EP2904005A1 - A method of purifying proteins - Google Patents
A method of purifying proteinsInfo
- Publication number
- EP2904005A1 EP2904005A1 EP13777203.4A EP13777203A EP2904005A1 EP 2904005 A1 EP2904005 A1 EP 2904005A1 EP 13777203 A EP13777203 A EP 13777203A EP 2904005 A1 EP2904005 A1 EP 2904005A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- haptoglobin
- hemopexin
- solution
- resin
- transferrin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- A61K38/1722—Plasma globulins, lactoglobulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/41—Porphyrin- or corrin-ring-containing peptides
- A61K38/42—Haemoglobins; Myoglobins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
- A61P33/06—Antimalarials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4717—Plasma globulins, lactoglobulin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates generally to a method of purifying proteins. More specifically, the present invention relates to a method of purifying haptoglobin and hemopexin from the same starting material, and uses thereof.
- Haemolysis is characterized by the destruction of red blood cells and is a hallmark of anaemic disorders associated with red blood cell abnormalities, such as enzyme defects, haemoglobinopathies, hereditary spherocytosis, paroxysmal nocturnal haemoglobinuria and spur cell anaemia, as well as extrinsic factors such as splenomegaly, autoimmune disorders (e.g., Hemolytic disease of the newborn), genetic disorders (e.g., Sickle-cell disease or G6PD deficiency), microangiopathic haemolysis, Gram-positive bacterial infection (e.g., Streptococcus, Enterococcus and Staphylococcus), parasite infection (e.g., Plasmodium), toxins and trauma (e.g., burns).
- Haemolysis is also a common disorder of blood transfusions, particularly massive blood transfusions and in patients using an extracorporeal cardio-pulmonary support.
- haemoglobin Hb
- heme iron and iron-containing compounds
- red blood cells red blood cells
- haemoglobin (Hb) and heme iron and iron-containing compounds
- released haemoglobin is bound by soluble proteins such as haptoglobin and transported to macrophages and hepatocytes.
- haptoglobin soluble proteins
- the buffering capacity of haptoglobin is overwhelmed.
- haemoglobin is quickly oxidised to ferri-haemoglobin, which in turn releases free heme (comprising protoporphyrin IX and iron).
- heme plays a critical role in several biological processes (e.g., as part of essential proteins such as haemoglobin and myoglobin), free heme is highly toxic. Free heme is a source of redox-active iron, which produces highly toxic reactive oxygen species (ROS) that damages lipid membranes, proteins and nucleic acids. Heme toxicity is further exacerbated by its ability to intercalate into lipid membranes, where it causes oxidation of membrane components and promotes cell lysis and death. [0004] The evolutionary pressure of continuous 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 haemolysis.
- ROS reactive oxygen species
- Hp Hb scavenger haptoglobin
- Hx heme scavenger proteins hemopexin
- al -microglobulin al -microglobulin
- the present invention provides a method of purifying Hp and Hx from the same starting material.
- the purified proteins can be used in compositions for treating conditions associated with haemolysis and aberrant Hb/heme levels.
- a method of purifying haptoglobin and hemopexin from a solution containing both proteins comprising:
- composition comprising the haptoglobin recovered by the methods disclosed herein.
- composition comprising the hemopexin recovered by the methods disclosed herein.
- composition comprising the transferrin recovered by the methods disclosed herein.
- composition comprising the haptoglobin recovered by the methods disclosed herein and the hemopexin recovered by the methods disclosed herein.
- composition comprising a haptoglobin content of at least 95% of total protein.
- composition comprising a hemopexin content of at least 80% of total protein.
- composition comprising a combined hemopexin and haptoglobin content of at least 80% of total protein.
- composition of the present invention as disclosed herein, and a pharmaceutically acceptable carrier.
- a method of treating a condition associated with haemolysis comprising administering to a subject in need thereof the composition or the formulation of the present invention, as disclosed herein.
- compositions or formulations of the present invention as disclosed herein, in the manufacture of a medicament for treating a condition associated with haemolysis.
- Figure 1 is a flow diagram of a Cohn Fractionation Process.
- process parameters e.g. pH, ethanol concentration, temperature, etc.
- process parameters e.g. pH, ethanol concentration, temperature, etc.
- FIG. 2 shows the recovery of transferrin (TRF), albumin (Alb), hemopexin (HPX) and haptoglobin (HAP) in the remaining filtrate following precipitation in the presence of 2.0M, 2.5M, 3.0M, 3.5M and 4.0M ammonium sulfate.
- TRF transferrin
- Alb albumin
- HPX hemopexin
- HAP haptoglobin
- Figure 3 shows a desirability plot of the design of experiment (DOE), showing the desirable conditions that would precipitate the haptoglobin from the plasma fraction, while keeping the hemopexin in solution.
- DOE design of experiment
- a factorial mathematical design was used to design and analyze the data. The desirability plot depicted in Figure 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.
- FIG 4 shows SDS-PAGE electrophoresis of hemopexin recovered following the various steps in the purification process disclosed herein.
- SDS-PAGE analysis was performed using pre-cast 10% Tris-Glycine gels (Novex, EC6075). All samples were diluted to a concentration of 0.1 mg/mL in Tris-Glycine SDS sample buffer (LC2676) and 20 ⁇ ⁇ of each sample was loaded into the sample well of the gel. Run time and voltage were set to the gel manufacturer's recommendations (125V constant). Each gel was stained with an easy to use type of Coomassie Brilliant Blue stain solution (Novex, Simply Blue Safestain, LC6065). Lane 1 (LMWS) contains Novex Sharp Unstained Protein Standards, LC5801.
- Figure 5 shows an SDS-PAGE electrophoresis (using the method described above in Figure 4) of haptoglobin intermediates recovered following the various steps in the purification process disclosed herein.
- the haptoglobin standard in lane 2 is from Benesis (T043HPX).
- Figure 6 shows a Western Blot of the Capto Q Eluate on a 10% Tris-Glycine Non-Reduced SDS-PAGE electrophoresis gel (using the method described in Figure 4).
- the separated proteins are then transferred to a nitrocellulose membrane and the membrane is blocked to prevent any non-specific binding of antibody.
- the nitrocellulose membrane is then incubated with a solution containing antibodies to Human Haptoglobin (rabbit anti-human haptoglobin, Sigma H8636).
- a secondary antibody linked to horseradish peroxidase (HRP) is then incubated with the nitrocellulose membrane (goat anti-rabbit HRP, Sigma A6154).
- the nitrocellulose is then developed with a solution containing peroxide thereby only visualizing the protein bands that specifically contain human haptoglobin.
