Compositions for pulmonary administration, kits, and methods for treatment

Abstract

Provided herein are compositions and methods for the treatment of hemolysis in pulmonary tissue. Also provided herein are methods of purifying proteins including apo-transferrin, haptoglobin, hemopexin, and combinations thereof.

Description

[0001] Compositions for Pulmonary Administration, Kits, and Methods for Treatment

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of priority of U.S. Provisional Application No. 63 / 669,027, filed July 9, 2024, which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under grant number R01 HL158076 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Hemolysis refers to the breakdown of red blood cells (RBCs), leading to the release of hemoglobin and other intracellular contents into the surrounding plasma. Hemolysis in the pulmonary vasculature has been increasingly recognized as a significant contributor to various pathophysiological conditions, including pulmonary hypertension, acute respiratory distress syndrome (ARDS), and transfusion-related acute lung injury (TRALI).

[0008] Free hemoglobin in the pulmonary circulation can scavenge nitric oxide (NO), leading to vasoconstriction, oxidative stress, and endothelial dysfunction. In addition, the release of free heme and iron exacerbates local inflammation and promotes tissue injury. These effects can impair gas exchange, increase pulmonary vascular resistance, and ultimately compromise respiratory function. Conditions such as sickle cell disease, sepsis, mechanical trauma from extracorporeal membrane oxygenation (ECMO), and blood transfusions are commonly associated with elevated levels of hemolysis in the lungs.

[0009] Current strategies for managing hemolysis are largely supportive and systemic in nature, such as the use of antioxidants, iron chelators, and NO donors. However, these approaches often lack specificity for the pulmonary tissue and may be associated with off- target effects or limited efficacy in targeting local hemolytic damage. There remains a need for improved methods that selectively target or mitigate the adverse effects of hemolysis within the pulmonary environment.

[0010] SUMMARY

[0011] Provided herein are compositions that comprise a therapeutically effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof to treat hemolysis in pulmonary tissue in a subject. In some embodiments, the composition comprises at least two of apo-transferrin, haptoglobin, and hemopexin. In certain embodiments, the composition comprises apo-transferrin, haptoglobin, and hemopexin.

[0012] In some embodiments, the hemolysis in pulmonary tissue is characterized by elevated levels of hemoglobin in the pulmonary tissue, elevated levels of free iron in pulmonary tissue, elevated levels of heme in the pulmonary tissue, or a combination thereof.

[0013] In some embodiments, the composition comprises an effective amount of apotransferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apohemoglobin-haptoglobin complex, or a combination thereof to reduce a level of hemoglobin in the pulmonary tissue of the subject, reduce a level of free iron in the pulmonary tissue, reduce a level of heme in the pulmonary tissue, or a combination thereof.

[0014] In some embodiments, the hemolysis in the pulmonary tissue in the subject is associated with pulmonary hypertension associated with sickle cell disease, acute respiratory distress syndrome (ARDS), and transfusion-related acute lung injury (TRALI), inhalation of a toxic or irritating agent that induces hemolysis (e.g., ozone, ionizing radiation, smoking, vapes, asbestos, silica, etc.), mechanical ventilation, lung transplantation, lung surgery, or a combination thereof.

[0015] In some embodiments, the composition is formulated for pulmonary administration to the subject.

[0016] In some embodiments, the composition comprises an aqueous solution or suspension.

[0017] In some embodiments, the composition comprises a dry powder formulation. In certain embodiments, the dry powder formulation comprises particles having a tap density of less than 0.4 g / cm, a mass median aerodynamic diameter (MMAD) of between 0.5 micron and 5.0 microns, or a combination thereof.

[0018] In some embodiments, the composition comprises an aerosol comprising liquid droplets or solid particles suspended in a gas. In certain embodiments, the aerosol comprises liquid droplets having a droplet diameter of less than 5 microns MMAD suspended in the gas. In certain embodiments, the aerosol comprises solid particles having a tap density of less than 0.4 g / cm, a mass median aerodynamic diameter (MMAD) of between 0.5 micron and 5.0 microns, or a combination thereof suspended in the gas.

[0019] In some embodiments, the composition comprises apo-transferrin. The apotransferrin can comprise human apo-transferrin or recombinant apo-transferrin.

[0020] In certain embodiments, the apo-transferrin is purified by ultrafiltration, such as tangential flow filtration (TFF). In certain embodiments, the apo-transferrin is purified by a process that comprises obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50-100 kDa; isolating transferrin from the protein cocktail using chromatography; and contacting the transferrin with a chelating agent to remove transferrin-bound iron, thereby isolating the apo-transferrin. In certain embodiments, the chromatography comprises ion exchange chromatography, such as anion exchange chromatography.

[0021] In some embodiments, the composition comprises haptoglobin. The haptoglobin can comprise human haptoglobin or recombinant haptoglobin.

[0022] In certain embodiments, the haptoglobin is purified by ultrafiltration, such as tangential flow filtration (TFF). In certain embodiments, the haptoglobin is purified by a process that comprises obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50 kDa and 0.2 microns (e.g., 50-500 kDa, 50-100 kDa, 100-500 kDa, 200-500 kDa, 300-500 kDa, 100 kDa-0.2 microns); and isolating haptoglobin from the protein cocktail. In certain embodiments, isolating haptoglobin from the protein cocktail comprises chromatography, such as hydrophobic interaction chromatography (e.g., using an alkyl ligand or an aryl ligand such as a phenyl ligand). In certain embodiments, isolating haptoglobin from the protein cocktail comprises ammonium sulfate precipitation, polyethylene glycol (PEG) precipitation, or a combination thereof.

[0023] In some embodiments, the composition comprises hemopexin. The hemopexin can comprise human hemopexin or recombinant hemopexin.

[0024] In certain embodiments, the hemopexin is purified by ultrafiltration, such as tangential flow filtration (TFF). In certain embodiments, the hemopexin is purified by a process that comprises obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50-100 kDa; and isolating hemopexin from the protein cocktail using chromatography. In certain embodiments, the chromatography comprises immobilized metal affinity chromatography, such as nickel (Ni2+) affinity chromatography.

[0025] Also provided herein are methods of treating hemolysis in pulmonary tissue in a subject comprising administering a composition comprising a therapeutically effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apohemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof to the subject.

[0026] In some embodiments, the hemolysis in pulmonary tissue is characterized by elevated levels of hemoglobin in the pulmonary tissue, elevated levels of free iron in pulmonary tissue, elevated levels of heme in the pulmonary tissue, or a combination thereof.

