haptoglobin loading solution by addition of water (eg, water for injection (WFI)) to reduce ionic strength. For example, at 1:4 to 1:5 dilution with WFI, a haptoglobin solution comprising 50 mM sodium acetate at pH 5.3 to 5.7 can have a final sodium acetate concentration of about 10 to 13 mM at pH 5.3 to 5.7. And may have a conductivity of less than 5.0 mS/cm. This generally allows the binding of haptoglobin to the anion exchange chromatography resin.

If pH adjustment is required beyond the buffering range of the buffer/buffers in the haptoglobin solution, additional buffer may be added to the haptoglobin solution. Additional buffers will typically have a concentration of about 5 mM to about 100 mM, and a pH range of about 5 to about 9. In certain embodiments, additional buffering agents will be in the range of about 10 mM to about 60 mM at a pH range of about 5 to about 9. An example of an additional buffer is sodium acetate. Certain embodiments use 50 mM sodium acetate in a pH range of 5.0 to 6.0. In a preferred embodiment, the buffer is 50 mM sodium acetate at a pH in the range of pH 5.3 to pH 5.7.

In addition, suitable buffers for eluting haptoglobin from the resin will be known to those skilled in the art. In certain embodiments, suitable buffering agents will have a concentration within the range of about 5 mM to about 100 mM. In certain embodiments, the buffering agent will be in the range of about 10 mM to about 60 mM. One example includes sodium acetate. Certain embodiments use 50 mM sodium acetate at a pH of 5.0 to 6.0. Another embodiment uses about 10 to about 40 mM sodium acetate buffer at a pH of about 5.0 to about 6.0. In a preferred embodiment, the buffer is about 50 mM sodium acetate to a pH of about pH 5.3 to about pH 5.7.

In a further embodiment, haptoglobin is eluted from the anion exchange resin using an elution buffer comprising 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 certain embodiments, haptoglobin elutes in the presence of about 150-170 mM NaCl. In a preferred embodiment, haptoglobin elutes in the presence of about 160 mM NaCl. However, those skilled in the art, the NaCl concentration of the elution buffer will depend on the protein load applied to the column, and adjustments beyond the limits described to achieve the necessary recovery and purity of haptoglobin eluted from the resin may be required. Will know.

In one embodiment, the eluted haptoglobin is recovered and stored separately for future use. In other embodiments, the eluted haptoglobin can be concentrated and dialyzed through, for example, the eluted haptoglobin through an ultrafiltration membrane and/or sterile filtered concentrated and/or dialysis filtered haptoglobin as needed. It is further purified.

In one embodiment, the method comprises:

(i) passing a solution containing the hemopexin through a hydrophobic interaction chromatography resin under conditions that cause the hemopexin to bind to a hydrophobic interaction chromatography resin;

(ii) collecting the flow-through fraction from step (i);

(iii) optionally washing the resin after step (ii) and collecting the flow-through washing fraction;

(iv) eluting the hemopexin from the resin after step (ii) and/or step (iii); And

(v) recovering the eluted hemopexin

It further includes.

Hydrophobic interaction chromatography (HIC) is a frequently used chromatographic technique for separation of proteins based on hydrophobic interactions between stationary phases and isolated proteins. The hydrophobicity level of the target protein will often influence the type of HIC resin used. During HIC, a high amount of salt is typically added to the solution to reduce the solubility of the target protein, thus target protein with HIC resin functionalized with suitable hydrophobic groups ( eg , phenyl, butyl and octadecyl groups). Increases the interaction of Suitable salts typically include sodium sulfate, potassium sulfate, ammonium sulfate, ammonium chloride, sodium chloride, sodium bromide, and sodium thiocyanate. Suitable hydrophobic interaction chromatography resins may be familiar to those skilled in the art. Examples include octyl sepharose and capto octyl chromatography resin. The conditions that cause hemopexin to bind to the resin will be known to those skilled in the art and will depend, for example, on the type of resin used and the hydrophobicity of the target protein ( ie , hemopexin).

The hemopexin load solution contains ammonium sulfate, and thus, a buffer suitable for column equilibration includes a buffer at a concentration of about 5 mM to about 100 mM, which can maintain a pH of about 6 to about 8, approximately 2 to 2.5M Ammonium sulfate. These conditions may enable the binding of hemopexin to hydrophobic interaction chromatography media. Another embodiment uses 2.2 to 2.5 M ammonium sulfate at pH 7.0 to 8.0. In certain embodiments, the buffer is 50 mM Tris containing 2.5 M ammonium sulfate at a pH of 7.4.

Hemopexin load solutions typically contain from about 2.0 to about 2.5 M ammonium sulfate buffered to a pH of about 6 to about 8. These conditions allow binding of hemopexin to the hydrophobic interaction medium. In another embodiment, the hemopexin load solution contained 2.2M to 2.5M ammonium sulfate buffered to a pH of 7.0 to 8.0. In certain embodiments, the hemopexin load solution contains 50 mM Tris as buffer, and 2.5 M ammonium sulfate to a pH of 7.4.

Once the solution containing hemopexin is passed through a hydrophobic interaction chromatography (HIC) resin, as described herein, the flow-through fraction can be collected and stored for future use.

The bound hemopexin can be eluted from the resin by means known to those skilled in the art. Before eluting hemopexin from the resin, the resin can optionally be washed with a suitable wash or buffer under conditions that maintain hemopexin bound to the resin. Suitable washing liquids and conditions will be known to those skilled in the art. The wash liquor concentration depends on the degree to which the column is loaded, but typical wash liquors will have a buffering effect at a pH of about 6 to about 8, and further contain approximately 0.8M to 1.5M ammonium sulfate. In another embodiment, the wash liquor contains 0.9M to 1.3M ammonium sulfate to a pH of 7.0 to 8.0. In a specific embodiment, the wash solution contains 50 mM Tris as a buffer, 1.13 M ammonium sulfate at a pH of 7.4. In addition, the flow through wash fraction can be collected and stored for future use as needed.

The eluted hemopexin recovered from the resin can be stored for future use. In addition, the eluted hemopexin can be further purified to remove any impurities in the eluate. Thus, in one embodiment, the method comprises:

(i) passing the eluted hemopexin through a metal ion affinity chromatography resin under conditions such that the hemopexin binds to the metal ion affinity chromatography resin;

(ii) eluting the hemopexin from the resin; And

(iii) recovering the eluted hemopexin

It further includes.