- the lanes contain Capto Q ImpRes Eluate (T0209001) at different concentrations (Lane 2 - 1 : 10 dilution, 0.1 mg/mL, 20 ⁇ L ⁇ loaded; lane 3 - 1 :20; lane 4 - 1 :40; lane 5 - 1 :80; lane 6 - 1 : 160; lane 7 - 1 :320; lane 8 - 1 :640; lane 9 1 : 1280; and lane 10 - 1 :2560).
- the Western blot indicates that most of the bands present in the Capto Q Eluate are haptoglobin.
- FIG. 7 shows SDS-PAGE electrophoresis (using the method described in Figure 4) of transferrin recovered from ion-exchange chromatography (Capto DEAE) following the various steps in the purification process disclosed herein. Peak one (lane 4) is heavily loaded, but appears to be pure transferrin (compare to lane 2, human transferrin, Sigma T8158). This indicates that it is possible to purify transferrin from the Octyl Sepharose Wash fraction, which also means that it is possible to purify hemopexin, haptoglobin, and transferrin from the same starting material.
- Figure 8 shows a chromatogram of the transferrin linear gradient from the ion- exchange chromatographic column during the purification process disclosed herein. Peaks labelled 1 to 4 were analysed by SDS-PAGE in Figure 7.
- Figure 9 is a flow diagram of a hemopexin purification process in accordance with an embodiment disclosed herein.
- Figure 10 is a flow diagram of a haptoglobin purification process in accordance with an embodiment disclosed herein.
- Figure 11 is a flow diagram of a combined haptoglobin/hemopexin/transferrin purification process in accordance with an embodiment disclosed herein.
- the present invention is predicated, at least in part, on the finding that haptoglobin and hemopexin can be purified from the same starting material.
- a method of purifying haptoglobin and hemopexin from a solution containing both proteins comprising:
- Haptoglobin is a tetrachain ( ⁇ 2 ⁇ 2 ) glycoprotein synthesized by the adult liver and secreted into the plasma.
- the propeptide form of Hp is proteolytically cleaved into an a-chain and a ⁇ -chain.
- Two oc-subunits and two ⁇ -subunits of Hp protein are then joined by inter-chain disulfide bonds to form the mature peptide, which can be either an (aP)-dimer or an (aP)-multimer.
- Hemopexin is a 60-kD plasma ⁇ - IB -glycoprotein comprising a single 439 amino acid long peptide chain, which forms two domains joined by an inter-domain linker. It has the highest known affinity for heme (Kd ⁇ lpM) of any characterized heme-binding protein and binds heme in an equimolar ratio between the two domains of Hx in a pocket formed by the inter-domain linker.
- the method comprises precipitating the haptoglobin from the solution by adding about 2.0M to about 2.5M ammonium sulphate to the solution.
- the method comprises precipitating the haptoglobin from the solution by adding about 2.2M to about 2.5M ammonium sulphate to the solution.
- the method comprises precipitating the haptoglobin from the solution by adding about 2.4M ammonium sulphate to the solution.
- the ammonium sulphate concentration is 2.0M or 2.1M or 2.2M or 2.3 M or 2.4M or 2.5M.
- the method comprises precipitating the haptoglobin from the solution at a pH of less than or equal to 8.
- the method comprises precipitating the haptoglobin from the solution at a pH within the range of about 6 to about 8.
- the method comprises precipitating the haptoglobin from the solution at a pH of about 7.
- the method comprises precipitating the haptoglobin from the solution at a pH in 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.
- the pH is typically measured in the haptoglobin and hemopexin solution and then during the addition of the ammonium sulphate and the precipitation of the haptoglobin.
- the pH can be adjusted (typically the concentration of the acid (e.g. HC1) or base (e.g. NaOH) used to adjust the pH is in the range of 0.05M to 0.6M).
- the majority of haptoglobin from the starting material will be found within the ammonium sulfate precipitate and the majority of the hemopexin from the starting material will be found in the remaining solution (also referred to as the suspension).
- the precipitate may comprise some hemopexin (e.g., trace amounts of Hx) and that the remaining solution (or suspension) may comprise some haptoglobin (e.g., trace amounts of Hp).
- the remaining solution or suspension
- trace amounts of Hp and Hx that may be present in the suspension and precipitate, respectively may be acceptable, for example, where both proteins will end up in the same composition.
- any solution comprising both haptoglobin and hemopexin can be used as the starting material in the method of the present invention, disclosed herein.
- the solution containing both haptoglobin and hemopexin further comprises transferrin.
- Suitable starting material would be known to persons skilled in the art, examples of which include plasma fractions such as various supernatants and precipitates derived from ethanol fractionation processes. Examples of such ethanol fractionation processes include Cohn fractionation and Kistler-Nitschmann fractionation. Examples of suitable plasma fractions include those derived from a Cohn fraction I, II, III, II+III, I+II+III, IV and V (See Figure 1) or a Kistler-Nitschmann fraction such as a Precipitate A or B.
- the solution is a human plasma fraction.
- the solution is a Cohn Fraction IV.
- the solution is a Cohn Fraction IV 4 .
- the Cohn Fraction IV 4 is derived from a Cohn Fraction II+III or a Cohn Fraction I+II+III.
- the solution is derived from a Fraction IV 4 Precipitate.
- the buffering agent used to resolublise the precipitate can be any agent or combination of agents that has a buffering capacity at around pH 7 (examples can include ADA, PIPES, ACES, MOPSO, MOPS, BES, TRIS).
- the buffering agent will be at a concentration from about 5 mM to about lOOmM.
- the resolublization buffer is 50mM Tris, pH 7.
- the resolublisation buffer can be added at about 5 to about 30 grams per gram of the starting material.
- the resolubilisation buffer is added at 5 to 10 grams per gram of starting material, or added at 10 to 15 grams per gram of starting material, or added at 15 to 20 grams per gram of starting material, or added at 20 to 25 grams per gram of starting material, or added at 25 to 30 grams per gram of starting material.
- the methods of the present invention are suitable for the commercial/industrial scale purification of hemopexin, haptoglobin and, optionally, transferrin.
- employing the method of the present invention on a commercial/industrial scale may involve the use of a plasma fraction derived from at least about 500 kg of plasma. More preferably, the plasma fraction will be derived from at least about 5,000 kg, 7,500 kg, 10,000 kg and/or 15,000 kg of plasma per batch.
- plasma for fractionation is the liquid part of blood remaining after separation of the cellular elements from blood collected in a receptacle containing an anticoagulant, or separated by any other suitable means known to persons skilled in the art, such as by continuous filtration or centrifugation of anticoagulated blood in an apheresis procedure.