[0027] In some embodiments, the composition comprises an effective amount of apotransferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apohemoglobin-haptoglobin complex, or a combination thereof to reduce a level of hemoglobin in the pulmonary tissue of the subject, reduce a level of free iron in the pulmonary tissue, reduce a level of heme in the pulmonary tissue, or a combination thereof.

[0028] In some embodiments, the hemolysis in the pulmonary tissue in the subject is associated with pulmonary hypertension associated with sickle cell disease, acute respiratory distress syndrome (ARDS), and transfusion-related acute lung injury (TRALI), inhalation of a toxic or irritating agent that induces hemolysis (e.g., ozone, ionizing radiation, smoking, vapes, asbestos, silica, etc.), mechanical ventilation, lung transplantation, lung surgery, or a combination thereof.

[0029] Also provided herein are methods of reducing a level of hemoglobin in the pulmonary tissue of a subject, reducing a level of free iron in the pulmonary tissue of the subject, reduce a level of heme in the pulmonary tissue of the subject, or a combination thereof, the method comprising administering a composition comprising a therapeutically effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof to the subject.

[0030] Also provided herein are methods of treating pulmonary hypertension associated with sickle cell disease, a method of treating acute respiratory distress syndrome (ARDS), a method of treating transfusion-related acute lung injury (TRALI), a method of treating inhalation of a toxic or irritating agent that induces hemolysis (e.g., ozone, ionizing radiation, smoking, vapes, asbestos, silica, etc.), a method of mechanical ventilation, a method of lung transplantation, a method of lung surgery, or a combination thereof, the method comprising administering a composition comprising a therapeutically effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobinhaptoglobin complex, or a combination thereof to the subject.

[0031] In some embodiments of the above methods, the administration comprises pulmonary administration.

[0032] In some embodiments of the above methods, the composition is administered to the subject using a nebulizer, a dry powder inhaler, or a pressurized metered dose inhaler.

[0033] In some embodiments of the above methods, the composition comprises a dry powder formulation and the administration comprises inhalation of the dry powder formulation. In some embodiments of the above methods, the dry powder formulation comprises particles having a tap density of less than 0.4 g / cm, a mass median aerodynamic diameter (MMAD) of between 0.5 micron and 5.0 microns, or a combination thereof.

[0034] In some embodiments of the above methods, the composition comprises an aqueous solution or suspension and the administration comprises inhalation of an aerosol formed by nebulization of the composition. In certain embodiments of the above methods, the nebulization of the composition creates: (a) a mass median aerodynamic diameter (MMAD) of droplet size of the composition emitted from a nebulizer of from 0.5 microns to 5 microns; (b) a volumetric mean diameter (VMD) droplet size of the composition emitted from a nebulizer of from 0.5 microns to 5 microns; (c) a Geometric Standard Deviation (GSD) of emitted droplet size distribution of the composition emitted from a nebulizer of from 1.0 micron to 3.4 microns; (d) a fine particle fraction (FPF=% of aerosol particles less than or equal to 5 microns) of droplets emitted from a nebulizer of at least 30%; (e) an output rate of at least 0.1 mL / min from a nebulizer; or a combination thereof.

[0035] In some embodiments of the above methods, the administration comprises pulmonary administration, and wherein pulmonary administration locally administers the apo-transferrin, the haptoglobin, the hemopexin, the apo-hemoglobin, the PEGylated apo-hemoglobin, the apo-hemoglobin-haptoglobin complex, or the combination thereof to the pulmonary tissue of the subject. In certain embodiments of the above methods, the administration comprises pulmonary administration, and wherein pulmonary administration of apo-transferrin, the haptoglobin, the hemopexin, the apo-hemoglobin, the PEGylated apo-hemoglobin, the apohemoglobin-haptoglobin complex, or the combination thereof does not substantially increase levels of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo- hemoglobin, apo-hemoglobin-haptoglobin complex, or a combination thereof circulation in a blood stream of the subject.

[0036] Also provided herein are methods of purifying apo-transferrin comprising obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50-100 kDa; isolating transferrin from the protein cocktail using chromatography; and contacting the transferrin with a chelating agent to remove transferrin-bound iron, thereby isolating the apo-transferrin. In certain embodiments, the chromatography comprises ion exchange chromatography, such as anion exchange chromatography.

[0037] Also provided herein are methods of purifying haptoglobin comprising obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50 kDa and 0.2 microns (e.g., 50-500 kDa, 50-100 kDa, 100-500 kDa, 200-500 kDa, 300-500 kDa, 100 kDa-0.2 microns); and isolating haptoglobin from the protein cocktail. In certain embodiments, isolating haptoglobin from the protein cocktail comprises chromatography, such as hydrophobic interaction chromatography (e.g., using an alkyl ligand or an aryl ligand such as a phenyl ligand). In certain embodiments, isolating haptoglobin from the protein cocktail comprises ammonium sulfate precipitation, polyethylene glycol (PEG) precipitation, or a combination thereof.

[0038] Also provided herein are methods of purifying hemopexin that comprise obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50-100 kDa; and isolating hemopexin from the protein cocktail using chromatography. In certain embodiments, the chromatography comprises immobilized metal affinity chromatography, such as nickel (Ni2+) affinity chromatography.

[0039] DESCRIPTION OF DRAWINGS

[0040] Figure 1. Process flow diagram of the Tf purification process. The protein cocktail (50 - 100 kDa) derived from Cohn fraction IV was applied onto a HiPrep Q FF 16 / 10 column to remove contaminant proteins. The final purified Tf was then treated with EDTA while stirring at 4°C overnight to yield apo-Tf.

[0041] Figures 2A-2B. Anion exchange chromatography of the protein cocktail derived from Cohn fraction IV to yield purified Tf. Figure 2A represents a typical chromatogram. The diluted protein cocktail was loaded onto a 20 mL HiPrep Q FF column. %B represents the amount of buffer B used to elute bound proteins. CV: column volume. Awash was performed at 10% buffer B to remove contaminant proteins. The absorbance was monitored at 280 nm. Figure 2B represents a typical SDS-PAGE of the Q FF purification of Tf from the diluted protein cocktail. Lane 1 : MW ladder. Lane 2: Protein cocktail. Lanes 3-5: Represent peak 1 fractions from Figure 2A. Lanes 6-11 : Represent peak 2 fractions. Lane 12: Consists of a 2 M NaCl cleaning wash.