Immobilized metal ion affinity chromatography (IMAC) is based on the covalent attachment of amino acids ( e.g. , histidine) to metals, which means that the protein is immobilized metal ion, e.g. zinc, cobalt, Try to have affinity for the metal ions retained in the column containing nickel or copper. Suitable metal ion affinity chromatography resins may be known to those skilled in the art. In one embodiment, the metal ion affinity chromatography resin is Ni-Sepharose.

Suitable buffers for equilibration and binding are designed to prevent non-specific binding to the chromatography medium, and are optimized to enhance the affinity of hemopexin while minimizing the binding of contaminant proteins. The buffer used for equilibration and binding can generally be in the pH range of about 6 to about 9, with the addition of a small amount (i.e. 1 to 50 mM) of histidine or imidazole, about 0.5M to about 1.0M sodium chloride It may include. In certain embodiments, the buffer may consist of about 0.5 mM to 100 mM sodium phosphate at a pH of about 7 to about 8, about 0.5 M to about 1.0 M sodium chloride, and about 1 mM to about 50 mM imidazole. In a specific embodiment, the equilibration and binding buffer consists of 20 mM sodium phosphate at pH 7.4, 0.5 M NaCl, and 30 mM imidazole. The loading solution (octyl eluate) can be adjusted to these concentrations through addition of a preferred solid excipient or by addition of a concentrated solution.

Once hemopexin is bound to a metal ion affinity chromatography (IMAC) resin, the resin can be washed to remove any remaining unbound or weakly bound impurities under conditions that maintain hemopexin bound to the resin. have. Suitable buffers for the washing step may be in the pH range of 6.0 to 9.0, and may include the addition of 0.5M to 1.0M sodium chloride with the addition of a small amount (i.e. 1 to 50mM) of histidine or imidazole. In some embodiments, the buffer may consist of about 0.5 mM to about 100 mM sodium phosphate from about pH 7 to about pH 8, about 0.5 M to about 1.0 M sodium chloride, and about 1 mM to about 80 mM imidazole. have. In certain embodiments, the wash buffer consists of 20 mM sodium phosphate to pH 7.4, 0.5 M NaCl, and 30 mM imidazole.

Bound hemopexin can be eluted from the resin by means known to those skilled in the art. Suitable buffers for elution can be in the pH range of about 6 to about 9, and can include the addition of about 0.5M to about 1.0M sodium chloride with histidine or imidazole at a concentration high enough to allow for elution. . In some embodiments, the elution buffer may consist of about 0.5mM to about 100mM sodium phosphate, from about 0.5M to 1.0M sodium chloride, and less than about 80mM imidazole from about pH 7.0 to about pH 8.0. In certain embodiments, the elution buffer consists of 20 mM sodium phosphate at pH 7.4, 0.5 M NaCl, and 100 mM imidazole. Alternatively, bound hemopexin can be eluted through application of a low pH buffer of about pH 4.0 to about pH 6.0. In some embodiments, about 5 mM to about 100 mM sodium acetate buffer at a pH of about 4.0 to about pH 6.0 as elution buffer can be used with about 0.5 M to about 1.0 M NaCl. The eluted hemopexin can be further purified, for example, by concentrating the hemopexin through an ultrafiltration membrane and dialysis filtration and/or sterile filtration of the concentrated and/or dialysis filtered hemopexin as necessary.

In addition, the inventors have discovered that any transferrin that may be present in the starting material remains in solution ( ie , in a solution containing hemopexin) after precipitation of haptoglobin in the presence of ammonium sulfate. Thus, the methods of the invention disclosed herein can also be used to purify transferrin from the same starting material. Thus, optimal conditions are provided to purify all three proteins (Hp, Hx and transferrin) from the same starting material. Thus, in one embodiment, a solution containing both haptoglobin and hemopexin ( eg , starting material) will further comprise transferrin.

In one embodiment, the method comprises:

(a) passing a solution containing the hemopexin through a hydrophobic interaction chromatography resin under conditions that allow the hemopexin to bind to a hydrophobic interaction chromatography resin;

(b) collecting a flow-through fraction from step (a);

(c) optionally washing the resin after the step (b) and collecting the flow-through washing fraction;

(d) the flow-through fraction from step (b) and/or the flow-through wash fraction from step (c), through anion exchange chromatography resin, under conditions that allow transferrin to bind to the anion exchange chromatography resin. Passing through; And

(e) recovering the transferrin from the resin

It further includes.

In certain embodiments, the anion exchange chromatography resin of step (ii) above is a strong anion exchange chromatography resin. In another specific embodiment, the anion exchange chromatography resin of step (ii) is a weak anion exchange chromatography resin.

Suitable strong anion exchange chromatography resins will be known to those skilled in the art. Examples of suitable anion exchange resins include those containing functional quaternary amine groups (Q) and/or diethylaminopropyl groups (ANX). In one embodiment, the strong anion exchange chromatography resin comprises functional quaternary amine groups ( eg , Capto Q ImpRes™).

Suitable weak anion exchange chromatography resins will be known to those skilled in the art. Examples include resins comprising tertiary or secondary amine functional groups, such as DEAE (diethylaminoethyl).

Those skilled in the art also, due to the ionic strength of the HIC wash buffer; It can be determined that dilution, dialysis filtration, chromatographic desalting, or other methods of buffer exchange/reduction of ionic strength may be required to allow transferrin to bind to the anion exchange resin. One method of such chromatographic desalination is that the HIC wash fraction (transferrin) containing from about 0.8M to about 1.5M of ammonium sulfate is bound directly onto a stronger hydrophobic ligand, such as a phenyl or butyl column. Includes. Once bound, transferrin can be eluted in WFI or low ionic strength buffers of formulations for anion exchange chromatography.

Buffers suitable for anion exchange chromatography equilibration and binding enable transferrin transfer to anion exchange media to facilitate chromatographic separation of transferrin from other impurities. Suitable buffers for equilibration of the chromatography medium have a concentration of about 5 mM to about 100 mM, a pH range of about 6 to about 9, and a conductivity of less than about 9.0 mS/cm. These conditions enable transferrin binding to anion exchange media. In certain embodiments, the buffering agent will be in the range of about 10mM to about 60mM, has a pH range of about 6 to about 9, and will have a conductivity of less than about 7.0mS/cm. Examples of suitable buffering agents include Tris and sodium phosphate. Certain embodiments use Trim of 50 mM to a pH range within the range of about 7 to about 9 with a conductivity of less than about 6.0 mS/cm. Another embodiment uses a Tris buffer of about 10 to about 40 mM to a pH of about 7.0 to about 9.0 with a conductivity of less than about 5.0 mS/cm.