- the precipitated haptoglobin and the solution containing hemopexin are recovered and stored separately before separately purifying the haptoglobin and/or hemopexin in one or more steps, in accordance with the present invention.
- the precipitated haptoglobin and/or the solution containing hemopexin are recovered and subjected immediately to further purification steps in accordance with the methods of the present invention; that is, separately purifying the haptoglobin and/or hemopexin in one or more steps.
- the method further comprises:
- the anion exchange chromatographic resin of step (ii) is a strong anion exchange chromatographic resin. In other embodiments the anion exchange chromatographic resin of step (ii) is a weak anion exchange chromatographic resin.
- Purification of proteins by chromatography can be performed using either axial flow columns, such as those available from GE Healthcare, Pall Corporation, Millipore and Bio-Rad, or using radial flow columns, such as those available from Proxcys or Sepragen. Chromatography can also be conducted using expanded bed technologies known to persons skilled in the art.
- Suitable solid supports would be familiar to persons skilled in the art and the choice will depend on the type of product to be purified.
- suitable solid supports include inorganic carriers, such as glass and silica gel, organic, synthetic or naturally occurring carriers, such as agarose, cellulose, dextran, polyamide, polyacrylamides, vinyl copolymers of bifunctional acrylates, and various hydroxylated monomers, and the like.
- the chromatography steps will generally be carried out under non-denaturing conditions and at convenient temperatures in the range of about -10°C to +30°C, more usually at about ambient temperatures.
- the chromatographic steps may be performed batch-wise or continuously, as convenient. Any convenient method of separation may be employed, such as column, centrifugation, filtration, decanting, or the like.
- Buffers that are suitable for dissolving the haptoglobin precipitate would be familiar to persons skilled in the art and may depend on the conditions required for performing the chromatographic purification step.
- the buffer will have a concentration of the buffering agent (i.e. Tris) from about 5 mM to about 100 mM.
- the buffering agent is from about 10 mM to about 60 mM.
- the buffer may comprise more than one buffering agent.
- suitable buffers are sodium acetate and Tris with a pH range of 5.5 to 9.0. Particular embodiments utilize a pH of 7.5 to 9.0.
- the pH of the buffer used to dissolve the haptoglobin precipitate is from 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.
- the buffer is about 50mM Tris at a pH of about pH 8.4 to about pH 8.6.
- the lipid content of the extracted precipitate comprising haptoglobin is reduced by exposure to a lipid removal agent under conditions that allow the lipid to bind to the lipid removal agent.
- lipid removal agents include fumed silica such as Aerosil.
- the lipid removal agent is a fumed silica.
- the fumed silica is an Aerosil (e.g. Aerosil 380).
- the lipid removal agent such as Aerosil can be added to the extracted precipitate comprising haptoglobin at about 0.5 g to about 4 g per liter of plasma equivalent.
- the lipid removal agent such as Aerosil is added at 1 to 2 g per liter plasma equivalent to the extracted precipitate comprising haptoglobin.
- the lipid removal agent such as fumed silica e.g. Aerosil
- the lipid removal agent such as Aerosil is added at 1.8 to 2.0 g per liter of plasma equivalent to the extracted precipitate comprising haptoglobin.
- the lipid removal agent such as Aerosil is added at 1.6 g per liter of plasma equivalent to the extracted precipitate comprising haptoglobin. It was determined that lipid removal is most effective within a specific pH range.
- a pH range of 5.5 to 9.0 was found to be effective in conjunction with Aerosil.
- the pH range is 6.5 to 8.6.
- the pH during lipid removal step is from 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.
- the preferred embodiment utilizes a pH range of 8.4 to 8.6.
- the lipid removal agent can be removed using methods such as filtration and or centrifugation.
- the lipid removal agent is removed by depth filtration.
- An example of a depth filter for use in this application is a Cuno 70CA filter or one of similar or smaller particle size retention capabilities.
- any anion exchange chromatographic resin can be used to separately purify haptoglobin from the haptoglobin solution, as long as the haptoglobin is capable of binding to the chromatographic resin while allowing some impurities in the solution to pass though the resin.
- the anion exchange chromatographic resin is a strong anion exchange resin.
- the anion exchange chromatographic resin is a weak anion exchange resin.
- Suitable resins would be known to persons skilled in the art.
- suitable anion exchange resins are ones comprising a functional quaternary amine group (Q) and/or a diethylaminopropyl group (ANX).
- the strong anion exchange chromatographic resin comprises a functional quaternary amine group (e.g., Capto Q ImpRes ).
- Solutions that are suitable for the equilibration (often referred to as an equilibration buffer) of the chromatography media usually have a concentration of a buffering agent of about 5mM to about 100 mM.
- the pH of the equilibration buffer will normally be in the range of about 5 to about 9 and the conductivity is typically less than about 9 mS/cm. These conditions generally allow for the binding of haptoglobin to the anion exchange media.
- the buffering agent will be in the concentration range from about 10 mM to about 60 mM.
- the pH of the equilibration buffer in particular embodiments is in a pH range from about 5.0 to about 9.0.
- the pH is in the range from about 5.0 to about 7.0, or from about 5.0 to about 6.0, or from about 5.3 to about 5.7.
- 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.
- the conductivity of the equilibration buffer in some embodiments is less than about 7.0 mS/cm. In particular 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 buffering agent for an equilibration buffer is sodium acetate.
- Particular embodiments utilize 50mM sodium acetate at a pH in the range 5.0 to 6.0, with a conductivity of less than 6.0 mS/cm.
- Other embodiments utilize about 10 to about 40 mM sodium acetate at a pH of about 5 to about 6.
- the equilibration buffer comprises 50mM 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 to be passed across the anion exchange chromatographic resin may initially require buffer exchange, desalting or dilution to reduce the ionic strength in order to enable binding to the resin.
- Suitable solutions for buffer exchange, desalting or dilution include those having a buffering agent at a concentration of about 5mM to about 100 mM.
- the pH range of these solutions is typically in the range from about pH 5 to about 9. Whilst the conductivity is usually less than about 8.0 mS/cm.
- the solution for buffer exchange, desalting or dilution will include a buffering agent 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.
- a solution for buffer exchange, desalting or dilution is sodium acetate.
- Particular embodiments utilize 50mM sodium acetate at a pH range 5.0 to 6.0, with a conductivity of less than 6.0 mS/cm.
- Other embodiments utilize about 10 to about 40 mM sodium acetate at a pH of about 5 to about 6.