[0042] Figures 3A-3B. MALDI-TOF analysis of the protein cocktail and purified Tf. Figure 3 A represents a typical MALDI-TOF spectra of the protein cocktail, which was used as the starting material for the purification of Tf. The protein cocktail primarily consists of HSA with a MW of 66.5 kDa and Tf with a MW of 78.4 kDa. Figure 3B represents a typical MALDI-TOF spectra of the purified Tf product with a MW of 78.4 kDa.

[0043] Figures 4A-4B. Trypsin digest mass spectrometry analysis of the protein cocktail and purified Tf. Both figures show the ten most abundant proteins and cover > 99% of all proteins in the sample. Figure 4A represents the protein composition of the protein cocktail that was used as the starting material for the purification of Tf. The error bars were derived from 3 separate purifications. Figure 4B represents the protein composition of the purified Tf. The error bars were derived from 3 separate purifications. TF: transferrin; ALB: albumin; HP: haptoglobin; GC: vitamin D-binding protein; HPX: hemopexin; APOH: beta-2-gly coprotein; HPR: haptoglobin related protein; SERPINA1 : alpha- 1 -antitrypsin; CP: ceruloplasmin; HBA1 : hemoglobin subunit alpha; HBB: hemoglobin subunit beta; APOA1 : apolipoprotein A-I; APOA2: apolipoprotein A-II; RBP4: retinol-binding protein 4; IGHG4: immunoglobulin heavy constant gamma 4; IGG1 : immunoglobulin gamma-1 heavy chain; and CFI: complement factor I.

[0044] Figure 5A. Ferric Fe binding capacity of purified apo-Tf. Purified Tf and FeNTA were incubated together for 2 hrs at 25 °C and the absorbance spectra was measured using UV-visible spectrometry. The spectra of FeNTA, Tf, and Tf + FeNTA were experimentally measured, and the apo-Tf spectra was calculated by subtracting the FeNTA and Tf spectra from the Tf + FeNTA spectra. The absorbance peak at 470 nm was used to determine the amount of iron bound to purified Tf.

[0045] Figure 5B. Fe titration of purified Tf. Purified Tf and increasing concentrations of FeNTA were incubated for 1 hr at 25 °C. Samples were aliquoted into a 96 well plate at a volume of 350 pL. The absorbance was measured at 450 nm and fit to 2 linear functions to determine the Fe saturation point of the purified Tf.

[0046] Figure 6. Auto-oxidation of Hb in the presence or absence of free ferric Fe and Tf. Absorbance changes were measured over a 4 hr timeframe at 25°C with a time point taken every 10 minutes.

[0047] Figure 7. Far UV CD spectra of apo-Tf in 50 mM PB, at pH 7.4.

[0048] Figures 8A-8B. Kinetics of Fe binding to Tf. Figure 8A shows stopped flow kinetic traces of fluorescence quenching obtained upon binding of 5 pM Tf with increasing concentrations of FeNTA from lO.OpM - 80.0 pM. Each kinetic trace represents an average of 10 scans and were fit to a biexponential function to obtain kapparent (pseudo 1storder rate constant). Figure 8B shows a linear fit of kapparent as a function of Tf concentration for regression of the fast and slow second order rate constant for the binding of FeNTA with apo- Tf.

[0049] Figure 9. EPR spectra of holo-Tf. Purified apo-Tf was incubated with FeNTA for 2 hours at room temperature on a shaker at 100 rpm. EPR measurements were taken at 20 K on an X-Band (9.4 Ghz) EMXPlus with the center field at 2600 G and sweep width of 4000 G. The receiver gain was set at 30 dB, modulation amplitude at 10 G, microwave attenuation at 20 dB, and microwave power at 2 mW. Five measurements were averaged to increase the signal to noise ratio.

[0050] Figure 10. Qualitative 3D reconstruction of whole lung using light sheet microscopy of Tf distribution. Panel A. Light sheet microscopy of Berk-SS mouse whole lung showing auto-fluorescence (AF, in green) after laser excitation at 488 nm. Panel B. shows a section of the right superior lung lobe (box 1) to highlight distribution of Alexa fluor 647 conjugated Tf fluorescence after laser excitation at 638 nm (red) merged with auto-fluorescence. Panel C. Shows the right middle lung lobe (box 2) with Alexa fluor 647 conjugated Tf fluorescence merged with auto-fluorescence. Panel D. Shows the left lung lobe (box 2) with Alexa fluor 647 conjugated Tf fluorescence merged with auto-fluorescence. All images were captured using a 5* detection objective and each illumination collar is set at 1.52 to match the refractive index of the Cubic solutions. All scale bars = 500 pm

[0051] Figure 11. Time course of lung vascular Tf distribution after intrapulmonary dosing. Representative images of tissue sections prepared from the left lung lobe are shown for nontreated (NT-Not treated), 2, 4, 8, 12 and 24 hours after intrapulmonary dosing. Column A. Shows DAPI (4',6-diamidino-2-phenylindole) nuclear staining (Blue). Column B. Shows Tf immunofluorescence in and around blood vessels (Red). Column C. Shows the merged image of DAPI with Tf. Column D. Shows the brightfield merge to allow for visualization of structural features. Lung vascular smooth muscle tissue images were obtained at 63* objective magnification, scale bars = 50 gm. Arrows indicate the location of blood vessels.

[0052] Figures 12A-12E. Tf distribution to murine lung lobes after intra-pulmonary dosing. Figures 12A-12D show the mean fluorescence intensity for Tf derived immunofluorescence staining area for the left, right superior, right middle, and right inferior lung lobes, respectively. Figure 12E shows the mean fluorescence intensity for Tf comparing fluorescence intensity between lung lobes over 24 hours post dosing. Image immunofluorescence intensity quantitation was determined for each of the four primary mouse lung lobes (N=4 mice / lobe). A minimum of 5 images per lobe per animal were used in the analysis. All tissue images were analyzed using ImageJ software. Data shown as mean ± SD, significance in this data set are shown as P<0.05*, P<0.01**, P< 0.001***, P < 0.0001 ****

[0053] Figures 13A-13B. Tf plasma concentrations. Figure 13 A shows the plasma concentrations of human plasma derived Tf after intra-pulmonary dosing (open circles) and subcutaneous dosing (grey circles). Time points were before dosing (0 hours), 1, 2, 4, 8, 12 and 24 hours after dosing (N=4 mice per time point). Figure 13B shows the AUC values for subcutaneous dosing and intra-pulmonary dosing AUC values were calculated using the linear trapezoidal rule from time 0-24 hours. Data are shown as the mean ± SD, significance in this data set are shown as P < 0.0001 ****.