In addition, buffers suitable for eluting transferrin from the resin will also be known to those skilled in the art. In certain embodiments, suitable buffering agents will have a concentration ranging from about 5 mM to about 100 mM. In certain embodiments, the buffering agent will be in the range of about 10 mM to about 60 mM. Examples of suitable buffering agents include Tris or sodium phosphate. Certain embodiments use 50 mM Tris to a pH in the range of about 7 to about 9. Another embodiment uses about 10 to about 40 mM sodium acetate buffer at a pH of about 7 to about 9. Those skilled in the art will appreciate that the concentration of elution buffer NaCl depends on the degree of protein loading applied to the chromatography resin. The elution method is a concentration gradient step in which the NaCl concentration in the buffer is increased in a stepwise fraction to elute transferrin, or the NaCl in the buffer gradually increases in a linear manner, and a linear concentration gradient monitoring the column eluate to ensure collection of transferrin. It can be made using.

Once transferrin is recovered from the anion exchange chromatography resin, it can be further purified, for example, by concentrating transferrin through an ultrafiltration membrane and dialysis filtration and/or sterile filtration of concentrated and/or dialysis filtered transferrin as needed. have.

When solutions containing haptoglobin and/or hemopexin and/or transferrin are used in clinical or veterinary applications ( e.g., for administration to subjects with conditions associated with hemolysis), those skilled in the art, It will be appreciated that it may be desirable to reduce the level of active virus content in the solution (viral titer) and other potentially infectious agents (eg, prions). This may be particularly desirable if the feedstock ( ie starting material) comprising haptoglobin and/or hemopexin and/or transferrin is derived from plasma. Methods for reducing viral titers in solution will be known to those skilled in the art. Examples are in the presence of high concentrations of stabilizers and/or other selected excipients or salts, such as pasteurization (eg, glycine (eg 2.75M) and sucrose (eg 50%)). Incubation at 60° C. for 10 hours), dry heat treatment, virus filtration (pass solution through nano-filter; eg 20 nm cut-off) and/or under conditions to inactivate virus in solution Treatment of the solution with a suitable organic solvent and detergent for a period of time is included. Solvent detergents have been used over 20 years, particularly to inactivate envelope viruses in plasma-derived products. Thus, it can be carried out using various reagents and methods known in the art (see, eg, US 4540573 and US 4764369, incorporated herein by reference). Suitable solvents include tri-n-butyl phosphate (TnBP) and ether, preferably TnBP (typically about 0.3%). Examples of suitable detergents include polysorbate (Tween) 80, polysorbate (Tween) 20 and Triton X-100 (typically about 0.3%). The choice of treatment conditions, including solvent and detergent concentrations, depends in part on the properties of the feedstock to a less pure feedstock, which generally requires higher reagent concentrations and more extreme reaction conditions. Preferred detergents are polysorbate 80, particularly preferred combinations are polysorbate 80 and TnBP. The feedstock, along with solvent and detergent reagent, can be stirred at a constant temperature and for a period of time sufficient to inactivate any enveloped virus that may be present. For example, solvent detergent treatment may be performed at 25° C. for about 4 hours. Subsequently, for example, adsorption on a chromatography medium, for example, C-18 hydrophobic resin, or eluting them in a drop-through fraction of the ion exchange resin under conditions to adsorb the desired protein. This can remove solvent detergent chemicals.

The virus inactivation step can be performed with any suitable step of the methods disclosed herein. In one embodiment, the feedstock comprising haptoglobin and/or hemopexin and/or transferrin undergoes a virus inactivation step prior to step (ii) from the first described aspect. In another embodiment, a solution comprising haptoglobin and/or hemopexin and/or transferrin, recovered from the ammonium sulfate precipitation step ( ie , from steps (ii) and/or (iii) above), is a virus inactivation step. To perform. In another embodiment, the virus inactivation step is performed after step (iii) above.

In one embodiment disclosed herein, the virus inactivation step includes pasteurization and/or treatment with organic solvents and detergents. In one embodiment, the method of the present invention further comprises heating the solution comprising haptoglobin and/or hemopexin and/or transferrin at 55°C to 61°C for about 30 minutes to about 12 hours. In certain embodiments, the solution is heated for about 10 to about 10.5 hours. In other embodiments disclosed herein, the virus inactivation step includes virus filtration. In certain embodiments, the methods of the present invention further comprise filtering the solution comprising hemopexin and/or transferrin through a virus filter having a pore size ranging from 15 mm to 35 mm. When viral filtration is used, we add that the addition of free amino acids (e.g., arginine) prior to the filtration step is the flux rate of haptoglobin and/or hemopexin and/or transferrin through the filter and It has been found that recovery can be significantly improved. Examples of such methods are described in US7919592.

In embodiments disclosed herein, the feedstock, or solution comprising haptoglobin and/or hemopexin and/or transferrin, undergoes a virus inactivation step prior to passage through the chromatography resin. The advantage of using a virus inactivation step, e.g., solvent detergent treatment, before passing the treated solution or feedstock through the chromatography resin, e.g. anion exchange resin, i.e., haptoglobin to the resin And/or conditions that promote the binding of hemopexin and/or transferrin and the removal of organic solvent and detergent into the flow through (droplet) fraction, to enable removal of organic solvent and detergent from the treated solution.

Pasteurization can produce protein aggregates and polymers. Thus, in some examples, it may be desirable to reduce the level of aggregates/polymers in the pasteurized solution. This can be achieved by any means known to those skilled in the art, but can be conveniently achieved by further chromatographic purification. In one embodiment disclosed herein, the pasteurized solution or feedstock is directed to haptoglobin and/or hemopexin and/or transferrin so that any aggregates or polymers are removed in a flow through (droplet) fraction. Pass through the anion exchange chromatography resin in a positive manner.

In certain embodiments, unless specifically stated otherwise, purification methods of haptoglobin and/or hemopexin and/or transferrin are generally performed in a temperature range of about 18°C to about 26°C.