- Other embodiments include dilution of the haptoglobin load solution by addition of water (such as water for injection (WFI)) to reduce ionic strength.
- water such as water for injection (WFI)
- WFI water for injection
- the haptoglobin solution comprising 50mM sodium acetate at pH 5.3-5.7 would have a final sodium acetate concentration of about 10-13mM, pH 5.3 to 5.7, and conductivity of less than 5.0 mS/cm. This generally allows for binding of the haptoglobin to the anion exchange chromatographic resin.
- an additional buffering agent can be added to the haptoglobin solution.
- the additional buffering agent will typically have a concentration of about 5mM to about 100 mM, and a pH range of about 5 to about 9. In particular embodiments the additional buffering agent will be in the range of about 10 mM to about 60 mM, with a pH range of about 5 to about 9.
- An example of an additional buffering agent is sodium acetate. Particular embodiments utilize 50mM sodium acetate at a pH range 5.0 to 6.0. In a preferred embodiment the buffer is 50mM sodium acetate at a pH in the range of pH 5.3 to pH 5.7.
- Buffers that are suitable for eluting the haptoglobin from the resin will also be known to persons skilled in the art.
- the suitable buffer agent will have a concentration in the range of about 5 mM to about 100 mM.
- the buffering agent will be in the range of about lOmM to about 60mM.
- An example includes sodium acetate.
- Particular embodiments utilize 50mM sodium acetate at a pH of 5.0 to 6.0.
- Other embodiments utilize about 10 to about 40 mM sodium acetate buffers at a pH of about 5.0 to about 6.0.
- the buffer is about 50mM sodium acetate at a pH of about pH 5.3 to about pH 5.7.
- the haptoglobin is eluted from the anion exchange resin with an elution buffer comprising from about lOOmM to about 200mM NaCl. This equates to an elution buffer having a conductivity range of about lOmS/cm (lOOmM NaCl) to about 18mS/cm (200mM NaCl).
- the haptoglobin is eluted in the presence of about 150 to 170 mM NaCl.
- the haptoglobin is eluted in the presence of about 160 mM NaCl.
- the eluted haptoglobin is recovered and stored separately for future use.
- the eluted haptoglobin is further purified, for example, by concentrating and diafiltering the eluted haptoglobin through an ultrafiltration membrane and/or sterile filtering the concentrated and/or diafiltering haptoglobin, as required.
- the method further comprises:
- Hydrophobic Interaction Chromatography is a chromatographic technique frequently used for the separation of proteins on the basis of a hydrophobic interaction between the stationary phase and the protein to be separated.
- the level of hydrophobicity of the target protein will often dictate the type of HIC resin to be used.
- a high amount of salt is typically added to the solution to reduce the solubility of the target protein and thus increase the interaction of the target protein with the HIC resin functionalized with a suitable hydrophobic group (e.g., phenyl, butyl and octadecyl groups).
- Suitable salts typically include sodium sulfate, potassium sulfate, ammonium sulfate, ammonium chloride, sodium chloride, sodium bromide, and sodium thiocyanate.
- Suitable hydrophobic interaction chromatographic resins would be familiar to persons skilled in the art. Examples include octyl sepharose and capto octyl chromatographic resins. The conditions that allow the hemopexin to bind to the resin will be known to persons skilled in the art and will be dictated, for example, by the type of resin used and the hydrophobicity of the target protein (i.e., hemopexin).
- the hemopexin load solution contains ammonium sulfate, therefore suitable buffers for column equilibration include buffering agents, at a concentration of about 5mM to about lOOmM, which would maintain a pH of about 6 to about 8 and contain approximately 2 to 2.5 M ammonium sulfate. These conditions would allow for the binding of hemopexin to the hydrophobic interaction chromatography media.
- buffering agents at a concentration of about 5mM to about lOOmM, which would maintain a pH of about 6 to about 8 and contain approximately 2 to 2.5 M ammonium sulfate. These conditions would allow for the binding of hemopexin to the hydrophobic interaction chromatography media.
- Another embodiment utilizes an ammonium sulfate concentration of 2.2 to 2.5M, pH 7.0 to 8.0.
- the buffer solution is 50mM Tris, containing 2.5M ammonium sulfate, at a pH of 7.4.
- the hemopexin load solution typically contains about 2.0 to about 2.5M ammonium sulfate buffered at pH of about 6 to about 8. These conditions allow for the binding of hemopexin to the hydrophobic interaction media.
- the hemopexin load solution contained 2.2M to 2.5M ammonium sulfate, buffered at a pH of 7.0 to 8.0.
- the hemopexin load solution contains 50mM Tris, as a buffering agent, and 2.5M ammonium sulfate at a pH of 7.4.
- the bound hemopexin can be eluted from the resin by means known to persons skilled in the art. Prior to eluting the hemopexin from the resin, the resin can optionally be washed with a suitable wash solution or buffer under conditions that retain the hemopexin bound to the resin. Suitable wash solutions and conditions will be known to persons skilled in the art. Wash solution concentrations depend to a certain degree on column load, however typical wash solutions will possess a buffering effect at a pH of about 6 to about 8 and additionally contain approximately 0.8M to 1.5M ammonium sulfate. In another embodiment, the wash solution contains 0.9M to 1.3M ammonium sulfate at a pH of 7.0 to 8.0. In a particular embodiment, the wash solution contains 50mM Tris, as the buffering agent, 1.13M ammonium sulfate, at a pH of 7.4. The flow through wash fraction can also be collected and stored for future use, as necessary.
- the eluted hemopexin that is recovered from the resin can be stored for future use.
- the eluted hemopexin may also be subjected to further purification to remove any impurities in the eluate.
- the method further comprises: (i) passing the eluted hemopexin through a metal ion affinity chromatographic resin under conditions that allow the hemopexin to bind to the resin; and
- Immobilized metal ion affinity chromatography is based on the covalent attachment of amino acids (e.g., histidine) to metals, allowing proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions, such as zinc, cobalt, nickel or copper.
- Suitable metal ion affinity chromatographic resins would be known to persons skilled in the art.
- the metal ion affinity chromatographic resin is Ni-Sepharose.
- Buffers suitable for equilibration and binding are designed to prevent nonspecific binding to the chromatography media and optimized to promote affinity for hemopexin while minimizing binding of contaminate proteins.
- Buffers used for equilibration and binding would be generally within a pH range of about 6 to about 9 and would include the addition of about 0.5M to about 1.0M sodium chloride along with the addition of a small amount (i.e. 1 to 50mM) histidine or imidazole.