[0054] Figures 14A-14B. Affinity chromatography of the protein cocktail to yield Hpx. Figure 14A: Typical chromatogram. Diluted protein cocktail was loaded onto a 20 mL HisPrep FF column loaded with Ni. %B represents the amount of buffer B used to elute bound proteins. A wash step was performed with 5% B to remove unbound proteins. Additional isocratic steps at 10%, and 25% B were used to remove contaminants bound to the column. Purified protein was obtained at 50% B. Figure 14B: Represents a typical SDS- PAGE gel of the Ni-NTA purification of Hpx from the protein cocktail. Lane 1 : MW ladder. Lane 2: Protein cocktail. Lane 3: Sample loading. Lane 4: Column wash. Lane 5: Peak 1. Lane 6: Peak 2. Lane 7-12: Peak 3.

[0055] Figures 15A-15B. MALDI-TOF analysis of the protein cocktail and purified Hpx. Figure 15 A: A representative MALDI-TOF spectra of the protein cocktail which was used as the starting material for the purification of Hpx. The protein cocktail consists primarily of HSA and Tf. Figure 15B: A representative MALDI-TOF spectra of the purified Hpx product with a MW of - 60 kDa.

[0056] Figures 16A-16B. CD Spectra. Figure 16A: Far UV CD spectra of purified Hpx. The CD spectra was collected for both apo-Hpx and holo-Hpx at a protein basis of 9 pM combined with equimolar heme. Figure 16B: Temperature induced protein unfolding.

[0057] Figures 17A-17D. Hpx activity. Figure 17A: Stopped flow kinetics measurements. Hpx (5 pM) was mixed with increasing concentrations of heme (10 - 40 pM) and the absorbance change at 414 nm was measured and normalized. Figure 17B: Plot of kapparent versus heme concentration. Figure 17C: Hpx binding to heme measured at 414 nm with increasing heme:Hpx molar ratio (L / P). Figure 17D: Ferric heme oxidation in the presence of H2O2.

[0058] Figure 18. EPR spectrum of holo-Hpx. Purified apo-Hpx at a protein basis of 200 pM was incubated with a L / P = 0.9 ratio of heme.

[0059] Figure 19. HIC chromatography of Hp. General chromatogram of the Hp purification process. The absorbance was monitored at 280 nm. Diluted protein sample (10x was loaded onto a 1 mL PhenylHP column in PBS with 1.5 M AS (pH 7.00) at a flow rate of 1.00 mL / min. The column was immediately washed with 100% buffer A for 5 column volume (CVs). An isocratic purification scheme was used to purify the Hp by increasing the %B to 20%, 50% and finally 100%.

[0060] Figures 20A-20D. Protein quantification. Figure 20A: Representative SDS-PAGE gel of the PhenylHP purification process under reducing conditions. Lane 1 : MW ladder. Lane 2: starting sample. Lane 3: wash step. Lanes 4-6: peak 1. Lanes 7-11 : peak 2. Lane 12: peak 3. Figure 20B: Representative SDS-PAGE gel of peak 2 fractions under non-reducing and reducing conditions. Lanes 1-5 are under non-reducing conditions. Lane 1 : starting sample. Lanes 2-5: peak 2. Lane 6: MW ladder. Lanes 7-11 are under reducing conditions. Lane 7: starting sample. Lanes 8-11 : peak 2. Figure 20C: Tryptic digest mass spectrometric analysis of the starting material (i.e. Hp rich material) bracketed between 100 - 500 kDa using TFF (n=l). Figure 20D: Tryptic digest mass spectrometric analysis of the purified Hp sample (n=3). Abbreviations for the trypsin digest LC-MS / MS analysis are as follows: HP: Haptoglobin, HPR: Haptoglobin related protein, HBB: Hemoglobin subunit beta, ALB: Albumin, HBA1 : Hemoglobin subunit alpha, HBD: Hemoglobin subunit delta, TF: Transferrin, A2M: alpha-2 macroglobulin, HPX: Hemopexin, SERPIND1 : Heparin cofactor 2, APOA1 : Apolipoprotein Al, IGHG1 : Immunoglobin heavy chain gamma 1 Figure 21. Hb binding capacity of Hp determined via SEC-HPLC. The absorbance was monitored at 413 nm (Soret peak of Hb).

[0061] Figures 22A-22B. Titration of Hp with Hb monitored via SEC-HPLC at an absorbance of 413 nm (Soret peak of Hb). Figure 22A: Titration of 5 pM Hp with increasing concentrations of Hb measured via SEC-HPLC. Figure 22B: Intensity change of the Hp elution peak at 8.1 minutes with increasing Hb concentration

[0062] Figures 23A-23B. MALDI-TOF analysis. Figure 23 A: Representative MALDI-TOF spectra of the starting sample (Hp rich fraction purified via TFF), and the purified Hp sample in the range 3,000 - 20,000 m / z. Figure 23B: Representative MALDI-TOF spectra of the starting sample, and the purified Hp sample in the range 3,000 - 70,000 m / z.

[0063] Figure 24. Far UV CD spectra showing the interaction between Hp and Hb. Hp was added in molar excess (1 :2 Hb:Hp molar ratio) at a 2.5 pM Hb tetrameric molar basis

[0064] Figures 25A-25B. Hp binding kinetics to Hb monitored via UV-visible stoppled flow spectrometry. Figure 25 A: Representative time course for binding of 0.25 pM Hp with increasing concentrations of Hb (5 pM, 10 pM, 15 pM and 20 pM, Hb tetramer basis). Data were fit to a single exponential function to determine the pseudo-first order rate constant (kobs) at each Hb concentration. Figure 25B: kobs values were plotted as function of Hb concentration to determine the second order rate constant for Hp binding to Hb

[0065] Figures 26A-26B. O2 equilibrium curve and offloading kinetics for Hb and the Hb- Hp complex. Figure 26A. Oxygen equilibrium curve of Hb and the Hb-Hp complex. Figure 26B. Deoxygenation kinetics time course for Hb and the Hb-Hp complex monitored at 437.5 nm at room temperature.

[0066] DETAILED DESCRIPTION

[0067] Definitions

[0068] As used herein, the term "tangential-flow filtration" refers to a process in which the fluid mixture containing the components to be separated by filtration is recirculated at high velocities tangential to the plane of the filtration membrane to reduce fouling of the filter. In such filtrations a pressure differential is applied along the length of the filtration membrane to cause the fluid and filterable solutes to flow through the membrane (i.e. filter).

[0069] This filtration is suitably conducted as a batch process as well as a continuous-flow process. For example, the solution may be passed repeatedly over the membrane while that fluid which passes through the filter is continually drawn off into a separate unit or the solution is passed once over the membrane and the fluid passing through the filter is continually processed downstream.