In another aspect of the present invention, a composition comprising haptoglobin recovered by the methods disclosed herein is provided. In one embodiment, the composition comprises a haptoglobin content of at least 80% of the total protein. In another embodiment, the composition comprises a haptoglobin content of at least 90% of the total protein. In another embodiment, the composition comprises a haptoglobin content of at least 95%. In another embodiment, the composition comprises a haptoglobin content of at least 98%. In certain embodiments, a composition comprising haptoglobin comprises less than 0.03 mg of IgA per mg of haptoglobin, as determined by immunoonephelometry. In certain embodiments, a composition comprising haptoglobin, if the haptoglobin concentration is 26 mg/mL, IgG less than 0.067 mg/mL, IgM less than 0.042 mg/mL, alpha-1 less than 0.050 mg/mL -Acid glycoprotein, pre-albumin less than 0.018 mg/mL, ceruloplasmin less than 0.021 mg/mL, hemopexin less than 0.051 mg/mL, apolipoprotein AI less than 0.053 mg/mL, 0.227 mg/mL Less than apolipoprotein B, less than 0.031 mg/mL antithrombin III, less than 0.1 mg/mL transferrin, less than 0.07 mg/mL alpha-1-antitrypsin, less than 0.1 mg/mL alpha-2-macroglobulin , Less than 0.7 mg/mL of IgA, and less than 0.15 mg/mL of albumin (as measured by immunoprecipitation).

In another aspect of the invention, a composition comprising hemopexin recovered by a method disclosed herein is provided. In one embodiment, the composition comprises a hemopexin content of at least 80% of the total protein. In another embodiment, the composition comprises a hemopexin content of at least 90% of the 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 another embodiment, the composition comprises a hemopexin content of at least 98%.

In certain embodiments, the composition comprising hemopexin is at least 98% pure, less than 0.067 mg/mL IgG, less than 0.066 mg/mL IgA, less than 0.042 mg/mL IgM, less than 0.048 mg/mL alpha- 1-antitrypsin, transferrin less than 0.090 mg/mL, alpha-1-acid glycoprotein less than 0.050 mg/mL, pre-albumin less than 0.018 mg/mL, ceruloplasmin less than 0.021 mg/mL, 0.053 mg apolipoprotein AI less than /mL, apolipoprotein B less than 0.227 mg/mL, antithrombin III less than 0.031 mg/mL, haptoglobin less than 0.17 mg/mL and albumin less than 0.045 mg/mL. (Measured by immunoturbidity measurement).

In certain embodiments, the composition comprising hemopexin is at least 97% pure, less than 1.7% haptoglobin, less than 0.4% transferrin, less than 0.2% albumin, and less than 0.1%, as determined by immunoturbination. Contains alpha-2 macroglobulin.

In another aspect of the invention, a composition comprising transferrin recovered by a method disclosed herein is provided. In one embodiment, the composition comprises a transferrin content of at least 80% of the total protein. In another embodiment, the composition comprises a transferrin content of at least 90% of the total protein. In another embodiment, the composition comprises a transferrin content of at least 95%. In another embodiment, the composition comprises a transferrin content of at least 98%.

In another aspect of the invention, a composition comprising haptoglobin recovered by a method disclosed herein and hemopexin recovered by a method disclosed herein is provided. In one embodiment, the composition comprises a combined haptoglobin and hemopexin content of at least 80% of the total protein. In another embodiment, the composition comprises a combined haptoglobin and hemopexin content of at least 90% of the total protein. In another embodiment, the composition comprises a combined haptoglobin and hemopexin content of at least 95% of the total protein. In another embodiment, the composition comprises a combined haptoglobin and hemopexin content of at least 98% of the total protein.

In one embodiment, the composition further comprises transferrin recovered by the method disclosed herein. In one embodiment, the composition comprises a combined haptoglobin, hemopexin and transferrin content of 80% or more of the total protein. In another embodiment, the composition comprises a combined haptoglobin, hemopexin and transferrin content of at least 90% of the total protein. In another embodiment, the composition comprises a combined haptoglobin, hemopexin and transferrin content of at least 95% of the total protein. In another embodiment, the composition comprises a combined haptoglobin, hemopexin and transferrin content of at least 98% of the total protein.

In another aspect of the invention, a composition comprising a haptoglobin content of at least 80%, 90%, 95%, or 98% of total protein is provided. In another aspect of the invention, a composition comprising a hemopexin content of at least 80%, 90%, 95%, or 98% of total protein is provided. In another aspect of the invention, a composition comprising a combined hemopexin and haptoglobin content of 80%, 90%, 95%, or 98% or more of total protein is provided. In another aspect of the invention, a composition comprising a combined hemopexin, haptoglobin and transferrin content of 80%, 90%, 95%, or 98% or more of total protein is provided.

Compositions comprising haptoglobin, hemopexin and/or transferrin recovered by the methods of the present invention disclosed herein, include other components ( e.g. , other plasma-derived proteins) than those they are usually related to. It will not contain substantially. Thus, in one embodiment, the composition comprising haptoglobin, hemopexin and/or transferrin is less than 20% of the total protein, preferably less than 10% of the total protein, more preferably less than 5% of the total protein. They will contain components ( ie impurities) that are different from those normally associated. Those skilled in the art will understand that the level of impurities present in the composition of the present invention may depend on the intended use of the composition. For example, when the composition is administered to a human subject in need of administration of the composition ( i.e. , for clinical use), it may be desirable for the composition to contain less than 5% impurities (of total protein). Can. Conversely, when the protein is used in vitro, it may be acceptable if the composition contains less than 5% impurities (of the total protein).

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

Suitable pharmaceutically acceptable carriers, diluents and/or excipients are known to those skilled in the art. Examples include solvents, dispersion media, antifungal and antibacterial agents, surfactants, isotonic agents and absorbents.

In addition, pharmaceutical formulations can be formulated by the addition of suitable stabilizers (or combinations thereof), such as amino acids, carbohydrates, salts, and detergents. In certain embodiments, stabilizers include mixtures of sugar alcohols and amino acids. Stabilizers can include mixtures of sugars (eg sucrose or trehalose), sugar alcohols (eg mannitol or sorbitol), and amino acids (eg proline, glycine and arginine). In a preferred embodiment, the formulation comprises an amino acid, for example arginine. In another embodiment, the formulation comprises a divalent metal ion at a concentration of 100 mM or less and a complexing agent as described in US7045601. In embodiments, the pH is preferably about 6.5 to 7.5, and the osmolality is at least 240 mosmol/kg.