- the buffer would consist of 0.5mM to lOOmM sodium phosphate, at about pH 7 to about 8, with about 0.5M to about l .OM sodium chloride, and about ImM to about 50mM imidazole.
- the equilibration and binding buffer consists of 20mM sodium phosphate, 0.5M NaCl, and 30mM imidazole, at pH 7.4.
- the load solution (Octyl Eluate) can be adjusted to these concentrations through addition of the desired solid excipients or by addition of a concentrated solution.
- the resin may be washed to remove any residual unbound or weakly bound impurities under conditions that retain the hemopexin bound to the resin.
- Buffers suitable for the wash step would be within a pH range of 6.0 to 9.0 and would include the addition of 0.5M to l .OM sodium chloride along with the addition of a small amount (i.e. 1 to 50mM) histidine or imidazole.
- the buffer would consist of about 0.5mM to about lOOmM sodium phosphate, at about pH 7 to about pH 8, with about 0.5M to about l .OM sodium chloride, and about ImM to about 80mM imidazole.
- the wash buffer consists of 20mM sodium phosphate, 0.5M NaCl, and 30mM imidazole, at pH 7.4.
- the bound hemopexin can be eluted from the resin by means known to persons skilled in the art.
- Buffers suitable for elution would be within a pH range of about 6 to about 9 and would include the addition of about 0.5M to about 1.0M sodium chloride along with histidine or imidazole at a concentration high enough to facilitate elution.
- the elution buffer would consist of about 0.5mM to about lOOmM sodium phosphate, at about pH 7.0 to about pH 8.0, with about 0.5M to 1.0M sodium chloride, and less than about 80mM imidazole.
- the elution buffer consists of 20mM sodium phosphate, 0.5M NaCl, and lOOmM imidazole, at pH 7.4.
- the bound hemopexin can be eluted through the application of a low pH buffer between about pH 4.0 and about pH 6.0. Some embodiments would utilize about 5mM to about lOOmM sodium acetate buffer, with about 0.5M to about 1.0M NaCl, at a pH between about 4.0 and about pH 6.0 as the eluting buffer.
- the eluted hemopexin can be further purified, for example, by concentrating and diafiltering the hemopexin through an ultrafiltration membrane and/or sterile filtering the concentrated and/or diafiltering hemopexin, as required.
- any transferrin that may be present in the starting material remains in solution (i.e., in the solution comprising hemopexin) following the precipitation of haptoglobin in the presence of ammonium sulfate.
- the methods of the present invention, disclosed herein can also be used to purify transferrin from the same starting material. Conditions are therefore provided that are optimal for purifying all three proteins (Hp, Hx and transferrin) from the same starting material.
- the solution containing both haptoglobin and hemopexin e.g., the starting material
- the method further comprises:
- step (b) collecting the flow-through fraction from step (a);
- step (c) optionally washing the resin following step (b) and collecting the flow- through wash fraction;
- step (d) passing the flow-through fraction from step (b) and/or the flow-through wash fraction from step (c) through a anion exchange chromatographic resin under conditions such that transferrin binds to the resin;
- the anion exchange chromatographic resin of step (ii) is a strong anion exchange chromatographic resin. In other particular embodiments the anion exchange chromatographic resin of step (ii) is a weak anion exchange chromatographic resin.
- Suitable strong anion exchange chromatographic resins will be known to persons skilled in the art.
- suitable anion exchange resins are ones comprising a functional quaternary amine group (Q) and/or a diethylaminopropyl group (ANX).
- the strong anion exchange chromatographic resin comprises a functional quaternary amine group (e.g., Capto Q ImpResTM).
- Suitable weak anion exchange chromatographic resins will be known to persons skilled in the art. Examples include resins comprising a tertiary or secondary amine functional group, such as DEAE (diethylaminoethyl).
- HIC Wash fraction due to the ionic strength of the HIC Wash fraction; dilution, diafiltration, chromatographic desalting, or other methods of buffer exchange / ionic strength reduction would be required to allow transferrin to bind to the anion exchange resin.
- One such method of chromatographic desalting includes binding the HIC Wash fraction (transferrin), which contains about 0.8M to about 1.5M ammonium sulfate, directly onto a stronger hydrophobic ligand, such as a phenyl or butyl column. Once bound, the transferrin can be eluted in WFI or a low ionic strength buffer in preparation for anion exchange chromatography.
- Buffers suitable for anion exchange chromatography equilibration and binding allow for the binding of transferrin to the anion exchange media to promote chromatographic separation of transferrin from other impurities.
- Buffers that are suitable for the equilibration of the chromatography media have a concentration of about 5mM to about 100 mM, a pH range of about 6 to about 9, with a conductivity of less than about 9.0 mS/cm. These conditions allow for the binding of transferrin to the anion exchange media.
- the buffering agent will be in the range of about 10 mM to about 60 mM, with a pH range of about 6 to about 9, with a conductivity of less than about 7.0 mS/cm.
- Suitable buffering agents include Tris and sodium phosphate. Particular embodiments utilize 50mM Tris at a pH range in the range from about 7 to about 9, with a conductivity of less than about 6.0 mS/cm. Other embodiments utilize about 10 to about 40 mM Tris buffers at a pH of about 7.0 to about 9.0, with a conductivity of less than about 5.0 mS/cm.
- Buffers that are suitable for eluting the transferrin from the resin will also be known to persons skilled in the art.
- the suitable buffer agent will have a concentration in the range of about 5 mM to about 100 mM.
- the buffering agent will be in the range of about lOmM to about 60mM.
- suitable buffering agents include Tris or sodium phosphate. Particular embodiments utilize 50mM Tris at a pH in the range of about 7 to about 9. Other embodiments utilize about 10 to about 40 mM sodium acetate buffers at a pH of about 7 to about 9. Persons skilled in the art would know elution buffer NaCl concentrations depend to an extent upon the protein load applied to the chromatographic resin.
- Methods of elution would consist of a step gradient, in which the NaCl concentration of the buffer is increased in a stepwise faction to elute off the transferrin, or by use of a linear gradient where the NaCl of the buffer is slowly increased in a linear fashion and the column effluent is monitored to ensure the collection of transferrin.
- the transferrin is recovered from the anion exchange chromatographic resin, it can be further purified, for example, by concentrating and diafiltering the transferrin through an ultrafiltration membrane and/or sterile filtering the concentrated and/or diafiltering transferrin, as required.