[0070] As used herein, the term "ultrafiltration" is used for processes employing membranes rated for retaining solutes having a molecular weight between about 1 kDa and 1000 kDa.

[0071] As used herein, the term "reverse osmosis" refers to processes employing membranes capable of retaining solutes of a molecular weight less than 1 kDa such as salts and other low molecular weight solutes.

[0072] As used herein, the term "microfiltration" refers to processes employing membranes in the 0.1 to 10 micron pore size range.

[0073] As used herein, the expression "transmembrane pressure" or "TMP" refers to the pressure differential gradient that is applied along the length of a filtration membrane to cause fluid and filterable solutes to flow through the filter.

[0074] The terms “isolating,” "purifying," and "separating," as used interchangeably herein, refer to increasing the degree of purity of a polypeptide or protein of interest or a target protein from a composition or sample comprising the polypeptide and one or more impurities (e.g., additional proteins or polypeptides).

[0075] The term "haptoglobin" as used herein refers to a protein that is synthesized and secreted mainly in the liver. In blood plasma, haptoglobin binds to cell-free hemoglobin released from erythrocytes with high affinity and thereby inhibits hemoglobin oxidative activity. The haptoglobin-hemoglobin complex is then removed by the reticuloendothelial system (mostly in the spleen). Haptoglobin, in its simplest form, consists of two alpha and two beta chains, connected by disulfide bridges. The chains originate from a common precursor protein, which is proteolytically cleaved during protein synthesis. Hp exists in two allelic forms in the human population, so-called Hpl and Hp2, the latter one having arisen due to partial duplication of the Hpl gene. Three genotypes of Hp, therefore, are found in humans: Hpl-1, Hp2-1, and Hp2-2. Hp of different genotypes have been shown to have similar effects in vivo in attenuating Hb-mediated toxicity. Furthermore, a protein with >90% sequence identity to the Hpl gene, called haptoglobin related protein (Hpr) also has high affinity for Hb.The term “haptoglobin” thus encompasses all Hp phenotypes (Hpl-l,Hp2-2 and Hp2-1).

[0076] "Pulmonary administration", as used herein, refers to administration of a pharmaceutical formulation containing an active agent into the lungs by inhalation. As used herein, the term "inhalation" refers to intake of air to the alveoli. The intake of air can occur through the mouth or nose. The intake of air can occur by self-admini strati on of a formulation while inhaling, or by administration via a respirator to a patient on a respirator.

[0077] "Inhalation Device", as used herein, refers to a device which facilitates delivery of an active agent via inhalation. Inhalation devices include, but are not limited to, dry powder inhalers, pressurized metered dose inhalers, breath actuated pressurized metered dose inhalers, nebulizers including vibrating mesh, ultrasonic and jet nebulizers, and soft mist inhalers, atomizer, or vaporizer.

[0078] "Pharmaceutically acceptable", as used herein, refers to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio, in accordance with the guidelines of agencies such as the Food and Drug Administration.

[0079] "Mass Median Aerodynamic Diameter" (MMAD), as used herein, refers to the median aerodynamic size of a plurality of particles. The "aerodynamic diameter" is the diameter of a unit density sphere having the same settling velocity, generally in air, as a powder and is therefore a useful way to characterize an aerosolized powder or other dispersed particle or particle formulation in terms of its settling behavior. The aerodynamic diameter encompasses particle or particle shape, density, and physical size of the particle or particle. MMAD can be experimentally determined by methods known in the art, such as by cascade impaction.

[0080] "Tap Density", as used herein, refers to a measure of the density of a powder. Tap density can be determined using the method of USP Bulk Density and Tapped Density, United States Pharmacopia convention, Rockville, Md., 10th Supplement, 4950-4951, 1999. Features which can contribute to low tap density include irregular surface texture and porous structure.

[0081] "Monodisperse" and "homogeneous size distribution", are used interchangeably herein and describe a plurality of nanoparticles or microparticles where the particles are the same or nearly aerodynamic diameter. As used herein, a monodisperse distribution refers to particle distributions in which 90% of the distribution lies within 5% of the mass median aerodynamic diameter.

[0082] "Co-administration", as used herein, refers to simultaneous and sequential administration of two or more different active agents. The two or more active agents can be included in the same or different pharmaceutical formulation. The two or more active agents can be intended to achieve the same or different clinical benefit. An appropriate time course for sequential administration may be chosen by the physician, according to such factors as the nature of a patient's illness, and the patient's condition.

[0083] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of hemolysis in pulmonary tissue.

[0084] In some embodiments, the treatment or prevention provided by the inventive method can include treatment or prevention of a condition associated with elevated levels of hemoglobin in the pulmonary tissue, elevated levels of free iron in pulmonary tissue, elevated levels of heme in the pulmonary tissue, or a combination thereof. In certain embodiments, the treatment or prevention provided by the inventive method can reduce a level of hemoglobin in the pulmonary tissue of the subject, reduce a level of free iron in the pulmonary tissue, reduce a level of heme in the pulmonary tissue, or a combination thereof.

[0085] In some embodiments, the hemolysis in the pulmonary tissue in the subject can be associated with pulmonary hypertension associated with sickle cell disease, acute respiratory distress syndrome (ARDS), and transfusion-related acute lung injury (TRALI), inhalation of a toxic or irritating agent that induces hemolysis (e.g., ozone, ionizing radiation, smoking, vapes, asbestos, silica, etc.), mechanical ventilation, lung transplantation, lung surgery, or a combination thereof.

[0086] In certain embodiments, the treatment or prevention provided by the inventive method can comprise a method of treating pulmonary hypertension associated with sickle cell disease, a method of treating acute respiratory distress syndrome (ARDS), a method of treating transfusion-related acute lung injury (TRALI), a method of treating inhalation of a toxic or irritating agent that induces hemolysis (e.g., ozone, ionizing radiation, smoking, vapes, asbestos, silica, etc.), a method of mechanical ventilation, a method of lung transplantation, a method of lung surgery, or a combination thereof.

[0087] Compositions

[0088] Provided herein are compositions that comprise a therapeutically effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof to treat hemolysis in pulmonary tissue in a subject. In some embodiments, the composition comprises at least two of apo-transferrin, haptoglobin, and hemopexin. In certain embodiments, the composition comprises apo-transferrin, haptoglobin, and hemopexin.

[0089] In some embodiments, the hemolysis in pulmonary tissue is characterized by elevated levels of hemoglobin in the pulmonary tissue, elevated levels of free iron in pulmonary tissue, elevated levels of heme in the pulmonary tissue, or a combination thereof.