In addition, pharmaceutical formulations can be sterilized by inn before dispensing and long-term storage. Preferably, the formulation will substantially retain its original stability properties for at least 2, 4, 6, 8, 10, 12, 18, 24, 36 months, or longer. For example, formulations stored at 2-8° C. or 25° C. can typically retain substantially the same molecular size distribution as measured by HPLC-SEC when stored for more than 6 months. Certain embodiments of the pharmaceutical formulations are stable for at least 6 months, 12 months, 18 months, 24 months, 36 months or more when stored at 2-8° C. and/or room temperature and may be suitable for commercial pharmaceutical use. .

The compositions described herein can be formulated in a number of possible dosage forms, eg, injectable formulations. Formulations and their subsequent administration (dosing) are within the skill of those skilled in the art. Dosing depends on the subject's responsiveness to treatment, but will continue unchanged as long as the desired effect (eg, a decrease in the level of free Hb/hem) is required. Those skilled in the art can easily determine the optimal dose, dosing method and repetition rate.

In one embodiment disclosed herein, the pharmaceutical formulation of the present invention is a solution having a volume of at least 5 mL and comprising at least 5 mg/mL of haptoglobin and/or hemopexin and/or transferrin. In another embodiment, the pharmaceutical formulation has a volume of at least 5 mL and comprises at least 20 mg/mL of haptoglobin and/or hemopexin and/or transferrin. In certain embodiments, the pharmaceutical formulation has a volume of 5 mL or more, and contains about 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg of haptoglobin and/or hemopexin and/or transferrin. Included in concentrations of /mL, 50mg/mL, 55mg/mL, 60mg/mL, 65mg/mL, 70mg/mL, 75mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 150mg/mL or 200mg/mL do. In another aspect, a vessel containing at least 5 mL of a stable pharmaceutically acceptable haptoglobin and/or hemopexin and/or transferrin solution is provided, wherein the haptoglobin and/or hemopexin and/or transferrin The concentration of is 20mg/mL or more.

In another embodiment of the invention, the pharmaceutical formulation comprising haptoglobin and/or hemopexin and/or transferrin is lyophilized. Due to the presence of lyophilized stabilizers such as sugars (eg sucrose), sugar alcohols (eg mannitol), and amino acids (eg glycine or proline) or combinations thereof, lyophilization is A stable powder with a long shelf life is obtained. Such powders can be stored, used directly or after storage as a powder, or after rehydration to prepare a pharmaceutical formulation. The lyophilized pharmaceutical formulations of the present invention can be prepared using any lyophilization method known in the art, including but not limited to freeze drying, i.e., haptoglobin and/or Alternatively, the hemopexin and/or transferrin-containing formulation is frozen, followed by evaporation under reduced pressure. The lyophilized formulations provided can substantially retain their original stability properties for at least 2, 4, 6, 8, 10, 12, 18, 24, 36 months or longer. For example, lyophilized formulations stored at 2-8° C. or 25° C. can typically retain substantially the same molecular size distribution as measured by HPLC-SEC when stored for more than 6 months. Certain embodiments of the haptoglobin and/or hemopexin and/or transferrin pharmaceutical formulations are at least 6 months, 12 months, 18 months, 24 months, 36 months or longer when stored at 2-8° C. and/or room temperature. While stable and suitable for commercial pharmaceutical use. In certain embodiments, the lyophilized pharmaceutical formulation comprises hemopexin. The present invention can be used for large-scale production of lyophilized pharmaceutical formulations. The lyophilized product can be prepared as a bulk formulation or, alternatively, can be distributed in smaller containers (e.g., single dose units) prior to lyophilization, and these smaller units can be used as sterile unit dosage forms. . The lyophilized formulation can be reconstituted to obtain a solution or suspension of protein. The lyophilized powder is rehydrated in a suitable volume using an aqueous solution. Preferred aqueous solutions are water for injection (WFI), phosphate-buffered saline or physiological saline. The mixture can be stirred to facilitate rehydration. Preferably, the reconstitution step is performed at room temperature.

In another aspect of the invention, a method of treating a condition related to hemolysis is provided, comprising administering a composition or formulation of the invention disclosed herein to a subject in need thereof.

As used herein, the term “subject” refers to an animal that includes a primate (lower or higher primate). Higher primates include people. While the present invention has specific application to target conditions in humans, those skilled in the art will appreciate that non-human animals may also benefit from the compositions and methods disclosed herein. Thus, skilled recipients will understand that the present invention has both human and veterinary applications. For convenience, "animal" includes livestock and companion animals, such as cattle, horses, sheep, pigs, camelids, goats, donkeys, dogs and cats. As for horses, these include horses used in the racing industry, and horses used in the recreational or livestock industry.

Compositions or formulations of the invention can be administered to a subject in a number of ways. Examples of suitable routes of administration include intravenous, subcutaneous, intra-arterial or infusion routes.

If necessary, the methods of the present invention may further include administering a second therapeutic agent. The second therapeutic compound can be co-administered to the subject sequentially (before or after administration of the compositions or formulations disclosed herein) or simultaneously. In one embodiment, the second therapeutic agent is an iron chelating agent (eg, deferioxamine or deferiprone).

In another aspect of the invention, the use of a composition or formulation of the invention disclosed herein in the manufacture of a medicament for treating a condition related to hemolysis is provided. Such compositions or formulations are preferably suitable for use in human patients.

Conditions associated with hemolysis and at risk of hemoglobin/hem-mediated toxicity are known in the art. In one embodiment, the condition is selected from acute hemolytic condition and/or chronic hemolytic condition. In one embodiment, the condition is hemoglobinemia with hemolytic anemia, transfusion-induced hemolysis, hemolytic uremic syndrome, autoimmune disease, malaria infection, trauma, transfusion, open heart surgery with cardiopulmonary bypass surgery, and hemolysis after burns or Hemoglobinuria. In one embodiment, the condition consists of sickle cell anemia, hereditary globular anemia, hereditary elliptic anemia, mediterranean anemia, congenital angioblastic hematopoietic anemia and paroxysmal nocturnal hemoglobinuria, systemic lupus erythematosus, and chronic lymphocytic leukemia. It is selected from the group.