- haptoglobin and/or hemopexin and/or transferrin is to be used for clinical or veterinary applications (e.g., for administration to a subject with a condition associated with haemolysis)
- a solution comprising haptoglobin and/or hemopexin and/or transferrin is to be used for clinical or veterinary applications (e.g., for administration to a subject with a condition associated with haemolysis)
- virus titre active virus content
- other potential infectious agents for example prions
- Examples include pasteurization (for example, incubating the solution at 60°C for 10 hours in the presence of high concentrations of stabilisers such as glycine (e.g. 2.75M) and sucrose (e.g. 50%) and/or other selected excipients or salts), dry heat treatment, virus filtration (passing the solution through a nano-filter; e.g., 20nm cut-off) and/or subjecting the solution to treatment with a suitable organic solvent and detergent for a period of time and under conditions to inactivate virus in the solution.
- Solvent detergent has been used for over 20 years to inactivate enveloped viruses particularly in plasma-derived products.
- Suitable solvents include tri-n-butyl phosphate (TnBP) and ether, preferably TnBP (typically at about 0.3%).
- Suitable detergents include polysorbate (Tween) 80, polysorbate (Tween) 20 and Triton X- 100 (typically at about 0.3%).
- TnBP tri-n-butyl phosphate
- Suitable detergents include polysorbate (Tween) 80, polysorbate (Tween) 20 and Triton X- 100 (typically at about 0.3%).
- the selection of treatment conditions including solvent and detergent concentrations depend in part on the characteristics of the feedstock with less pure feedstocks generally requiring higher concentrations of reagents and more extreme reaction conditions.
- a preferred detergent is polysorbate 80 and a particularly preferred combination is polysorbate 80 and TnBP.
- the feedstock may be stirred with solvent and detergent reagents at a temperature and for a time sufficient to inactivate any enveloped viruses that may be present.
- the solvent detergent treatment may be carried out for about 4 hours at 25°C.
- the solvent detergent chemicals are subsequently removed by for example adsorption on chromatographic media such as C-18 hydrophobic resins or eluting them in the drop-through fraction of ion exchange resins under conditions which adsorb the protein of interest.
- the virus inactivation step can be performed at any suitable stage of the methods disclosed herein.
- the feedstock comprising haptoglobin and/or hemopexin and/or transferrin is subject to a viral inactivation step prior to step (ii) from the first described aspect.
- the solution comprising haptoglobin and/or hemopexin and/or transferrin that is recovered from the ammonium sulphate precipitation step i.e., from steps (ii) and/or (iii)
- a viral inactivation step is conducted after step iii).
- the viral inactivation step comprises pasteurisation and/or treatment with an organic solvent and detergent.
- the method of the invention further comprises heating a solution comprising either haptoglobin and/or hemopexin and/or transferrin at 55°C to 61°C for about 30 minutes to about 12 hours. In a particular embodiment the solution is heated for about 10 to about 10.5 hours.
- the virus inactivation step comprises virus filtration.
- the method of the invention further comprises filtering a solution comprising either hemopexin and/or transferrin through a virus filter having a pore size ranging from 15 nm to 35 nm.
- a free amino acid e.g. , arginine
- a free amino acid e.g. , arginine
- the feedstock or solution comprising haptoglobin and/or hemopexin and/or transferrin is subject to a viral inactivation step before it is passed through a chromatographic resin.
- a virus inactivation step such as solvent detergent treatment prior to passing the treated solution or feedstock through a chromatographic resin such as an anion exchange resin is that it allows for the removal of the organic solvent and detergent from the treated solution by utilizing conditions that promote binding of the haptoglobin and/or hemopexin and/or transferrin to the resin and removal of the organic solvent and detergent with the flow-through (drop- through) fraction.
- Pasteurization can generate protein aggregates and polymers. Therefore, it may be desirable in some instances to reduce the level of aggregates/polymers in a pasteurized solution. This can be achieved by any means known to persons skilled in the art, although conveniently can be achieved by further chromatographic purification.
- the pasteurized solution or feedstock is passed through an anion exchange chromatographic resin in positive mode with respect to the haptoglobin and/or hemopexin and/or transferrin such that any aggregates or polymers are removed with the flow-through (drop-through) fraction.
- the methods of purifying haptoglobin, and/or hemopexin and/or transferrin are conducted generally in the temperature range of about 18°C to about 26°C.
- composition comprising the haptoglobin recovered by the methods disclosed herein.
- the composition comprises a haptoglobin content of at least 80% of total protein.
- the composition comprises a haptoglobin content of at least 90% of total protein.
- the composition comprises a haptoglobin content of at least 95%.
- the composition comprises a haptoglobin content of at least 98%.
- the composition comprising haptoglobin comprises less than 0.03 mg of IgA per mg of haptoglobin as determined by immunonephelometry.
- the composition comprising haptoglobin when at a haptoglobin concentration of 26 mg/mL contains less than 0.067 mg/mL IgG, less than 0.042 mg/mL IgM, less than 0.050 mg/mL alpha- 1 -acid glycoprotein, less than 0.018 mg/mL pre-albumin, less than 0.021 mg/mL ceruloplasmin, less than 0.051 mg/mL hemopexin, less than 0.053 mg/mL apolipoprotein A-I, less than 0.227 mg/mL 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 IgA, and less than 0.15 mg/mL albumin (as determined by immunonephel
- a composition comprising the hemopexin recovered by the methods disclosed herein.
- the composition comprises a hemopexin content of at least 80% of total protein.
- the composition comprises a hemopexin content of at least 90% of total protein.
- the composition comprises a hemopexin content of at least 95%.
- the composition comprises a hemopexin content of at least 97%.
- the composition comprises a hemopexin content of at least 98%.
- the composition comprising hemopexin is at least 98% pure and comprises 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, less than 0.090 mg/mL transferrin, less than 0.050 mg/mL alpha- 1 -acid glycoprotein, less than 0.018 mg/mL prealbumin, less than 0.021 mg/mL ceruloplasmin, less than 0.053 mg/mL apolipoprotein A-I, less than 0.227 mg/mL apolipoprotein B, less than 0.031 mg/mL antithrombin III, less than 0.17 mg/mL haptoglobulin and less than 0.045 mg/mL albumin (as determined by immunonephelometry) .
- composition comprising hemopexin is at least 97% pure and contains 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 immunonephelometry.
- composition comprising the transferrin recovered by the methods disclosed herein.
- the composition comprises a transferrin content of at least 80% of total protein.
- the composition comprises a transferrin content of at least 90% of total protein.
- the composition comprises a transferrin content of at least 95%.
- the composition comprises a transferrin content of at least 98%.
- composition comprising the haptoglobin recovered by the methods disclosed herein and the hemopexin recovered by the methods disclosed herein.
- the composition comprises a combined haptoglobin and hemopexin content of at least 80% of total protein.