[0090] In some embodiments, the composition comprises an effective amount of apotransferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo- hemoglobin-haptoglobin complex, or a combination thereof to reduce a level of hemoglobin in the pulmonary tissue of the subject, reduce a level of free iron in the pulmonary tissue, reduce a level of heme in the pulmonary tissue, or a combination thereof.

[0091] In some embodiments, the hemolysis in the pulmonary tissue in the subject is associated with pulmonary hypertension associated with sickle cell disease, acute respiratory distress syndrome (ARDS), and transfusion-related acute lung injury (TRALI), inhalation of a toxic or irritating agent that induces hemolysis (e.g., ozone, ionizing radiation, smoking, vapes, asbestos, silica, etc.), mechanical ventilation, lung transplantation, lung surgery, or a combination thereof.

[0092] In some embodiments, the composition is formulated for pulmonary administration to the subject. Aspects of these pulmonary formulations are described in more detail below.

[0093] In some embodiments, the composition comprises an aqueous solution or suspension.

[0094] In some embodiments, the composition comprises a dry powder formulation. In certain embodiments, the dry powder formulation comprises particles having a tap density of less than 0.4 g / cm, a mass median aerodynamic diameter (MMAD) of between 0.5 micron and 5.0 microns, or a combination thereof.

[0095] In some embodiments, the composition comprises an aerosol comprising liquid droplets or solid particles suspended in a gas. In certain embodiments, the aerosol comprises liquid droplets having a droplet diameter of less than 5 microns MMAD suspended in the gas. In certain embodiments, the aerosol comprises solid particles having a tap density of less than 0.4 g / cm, a mass median aerodynamic diameter (MMAD) of between 0.5 micron and 5.0 microns, or a combination thereof suspended in the gas.

[0096] In some embodiments, the composition comprises apo-transferrin. The apotransferrin can comprise human apo-transferrin or recombinant apo-transferrin. In certain embodiments, the apo-transferrin is purified by ultrafiltration, such as tangential flow filtration (TFF). In certain embodiments, the apo-transferrin is purified by a process that comprises obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50-100 kDa; isolating transferrin from the protein cocktail using chromatography; and contacting the transferrin with a chelating agent to remove transferrin-bound iron, thereby isolating the apo-transferrin. In certain embodiments, the chromatography comprises ion exchange chromatography, such as anion exchange chromatography.

[0097] In some embodiments, the composition comprises haptoglobin. The haptoglobin can comprise human haptoglobin or recombinant haptoglobin.

[0098] In certain embodiments, the haptoglobin is purified by ultrafiltration, such as tangential flow filtration (TFF). In certain embodiments, the haptoglobin is purified by a process that comprises obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50 kDa and 0.2 microns (e.g., 50-500 kDa, 50-100 kDa, 100-500 kDa, 200-500 kDa, 300-500 kDa, 100 kDa-0.2 microns); and isolating haptoglobin from the protein cocktail. In certain embodiments, isolating haptoglobin from the protein cocktail comprises chromatography, such as hydrophobic interaction chromatography (e.g., using an alkyl ligand or an aryl ligand such as a phenyl ligand). In certain embodiments, isolating haptoglobin from the protein cocktail comprises ammonium sulfate precipitation, polyethylene glycol (PEG) precipitation, or a combination thereof.

[0099] In some embodiments, the composition comprises hemopexin. The hemopexin can comprise human hemopexin or recombinant hemopexin.

[0100] In certain embodiments, the hemopexin is purified by ultrafiltration, such as tangential flow filtration (TFF). In certain embodiments, the hemopexin is purified by a process that comprises obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50-100 kDa; and isolating hemopexin from the protein cocktail using chromatography. In certain embodiments, the chromatography comprises immobilized metal affinity chromatography, such as nickel (Ni2+) affinity chromatography.

[0101] In some embodiments, the composition comprises apo-hemoglobin and / or PEGylated apo-hemoglobin. Apo-hemoglobins and PEGylated apo-hemoglobins, as well as methods of isolating apo-hemoglobins and PEGylated apo-hemoglobins, are described, for example, in International Publication No. WO 2020 / 160505, which is incorporated herein by reference in its entirety.

[0102] In some embodiments, the composition comprises an apo-hemoglobin-haptoglobin complex. Apo-hemoglobin-haptoglobin complexes (apoHb-Hps) are described, for example, in International Publication No. WO 2020 / 236952, which is incorporated herein by reference in its entirety.

[0103] In some embodiments, the apoHb-Hp complex can comprise apohemoglobin (apoHb) and haptoglobin (Hp) at a weight ratio of at least 1 : 1 (e.g., at least 1 : 1.1, at least 1 : 1.2, at least 1 : 1.3, at least 1 : 1.4, at least 1 : 1.5, at least 1 : 1.6, at least 1 : 1.7, at least 1 : 1.8, at least 1 : 1.9, at least 1 :2, at least 1 :2.1, at least 1 :2.2, at least 1 :2.3, at least 1 :2.4, at least 1 :2.5, at least 1 :2.6, at least 1 :2.7, at least 1 :2.8, at least 1 :2.9 or at least 1 :3). In some embodiments, the apoHb-Hp complex can comprise apoHb and Hp at a weight ratio of 1 :3 or less (e.g., 1 :2.9 or less, 1 :2.8 or less, 1 :2.7 or less, 1 :2.6 or less, 1 :2.5 or less, 1 :2.4 or less, 1 :2.3 or less, 1 :2.2 or less, 1 :2. l or less, 1 :2 or less, 1 : 1.9 or less, 1 : 1.8 or less, 1 : 1.7 or less, 1 : 1.6 or less, 1 : 1.5 or less, 1 : 1.4 or less, 1 : 1.3 or less, 1 : 1.2 or less, or 1 : 1.1 or less).

[0104] The apoHb-Hp complex comprises apoHb and Hp at a weight ratio ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the apoHb-Hp complex can comprise apoHb and Hp at a weight ratio of from 1 : 1 to 1 :3 (e.g., from 1 : 1.5 to 1 :2.5, or from 1 : 1.7 to 1 :2.2, or from 1 :2.5 to 1 :3).

[0105] In some embodiments, the Hp can be prepared using the ultrafiltration methods described below. For example, Hp can be prepared from plasma or fraction thereof (e.g., plasma fraction IV, plasma fraction V, a fraction of precipitated plasma (from salting out, or equivalent) or a combination thereof).