Those skilled in the art will understand that the present invention described herein may allow for changes and modifications other than those specifically described. It should be understood that the present invention includes all such changes and modifications that are within the spirit and scope. In addition, the present invention, individually or collectively, and any two or more of any of the above steps or features, all or all of the steps, features, compositions, and compounds mentioned or represented herein. And all combinations.

Certain embodiments of the present invention will be described below with reference to the following examples, which are intended for purposes of explanation only and are not intended to limit the general scope described herein above.

Example

Example One

Starting material: Cone fraction IV ₄ precipitate was used as starting material for purification of haptoglobin, hemopexin, and transferrin (see Figure 1).

Sediment extraction: Sediment extraction was performed by introducing 20 grams of extraction buffer per gram of fraction IV ₄ precipitate (20x extraction ratio). The buffer and precipitate were mixed for at least 1 hour. The extraction buffer consisted of 50 mM Tris adjusted to a pH of 7.0 with concentrated HCl. Extraction buffer was prepared at a temperature of 20-25°C. In addition, the extraction of precipitates was also carried out at a temperature of 20 to 25 ℃. The pH during extraction was maintained between 7.0 and 8.0 (preferably 7.0) for the duration of the extraction time of 1 hour.

Ammonium sulfate precipitation: Solid sodium sulfate was added to the Fraction IV ₄ extract to achieve a final concentration of 2.0 to 2.5M (preferably 2.4M). Under stirring, ammonium sulfate was slowly added to the extract and allowed to mix continuously for at least 1 hour. Precipitation of ammonium sulfate was carried out at a temperature of 20 to 25°C.

Lipids were precipitated using an ammonium sulfate concentration of 2.5 M to clarify the fraction IV ₄ extract, while maintaining hemopexin and transferrin as soluble. Concurrently, this ammonium sulfate concentration resulted in the precipitation of a significant portion of the haptoglobin present in the fraction IV ₄ extract (see Figure 2). Precipitating haptoglobin while maintaining hemopexin soluble in 2.2M to 2.5M ammonium sulfate enables co-purification of hemopexin and haptoglobin from the same starting fraction.

To further narrow the range of acceptable ammonium sulfate concentrations, an experimental design (DOE) was performed taking into account the effect of pH and ammonium sulfate concentrations. 3 shows a preferred degree plot of DOE. This plot provides the most desirable conditions for precipitating the most haptoglobin while maintaining the most hemopexin soluble. The preferred degree plot shows that the pH maintained between 7 and 8 (preferably 7.0) and the ammonium sulfate concentration of 2.2M to 2.5M (preferably 2.4M) are optimal for separating both proteins from the same starting material. In addition, transferrin remains soluble at concentrations greater than 2.5M, so these conditions were optimal for purification of all three proteins from the same starting material.

Filtration: To prepare an ammonium sulfate treated extract for filtration, 10 grams of C1000 filter aid per liter of equivalent plasma used in the batch was added. The C1000 filter aid was allowed to mix for at least 15 minutes prior to filtration. Plate and frame filter presses are used for filtration to enable collection of haptoglobin-concentrated sediments. The plate and frame filter press was assembled with a sheet of 175 type filter paper in front of a 3M (Cuno) 70CA depth filtration filter sheet. For all, 3 L of equivalent plasma was placed in the batch, and 0.193 mL of the sediment collection site was required (3 L plasma/4 Ertel 4S filter frame). The ammonium sulfate treated extract was then pumped into the filter press through the use of a double diagram pressurized air operated pump.

After the end of the C1000 mixing time, the treated extract was pumped into a filter press, and the filtrate was collected after a single pass through the filter press. The filter press was then post-washed with a 1 to 2 press volume of 2.4M ammonium sulfate, 50mM Tris and adjusted to pH 7.0. Filtration proser was performed at a temperature of 20 to 25°C. The obtained filtrate contains hemopexin and can be stored at 2 to 8°C until further purification. The resulting precipitate contains haptoglobin and can be stored at -20°C or lower until further purification.

Filtrate From fraction Hemopexin (Hx) refine:

The Hx process scheme is highlighted in FIG. 9.

(a) Octyl Sepharose chromatography (HIC) : The filtrate obtained from the extraction of the fraction IV ₄ precipitate and the treatment with ammonium sulfate was further clarified using a 0.22 µm filter. The filtrate was then loaded onto an octyl Sepharose column (GE Lifesciences) equilibrated with 3 column volumes of 2.5M ammonium sulfate, 50mM Tris at pH 7.4. Alternatively, a capto octyl column can be used for this step. Filtrates of 8 to 12 column volumes (target = 10) were loaded onto an octyl Sepharose column. Impurities and any unbound proteins were washed away from the column with a column volume of 3 of 0.9M to 1.2M (target 1.13M) ammonium sulfate, 50mM Tris at pH 7.4 (wash). The wash fraction contained transferrin, which can be stored for further purification. The hemopexin-containing fraction (eluate) was then eluted from the column with 3 column volumes of water (WFI). The eluate was then stored at 2-8° C. until further transferrin purification. Octyl Sepharose (HIC) purification was performed at a temperature of 20-25°C.

(b) Ni - Sepharose chromatography ( IMAC ) : 20 mM sodium phosphate, 500 mM sodium chloride, and 30 mM imidazole were added to the octyl sepharose eluate. Once added, the pH of the octyl sepharose eluate was adjusted to 7.4. Next, 2 to 3 column volumes of octyl sepharose eluate were equilibrated with a buffer containing 20 mM sodium phosphate, 500 mM sodium chloride and optionally 30 mM imidazole and adjusted to pH 7.4 Ni-Sepharose (GE Lifesciences) ) Loaded on the column. The addition of imidazole to the octyl sepharose eluate (load) reduces the affinity of Ni-Sepharose for albumin and other impurities, while maintaining its binding affinity for hemopexin. Thus, during the loading step, impurities flowed through the column, while hemopexin was bound to the resin. After the end of the load, the column was washed with 2 column volumes of 20 mM sodium phosphate at pH 7.4, 500 mM sodium chloride and 30 mM imidazole to remove any unbound impurities. Hemopexin was then eluted from the column with 20 mM sodium phosphate, 500 mM sodium chloride and 100 mM imidazole at pH 7.4. Hemopexin present in the Ni-Sepharose eluate was estimated to be greater than 95% by SDS-PAGE (see Figure 4). The Ni-Sepharose chromatography (IMAC) process was performed at 20-25° C. and the resulting eluate was stored at -20° C. or lower until use.