- the composition comprises a combined haptoglobin and hemopexin content of at least 90% of total protein.
- the composition comprises a combined haptoglobin and hemopexin content of at least 95% of total protein.
- the composition comprises a combined haptoglobin and hemopexin content of at least 98% of total protein.
- the composition further comprises the transferrin recovered by the methods disclosed herein.
- the composition comprises a combined haptoglobin, hemopexin and transferrin content of at least 80% of total protein.
- the composition comprises a combined haptoglobin, hemopexin and transferrin content of at least 90% of total protein.
- the composition comprises a combined haptoglobin, hemopexin and transferring content of at least 95% of total protein.
- the composition comprises a combined haptoglobin, hemopexin and transferrin content of at least 98% of total protein.
- compositions comprising a haptoglobin content of at least 80%, 90%, 95%, or 98% of total protein.
- a composition comprising a hemopexin content of at least 80%, 90%, 95%, or 98% of total protein.
- a composition comprising a combined hemopexin, haptoglobin and transferrin content of at least 80%, 90%, 95%, or 98% of total protein.
- compositions comprising haptoglobin, hemopexin and/or transferrin recovered by the methods of the present invention disclosed herein will be substantially free of other components with which they are normally associated (e.g., other plasma- derived proteins).
- the composition comprising haptoglobin, hemopexin and/or transferrin will comprise 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 with which they are normally associated (i.e., impurities).
- impurities i.e., impurities
- compositions are to be administered to a human subject in need thereof (i.e., for clinical use), it would be desirable that the composition comprises less than 5% impurities (of total protein). Conversely, where the proteins are to be used in vitro, it may be acceptable if the composition comprises more than 5% of impurities (of total protein).
- composition of the present invention as 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 and absorption agents and the like.
- the pharmaceutical formulation may also be formulated by the addition of (or a combination of) suitable stabilisers, for example, an amino acid, a carbohydrate, a salt, and a detergent.
- the stabiliser comprises a mixture of a sugar alcohol and an amino acid.
- the stabilizer may comprise a mixture of a sugar (e.g. sucrose or trehalose), a sugar alcohol (e.g. mannitol or sorbitol), and an amino acid (e.g. proline, glycine and arginine).
- the formulation comprises an amino acid such as arginine.
- the formulation comprises divalent metal ions in a concentration up to lOOmM and a complexing agent as described in US7045601.
- the pH is preferably about 6.5 to 7.5 and the osmolality is at least 240 mosmol/kg.
- the pharmaceutical formulation may also be sterilised by filtration prior to dispensing and long term storage.
- the formulation will retain substantially its original stability characteristics for at least 2, 4, 6, 8, 10, 12, 18, 24, 36 or more months.
- 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 6 months or longer.
- Particular embodiments of the pharmaceutical formulation 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 when stored at 2-8°C and/or room temperature.
- compositions described herein may be formulated into any of many possible dosage forms such as injectable formulations.
- the formulations and their subsequent administration (dosing) are within the skill of those in the art. Dosing is dependent on the responsiveness of the subject to treatment, but will invariably last for as long as the desirable effect (e.g., a reduction in the level of free Hb/heme) is desired. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
- the pharmaceutical formulation of the present invention is a solution that has a volume of at least 5 mL and comprises at least 5 mg/mL 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 haptoglobin and/or hemopexin and/or transferrin.
- the pharmaceutical formulation has a volume of at least 5 mL and comprises haptoglobin and/or hemopexin and/or transferrin at a concentration of about 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 150 mg/mL or 200 mg/mL.
- a vessel containing at least 5 mL of a stable pharmaceutically acceptable haptoglobin and/or hemopexin and/or transferrin solution wherein the concentration of haptoglobin and/or hemopexin and/or transferrin is at least 20 mg/mL.
- the pharmaceutical formulation comprising haptoglobin and/or hemopexin and/or transferrin is lyophilized. Due to the presence of lyophilization stabilizer, like sugars (e.g. sucrose), sugar alcohols (e.g. mannitol), and an amino acid (e.g. glycine or proline) or combinations thereof, the lyophilisation yields in a stable powder having a long shelf life. This powder may be stored, used directly or after storage as a powder or used after rehydration to form the pharmaceutical formulation.
- the lyophilized pharmaceutical formulation of the present invention may be formed using any method of lyophilization known in the art, including, but not limited to, freeze drying, i.e.
- the haptoglobin and/or hemopexin and/or transferrin- containing formulation is subjected to freezing followed by reduced pressure evaporation.
- the lyophilized formulations that are provided can retain substantially their original stability characteristics for at least 2, 4, 6, 8, 10, 12, 18, 24, 36 or more months.
- 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 6 months or longer.
- Particular embodiments of the haptoglobin and/or hemopexin and/or transferrin pharmaceutical formulation 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 when stored at 2-8°C and/or room temperature.
- the lyophilised pharmaceutical formulation comprises hemopexin.
- the invention may be used for large scale production of lyophilised pharmaceutical formulations.
- the lyophilized. product may be prepared for bulk preparations, or alternatively, may be apportioned in smaller containers (for example, single dose units) prior to lyophilization, and such smaller units may be used as sterile unit dosage forms.
- the lyophilized formulation can be reconstituted in order to obtain a solution or suspension of the protein.
- the lyophilized powder is rehydrated with an aqueous solution to a suitable volume.
- Preferred aqueous solutions are water for injection (WFI), phosphate-buffer saline or a physiological saline solution.
- the mixture can be agitated to facilitate rehydration.
- the reconstitution step is conducted at room temperature.
- a method of treating a condition associated with haemolysis comprising administering to a subject in need thereof the composition or the formulation of the present invention, as disclosed herein.
- subject refers to an animal which includes a primate (a lower or higher primate).
- a higher primate includes human.
- the present invention has particular application to targeting conditions in humans, it would be understood by those skilled in the art that non-human animals may also benefit from the compositions and methods disclosed herein.
- an "animal” includes livestock and companion animals such as cattle, horses, sheep, pigs, camelids, goats, donkeys, dogs and cats. With respect to horses, these include horses used in the racing industry as well as those used recreationally or in the livestock industry.
- compositions or formulations of the present invention may be administered to the subject a number of ways.
- suitable routes of administration include intravenous, subcutaneous, intra-arterial or by infusion.
- the molecules are administered intravenously.
- the methods of the present invention may further comprise administering a second therapeutic agent.
- the second therapeutic compound may be coadministered to the subject sequentially (before or after administration of the compositions or formulations disclosed herein) or concurrently.
- the second therapeutic agent is an iron chelating agent (e.g., deferrioxamine or deferiprone).