[0106] In certain embodiments, the Hp can have an average molecular weight of at least 70 kDa (e.g., at least 80 kDa, at least 90 kDa, at least 100 kDa, at least 150 kDa, at least 200 kDa, at least 250 kDa, at least 300 kDa, at least 350 kDa, at least 400 kDa, at least 450 kDa, at least 500 kDa, at least 550 kDa, at least 600 kDa, at least 650 kDa, at least 700 kDa, at least 750 kDa, at least 800 kDa, at least 850 kDa, at least 900 kDa, or at least 950 kDa). In certain embodiments, the Hp can have an average molecular weight of 1,000 kDa or less (e.g., 950 kDa or less, 900 kDa or less, 850 kDa or less, 800 kDa or less, 750 kDa or less, 700 kDa or less, 650 kDa or less, 600 kDa or less, 550 kDa or less, 500 kDa or less, 450 kDa or less, 400 kDa or less, 350 kDa or less, 300 kDa or less, 250 kDa or less, 200 kDa or less, 150 kDa or less, or 100 kDa or less).

[0107] The Hp can have an average molecular weight ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the Hp can have an average molecular weight of from 70 kDa to 1,000 kDa (e.g., from 80 kDa to 1,000 kDa, from 90 kDa to 800 kDa, from 80 kDa to 1,000 kDa, or from 80 kDa to 800 kDa).

[0108] In some embodiments, the apoHb can be prepared using the ultrafiltration methods described below. The apoHb prepared by various methods possess the same chemical identity (primary structure) and primarily the same quaternary conformation compared to apoHb prepared by existing precipitation or liquid-liquid extraction methodologies. The apoHb produced by the ultrafiltration methods described herein can exist in aqueous solution primarily as an ab dimer without the use of reducing agents (2-mercaptoethanol, dithiothreitol). In contrast, previous methodologies may produce non-native tetramers (012P2) that require reducing agents to form ab dimers. Furthermore, the apoHb produced in the current methodology is stable for over a week at room temperature and stable at 4 °C, -80 °C and in lyophilized form. Previous methodologies produced apoHb that quickly precipitated (approximately 24 hours) when stored at room temperature. In certain embodiments, the apoHb can be characterized by a residual Soret peak having a maximum absorption ranging from 411-417 nm, such as 412 nm (after renaturation / neutralization, but before complexation with Hp). Previous methodologies produced apoHb which had a residual Soret peak at 402- 407 nm.

[0109] The apoHb-Hp complex can be formed by combining apoHb and Hp at an appropriate weight ratio. By way of example, since apoHb and Hp bind at a 1 : 1 molar ratio (apoHb ab dimer binds to an ab Hp dimer) that equates to 1 : 1 to 1 :3 mass ratio depending on the Hp preparation and / or phenotype. ApoHb-Hp complexes can be formed by mixing apoHb and Hp at a weight ratio of at least 1 : 1 (e.g., at least 1 : 1.5, at least 1 :2, or at least 1 :3). By mixing an excess of apoHb with Hp, saturation of Hp Hb-binding sites can be achieved. Following complexation, the apoHb-Hp complex can be purified using tangential flow filtration (e.g., diafiltration using a 70 kDa TFF module to remove excess apoHb).

[0110] The apoHb and Hp can be wild-type proteins, recombinant proteins, or mutants. In certain embodiments, the apoHb can comprise an apoHb mutant which exhibits enhanced stability. Such mutants are known in the art, and described for example in U.S. Patent No. 7,803,912 to Olson et al. which is incorporated herein by reference. In some examples, the apoHb can include one or more of the following amino acid mutations (the amino acids are specified by their helical location, i.e., A13 represents the thirteenth position along the A helix): a GlyA13 to Ala or Ser; a GlyB3 to Ala, Asp, Glu, or Asn; a CysGl 1 to Ser, Thr, or Vai; b GlyA13 to Ala or Ser; b ProD2 to Ala; b GlyD7 to Lys; b GlyE13 to Ala, Thr, or Asp; b CysG14 to Vai, Thr, Ser, or lie; b ProH3 to Glu, Ala, or Gin; b CysG14 to Thr; b HisG18 to lie, Leu, or Ala; b ProH3 to Glu; b TyrH8 to Trp or Leu; b ValHl 1 to Met, Leu, or Phe; or any combination thereof. Other apohemoglobins include, for example, a(H58L / V62F); b(H63E / U67R); aH87G; bH920; bN108K; aV96W; and combinations thereof. The apoHb- Hp complex can further include one or more active agents coordinated to the apoHb-Hp complex. In some cases, the active agent can be non-covalently associated with the apoHb- Hp complex. For example, in some cases, the active agent can be a hydrophobic active agent that non-covalently associates with the heme-binding region of apoHb. In other cases, the active agent can be covalently attached to the apoHb, covalently attached to the Hp, or a combination thereof.

[0111] Formulations for Pulmonary Administration

[0112] An effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof to treat hemolysis in pulmonary tissue can be formulated for systemic administration (i.e., enteral or parenteral administration). However, in certain embodiments, the apotransferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apohemoglobin-haptoglobin complex, or a combination thereof can be formulated to provide for administration of an effective amount of apo-transferrin, haptoglobin, hemopexin, or a combination thereof to treat hemolysis in pulmonary tissue directly to the lungs of a subject in need thereof.

[0113] Such formulations can contain an effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof in a pharmaceutical carrier appropriate for pulmonary administration. Pharmaceutical formulations and methods for the pulmonary administration of active agents are known in the art.

[0114] The respiratory tract is the structure involved in the exchange of gases between the atmosphere and the blood stream. The respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchi oli. The upper and lower airways are called the conducting airways. The terminal bronchioli then divide into respiratory bronchioli which then lead to the ultimate respiratory zone, the alveoli, or deep lung, where the exchange of gases occurs.

[0115] The alveolar surface area is the largest in the respiratory system and is where drug absorption occurs. The alveoli are covered by a thin epithelium without cilia or a mucus blanket and secrete surfactant phospholipids. Effective delivery of therapeutic agents via pulmonary routes requires that the active agent be formulated so as to reach the alveoli.

[0116] In the case of pulmonary administration, formulations can be divided into dry powder formulations and liquid formulations. Both dry powder and liquid formulations can be used to form aerosol formulations. The term aerosol as used herein refers to any preparation of a fine mist of particles, which can be in solution or a suspension, whether or not it is produced using a propellant.

[0117] Useful formulations, and methods of manufacture, are described by Caryalho, et al., J Aerosol Med Pulm Drug Deliv. 2011 Apr.; 24(2): 61-80. Epub 2011 Mar. 16, for delivery of chemotherapeutic drugs to the lungs.