(c) Concentration/dialysis filtration : Ni-Sepharose eluate was concentrated to the desired concentration (1-20% w/v), followed by dialysis filtration with 10 volumes of phosphate buffered saline per volume of concentrate. Concentration and dialysis filtration were performed using a 30 kD ultrafiltration membrane. Once diafiltration was finished, the concentrate was present at the desired concentration, hemopexin was optionally formulated with sugars and/or amino acids, sterile filtered, and stored below -20°C. Purified hemopexin may exist in this form in pre-clinical animal and cell studies.

The final yield of hemopexin recovered from the process was estimated to be approximately 0.151 g/L plasma.

The concentrated hemopexin formulation (approximately 2.3% w/v) was characterized by immunoturbination measurements. Plasma proteins such as IgG (detection limit, LOD = <0.067 mg/mL), IgA (LOD = <0.066 mg/mL), IgM (LOD = <0.042 mg/mL), alpha-1-antitrypsin ( LOD = 0.048mg/mL), transferrin (LOD = <0.090mg/mL), alpha-1-acid glycoprotein (LOD = 0.050mg/mL), pre-albumin (LOD = 0.018mg/mL), ceruloplasm Min (LOD = 0.021 mg/mL), apolipoprotein AI (LOD = <0.053 mg/mL), apolipoprotein B (LOD = <0.227 mg/mL) and antithrombin III (LOD = 0.031 mg/mL) It was below the detectable level. Only trace amounts were detected for other plasma proteins, such as haptoglobulin (0.165 mg/mL) and albumin (0.038 mg/mL). These results indicate that Hx accounted for more than 99% of the total protein in the formulation.

Additional batches of hemopexin were processed according to the method described above. The batch was concentrated with 35 mg/mL protein. Analysis of the batch by immunoturbination revealed a hemopexin purity of about 98% with impurities comprising 1.6% haptoglobin, 0.3% transferrin, 0.2% albumin, and 0.1% alpha-2 macroglobulin. Showed.

From 2.5M ammonium sulfate precipitate Haptoglobin (Hp) refine

The Hp process scheme is highlighted in FIG. 10.

(a) Sediment extraction and filtration : Haptoglobin was extracted from the sediment by introduction of 20 grams of extraction buffer per gram of sediment (20x ratio). The extraction buffer consisted of 50mM sodium acetate and adjusted to pH 5.5. The buffer and precipitate were mixed for at least 1 hour. After 15 minutes, the pH was adjusted within the range of 5.4 to 5.6. Low pH extraction buffer was used to reduce lipid extraction, while low pH was used during subsequent chromatography steps. The extract was then passed through a Cuno 70CA filter or equivalent depth filter to remove the remaining filter aids and undissolved proteins and lipids. Then, the filtrate was clarified using a 0.2 µm filter. The filtrate obtained was prepared for subsequent chromatography steps. The extraction buffer formulation, extraction process, and filtration process were all performed at 20-25°C.

In another embodiment, haptoglobin was extracted from the precipitate by introduction of 20 grams of extraction buffer per gram of precipitate (20x ratio). The extraction buffer consisted of 50 mM Tris and adjusted to pH 8.5. The buffer and precipitate were mixed for at least 1 hour. After 15 minutes, the pH was adjusted within the range of 8.4 to 8.6. To reduce lipid content and adjuvant in clarification of the extracted precipitate, a lipid adsorbent, Aerosil (fumed silica), was added at about 1.6 to about 1.8 grams per liter of equivalent plasma. Then, the Aerosil-treated extract was allowed to mix for at least 1 hour. The extract was then passed through a Cuno 70CA filter or other similar depth filter to remove the remaining filter aids and undissolved proteins and lipids. Then, the filtrate was clarified using a 0.2 µm filter. The filtrate obtained was prepared for subsequent chromatography steps. The extraction buffer formulation, extraction process, and filtration process were all performed at 20-25°C.

(b) Capto Q ImpRes chromatography step : Sodium acetate was added to the filtrate obtained from the above step to a final concentration of 50 mM, and the pH of the filtrate was adjusted to pH 5.5 using glacial acetic acid. Then, the pH-adjusted filtrate was diluted 1:4 to 1:5 with cold water (WFI), and at a temperature of about 2 to 8° C., GE Healthcare Capto Q ImpRes chromatography column equilibrated with 50 mM sodium acetate, pH 5.5 Phase. The load requires sodium acetate as a low pH buffer, and dilution with water is required to reduce the conductivity of the load so that haptoglobin can bind to the column. After the end of the load, the column was washed with 2 column volumes of 50 mM sodium acetate at pH 5.5 to remove any unbound contaminant proteins. Haptoglobin was then eluted with 50 mM sodium acetate, 100-200 mM NaCl (preferably 162 mM) at pH 5.5. Since the elution conditions depend in part on the load volume used, a wide NaCl concentration range in the elution buffer is required. Until the concentration/dialysis filtration, the eluate can be stored for a short time at 2 to 8°C, and can be stored for a long time at -20°C or less. The haptoglobin content of the Capto Q ImpRes eluate was estimated to be greater than 95% by SDS-PAGE (see Figures 5 and 6). The chromatography buffer and chromatography steps were all performed at 20-25°C.

(c) Concentration/dialysis filtration : The Capto Q ImpRes eluate was concentrated to a specific concentration, followed by dialysis filtration with 10 volumes of phosphate buffered saline per volume of concentrate. Concentration and dialysis filtration were performed using a 30 kD ultrafiltration membrane. Once diafiltration was finished, the concentrate was present at the desired concentration, and the purified haptoglobin solution was sterile filtered and stored at -20°C or lower.

Optionally, the lipid adsorption step can be performed before or after the anion exchange chromatography step, or after the concentration/dialysis filtration step. Examples of suitable lipid adsorbents are fumed silica such as Aerosil (eg Aerosil 380).

The final yield of haptoglobin recovered from the process was estimated to be approximately 0.285 g/L plasma input.