- compositions or formulations of the present invention in another aspect of the present invention, in the manufacture of a medicament for treating a condition associated with haemolysis.
- Such compositions or formulations are preferably suitable for use in human patients.
- the condition is selected from an acute haemolytic condition and/or a chronic haemolytic condition.
- the condition is selected from the group consisting of haemolytic anaemia, transfusion-induced haemolysis, haemolytic uraemic syndrome, an autoimmune disease, malaria infection, trauma, blood transfusion, open heart surgery using cardiopulmonary bypass and burns, including in the treatment of hemoglobinemia or hemoglobinuria accompanied with hemolysis after burn.
- the condition is selected from the group consisting of sickle cell anaemia, hereditary spherocytosis, hereditary elliptocytosis, thalassemia, congenital dyserythropoietic anemia and Paroxysmal nocturnal hemoglobinuria, systemic lupus erythematosus and chronic lymphocytic leukemia.
- Precipitate Extraction Extraction of the precipitate was performed by the introduction of 20 grams of Extraction Buffer per gram of Fraction IV 4 Precipitate (20x Extraction Ratio). The buffer and the precipitate were mixed for a minimum of 1 hour. Extraction Buffer consisted of 50mM Tris adjusted to a pH of 7.0 with Concentrated HC1. The Extraction Buffer was prepared at a temperature of 20-25 °C. The precipitate extraction was also performed at a temperature of 20-25°C. The pH during the extraction was maintained between 7.0 to 8.0 (preferably 7.0) for the duration of the 1 hour extraction time.
- Ammonium Sulfate Precipitation Solid ammonium sulfate was added to the Fraction IV 4 extract in order to achieve a final concentration of 2.0 to 2.5M (preferably 2.4M). Under agitation, the ammonium sulfate was slowly added to the extract and allowed to continually mix for a minimum of 1 hour. The ammonium sulfate precipitation was performed at a temperature of 20-25°C.
- the treated extract was pumped into the filter press and the filtrate was collected after a single pass through the filter press.
- the filter press was then post-washed with 1 to 2 press volumes of 2.4M ammonium sulfate, 50mM Tris, adjusted to pH 7.0.
- the filtration process was performed at a temperature of 20-25°C.
- the resulting filtrate contains hemopexin and can be stored at 2- 8°C until it is carried forward for further purification.
- the resulting precipitate contains haptoglobin and can be stored at less than or equal to -20°C until it is carried forward for further purification.
- Impurities and any unbound protein were washed off of the column with 3 column volumes 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 saved for further purification.
- the hemopexin containing fraction (Eluate) was then eluted off of the column with three column volumes of water (WFI). The Eluate was then stored at 2-8°C until used for further transferrin purification.
- the Octyl Sepharose (HIC) purification was performed at a temperature of 20-25°C.
- IMAC Ni-Sepharose Chromatography
- the 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 less than or equal to -20°C until use.
- the final yield of hemopexin recovered from the process was estimated to be approximately 0.151 g L plasma.
- the concentrated hemopexin preparation (approximately 2.3% w/v) was characterised by immune-nephelometry.
- a further batch of hemopexin was processed according to the methods described above.
- the batch was concentrated to 35 mg/mL protein.
- Haptoglobin Hp
- the Haptoglobin was extracted from the precipitate by the introduction of 20 grams of extraction buffer/gram of precipitate (20x ratio).
- the extraction buffer consisted of 50mM Tris, adjusted to pH 8.5.
- the buffer and the precipitate were mixed for a minimum of 1 hour.
- the pH was adjusted to within the range of 8.4 to 8.6.
- Aerosil fumed silica
- Aerosil treated extract was then allowed to mix for a minimum of 1 hour.
- the extract was passed through a Cuno 70CA filter or other similar depth filter.
- the filtrate was then clarified by use of a 0.2 ⁇ filter.
- the resulting filtrate was ready for the subsequent chromatography step.
- the extraction buffer preparation, the extraction process, and the filtration process were all performed at 20- 25°C.
- the column was washed with 2 column volumes of 50mM sodium acetate, pH 5.5 to remove any unbound contaminate proteins.
- the haptoglobin was then eluted with 4 column volumes of 50mM sodium acetate, 100-200 mM NaCl (preferably 162mM), pH 5.5.
- a wide NaCl concentration range of the elution buffer is required as the elution conditions are partially dependant on the load volume utilized.
- the eluate can be stored at 2-8°C, short term, and at less than or equal to -20°C, long term, until concentration/diafiltration.
- 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 buffers and chromatography steps were all performed at 20-25°C.
- a lipid adsorption step can be conducted before or after the anion exchange chromatography step or after the concentration/diafiltration step.
- An example of a suitable lipid adsorption agent is a fumed silica like Aerosil (e.g. Aerosil 380).
- the final yield of haptoglobin recovered from the process was estimated to be approximately 0.285 g/L plasma input.
- the concentrated haptoglobin preparation (approximately 2.6% w/v) was characterised by immune-nephelometry.
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AU2013203930A AU2013203930B2 (en) | 2012-10-03 | 2013-04-11 | A method of purifying proteins |
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ES2887588T3 (es) * | 2015-08-13 | 2021-12-23 | Kamada Ltd | Composiciones derivadas de pasta de fracción de Cohn y uso de las mismas |
KR20180129791A (ko) * | 2016-03-29 | 2018-12-05 | 교와 핫꼬 기린 가부시키가이샤 | 혈 중에서 합토글로빈에 결합하여 다가의 면역 복합체를 형성하는 항체를 유효 성분으로 하는 자기 면역 질환 치료제 |
KR20200038507A (ko) * | 2017-08-08 | 2020-04-13 | 체에스엘 베링 아게 | 헤모펙신 제형 |
US20200393472A1 (en) * | 2018-04-18 | 2020-12-17 | Sekisui Medical Co., Ltd. | Haemoglobin analysis method |
CA3138650A1 (en) | 2019-05-17 | 2020-11-26 | Universitaet Zuerich | Haptoglobin for use in treating an adverse secondary neurological outcome following a haemorrhagic stroke |
WO2022162218A1 (en) | 2021-02-01 | 2022-08-04 | Csl Behring Ag | Method of treating or preventing an adverse secondary neurological outcome following a haemorrhagic stroke |
US20240034749A1 (en) * | 2021-02-04 | 2024-02-01 | Green Cross Corporation | Method for purification of hemopexin and haptoglobin |
WO2024134608A1 (en) | 2022-12-22 | 2024-06-27 | Csl Behring Ag | Method of purifying hemopexin using mixed-mode chromatography |
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