Concentrated haptoglobin preparations (approximately 2.6% w/v) were characterized by immunoturbination measurements. Plasma proteins such as IgG (detection limit, LOD = <0.067 mg/mL), IgM (LOD = <0.042 mg/mL), alpha-1-acid glycoprotein (LOD = 0.050 mg/mL), pre- Albumin (LOD = 0.018 mg/mL), Ceruloplasmin (LOD = 0.021 mg/mL), Hemopexin (LOD = <0.051 mg/mL), Apolipoprotein AI (LOD = <0.053 mg/mL), Apo Lipoprotein B (LOD = <0.227 mg/mL) and antithrombin III (LOD = 0.031 mg/mL) were below detectable levels, while other plasma proteins such as transferrin (0.099 mg/mL), alpha Only trace amounts were detected for -1-antitrypsin (0.062 mg/mL), alpha-2-macroglobulin (0.086 mg/mL), IgA (0.65 mg/mL), and albumin (0.123 mg/mL). These results indicate that Hp accounted for over 96% of the total protein in the formulation.

Octyl From the 1.13M ammonium sulfate wash fraction Transferrin refine:

Before performing ion-exchange chromatography on the octyl wash fraction, ammonium sulfate was diafiltered and exchanged with a lower ionic strength buffer. Because of this, the octyl eluate was concentrated and dialyzed against 10 volumes of 50 mM Tris, pH 7.0 per eluate volume. Concentration and dialysis filtration were performed using a 30 kD ultrafiltration membrane.

The dialysis filtered wash fraction was then loaded onto a GE Healthcare Capto DEAE column equilibrated with 50 mM Tris, pH 7.0. To determine if it was feasible to obtain pure transferrin from this fraction, a linear concentration gradient was performed over 10 column volumes using 50 mM Tris, pH 7.0 as starting buffer and terminating with 50 mM Tris, 0.5 M NaCl, pH 7.0. (See Figure 8). Fractions corresponding to each peak on the chromatogram were collected. SDS-PAGE analysis was performed to determine if one of the peaks contained pure transferrin. As shown in Figure 7, peak 1 was overloaded, but was found to be pure transferrin. This indicates that it is possible to purify transferrin from the octyl sepharose wash fraction, and also it is possible to purify hemopexin, haptoglobin, and transferrin from the same starting material (see also FIG. 11).

Claims (25)

A method for purifying haptoglobin and hemopexin from a solution containing both haptoglobin and hemopexin, the method comprising:
(i) providing a solution containing both haptoglobin and hemopexin;
(ii) precipitating haptoglobin from the solution by adding ammonium sulfate to the solution;
(iii) separating the precipitated haptoglobin from the solution containing hemopexin; And
(iv) separately purifying haptoglobin and/or hemopexin in one or more steps
In this case, by adding 2.3M to 2.5M ammonium sulfate to the solution, haptoglobin precipitates from the solution at pH 6 to 8,
Step (iv) is
(v) dissolving the precipitated haptoglobin in a buffer to obtain a haptoglobin solution;
(vi) passing the haptoglobin solution through an anion exchange chromatography resin under conditions that allow the haptoglobin to bind to the anion exchange chromatography resin;
(vii) eluting the haptoglobin from the resin; And
(viii) recovering the eluted haptoglobin
Or
Step (iv) is
(ix) passing the solution containing hemopexin through a hydrophobic interaction chromatography resin, under conditions that allow the hemopexin to bind to a hydrophobic interaction chromatography resin;
(x) collecting the flow-through fraction from step (ix);
(xi) optionally washing the resin after step (x) and collecting the flow through wash fraction;
(xii) eluting the hemopexin from the resin after step (x) and/or step (xi); And
(xiii) recovering the eluted hemopexin
A method of purifying haptoglobin and hemopexin from a solution containing both haptoglobin and hemopexin. The method of claim 1 comprising precipitating haptoglobin from the solution by adding 2.4M ammonium sulfate to the solution. The method of claim 1 comprising precipitating haptoglobin from the solution at a pH of 7. The method of any one of claims 1 to 3, wherein the solution containing both haptoglobin and hemopexin further comprises transferrin. The method according to any one of claims 1 to 3, wherein the solution is a human plasma fraction. The method of claim 5, wherein the solution is a Cohn Fraction IV. The method of claim 6, wherein the solution is cone fraction IV ₄ . delete The method of claim 1, further comprising exposing the haptoglobin solution to a lipid removal agent under conditions that cause the lipid to bind to the lipid removal agent. delete According to claim 1,
(i) passing the eluted hemopexin through a metal ion affinity chromatography resin under conditions such that the hemopexin binds to the metal ion affinity chromatography resin;
(ii) eluting the hemopexin from the resin; And
(iii) recovering the eluted hemopexin
The method further comprising a. According to claim 4,
(xiv) the flow-through fraction from step (x) above and/or the flow-through washing fraction from step (xi) above through anion exchange chromatography resin under conditions that allow transferrin to bind to the anion exchange chromatography resin. Passing through; And
(xv) recovering the transferrin from the resin
The method further comprising a. Contain at least 97% hemopexin and less than 1.7% haptoglobin, less than 0.4% transferrin, less than 0.2% albumin and less than 0.1% alpha-2 macroglobulin, as determined by immunoonephelometry Composition comprising a. A pharmaceutical formulation for treating a condition related to hemolysis, comprising the composition of claim 13 and a pharmaceutically acceptable carrier. 15. The pharmaceutical formulation of claim 14, comprising at least one of amino acids, carbohydrates, salts and detergents. The pharmaceutical formulation of claim 15 comprising a mixture of sugar alcohols and amino acids. The pharmaceutical formulation of claim 15 comprising a mixture of sugars, optionally sucrose or trehalose, sugar alcohols, optionally mannitol or sorbitol, and amino acids, optionally proline, glycine and arginine. 16. The pharmaceutical formulation of claim 15 comprising arginine. The pharmaceutical formulation of claim 14, having a pH of 6.5 to 7.5 and an osmolarity of at least 240 mosmol/kg. 15. The pharmaceutical formulation of claim 14, which is lyophilized. 21. The pharmaceutical formulation of claim 20, having a substantially identical molecular size distribution as measured by HPLC-SEC when stored for 6 months or more. A solution comprising the pharmaceutical formulation of claim 14 having a volume of at least 5 mL and comprising at least 5 mg/mL of hemopexin. The solution of claim 22 comprising at least 20 mg/mL of hemopexin.
delete delete

Images