[0118] Dry Powder Formulations

[0119] Dry powder formulations are finely divided solid formulations containing one or more active agents which are suitable for pulmonary administration. In dry powder formulations, the one or more active agents can be incorporated in crystalline or amorphous form.

[0120] Dry powder formulations include, at a minimum, apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof. Such dry powder formulations can be administered via pulmonary inhalation to a patient without the benefit of any carrier, other than air or a suitable propellant. Preferably, however, the dry powder formulations contain apo-transferrin, haptoglobin, hemopexin, or a combination thereof in combination with a pharmaceutically acceptable carrier.

[0121] The pharmaceutical carrier may include a bulking agent, such as carbohydrates (including monosaccharides, polysaccharides, and cyclodextrins), polypeptides, amino acids, and combinations thereof. Suitable bulking agents include fructose, galactose, glucose, lactitol, lactose, maltitol, maltose, mannitol, melezitose, myoinositol, palatinite, raffinose, stachyose, sucrose, trehalose, xylitol, hydrates thereof, and combinations thereof. The pharmaceutical carrier may include a lipid or surfactant. Natural surfactants such as dipalmitoylphosphatidylcholine (DPPC) are the most preferred. This is commercially available for treatment of respiratory distress syndrome in premature infants. Synthetic and animal derived pulmonary surfactants include:

[0122] Synthetic Pulmonary Surfactants

[0123] • Exosurf — a mixture of DPPC with hexadecanol and tyloxapol added as spreading agents Pumactant (Artificial Lung Expanding Compound or ALEC) — a mixture of DPPC and PG

[0124] • KL-4 — composed of DPPC, palmitoyl-oleoyl phosphatidylglycerol, and palmitic acid, combined with a 21 amino acid synthetic peptide that mimics the structural characteristics of SP-B.

[0125] • Venticute — DPPC, PG, palmitic acid and recombinant SP-C

[0126] Animal Derived Surfactants

[0127] • Alveofact — extracted from cow lung lavage fluid

[0128] • Curosurf — extracted from material derived from minced pig lung

[0129] • Infasurf — extracted from calf lung lavage fluid

[0130] • Survanta — extracted from minced cow lung with additional DPPC, palmitic acid and tripalmitin

[0131] Exosurf, Curosurf, Infasurf, and Survanta are the surfactants currently FDA approved for use in the U.S.

[0132] The pharmaceutical carrier may also include one or more stabilizing agents or dispersing agents. The pharmaceutical carrier may also include one or more pH adjusters or buffers. Suitable buffers include organic salts prepared from organic acids and bases, such as sodium citrate or sodium ascorbate. The pharmaceutical carrier may also include one or more salts, such as sodium chloride or potassium chloride.

[0133] Dry powder formulations are typically prepared by blending apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobinhaptoglobin complex, or a combination thereof with a pharmaceutical carrier. Optionally, additional active agents may be incorporated into the mixture. The mixture is then formed into particles suitable for pulmonary administration using techniques known in the art, such as lyophilization, spray drying, agglomeration, spray coating, extrusion processes, hot melt particle formation, phase separation particle formation (spontaneous emulsion particle formation, solvent evaporation particle formation, and solvent removal particle formation), coacervation, low temperature casting, grinding, milling (e.g., air-attrition milling (jet milling), ball milling), high pressure homogenization, and / or supercritical fluid crystallization.

[0134] An appropriate method of particle formation can be selected based on the desired particle size, particle size distribution, and particle morphology. In some cases, the method of particle formation is selected so as to produce a population of particles with the desired particle size, particle size distribution for pulmonary administration. Alternatively, the method of particle formation can produce a population of particles from which a population of particles with the desired particle size, particle size distribution for pulmonary administration is isolated, for example by sieving.

[0135] It is known in the art that particle morphology affects the depth of penetration of a particle into the lung as well as uptake of the drug particles. As discussed above, drug particles should reach the alveoli to maximize therapeutic efficacy. Accordingly, dry powder formulations is processed into particles having the appropriate mass median aerodynamic diameter (MMAD), tap density, and surface roughness to achieve delivery of the one or more active agents to the deep lung. Preferred particle morphologies for delivery to the deep lung are known in the art, and are described, for example, in U.S. Pat. No. 7,052,678 to Vanbever, et al.

[0136] Particles having a mass median aerodynamic diameter (MMAD) of greater than about 5 microns generally do not reach the lung; instead, they tend to impact the back of the throat and are swallowed. Particles having diameters of about 3 to about 5 microns are small enough to reach the upper- to mid-pulmonary region (conducting airways), but may be too large to reach the alveoli. Smaller particles, (i.e., about 0.5 to about 3 microns), are capable of efficiently reaching the alveolar region. Particles having diameters smaller than about 0.5 microns can also be deposited in the alveolar region by sedimentation, although very small particles may be exhaled.

[0137] The precise particle size range effective to achieve delivery to the alveolar region will depend on several factors, including the tap density of particles being delivered. Generally speaking, as tap density decreases, the MMAD of particles capable of efficiently reaching the alveolar region of the lungs increases. Therefore, in cases of particles with low tap densities, particles having diameters of about 3 to about 5 microns, about 5 to about 7 microns, or about 7 to about 9.5 microns can be efficiently delivered to the lungs. The preferred aerodynamic diameter for maximum deposition within the lungs can be calculated. See, for example, U.S. Pat. No. 7,052,678 to Vanbever, et al.

[0138] In some embodiments, the dry powder formulation is composed of a plurality of particles having a median mass aerodynamic diameter between about 0.5 to about 10 microns, more preferably between about 0.5 microns to about 7 microns, most preferably between about 0.5 to about 5 microns. In some embodiments, the dry powder formulation is composed of a plurality of particles having a median mass aerodynamic diameter between about 0.5 to about 3 microns. In some embodiments, the dry powder formulation is composed of a plurality of particles having a median mass aerodynamic diameter between about 3 to about 5 microns. In some embodiments, the dry powder formulation is composed of a plurality of particles having a median mass aerodynamic diameter between about 5 to about 7 microns. In some embodiments, the dry powder formulation is composed of a plurality of particles having a median mass aerodynamic diameter between about 7 to about 9.5 microns.

[0139] In some cases, there may be an advantage to delivering particles larger than about 3 microns in diameter. Phagocytosis of particles by alveolar macrophages diminishes precipitously as particle diameter increases beyond about 3 microns. Kawaguchi, H., et al., Biomaterials 7: 61-66 (1986); Krenis, L. J. and Strauss, B., Proc. Soc. Exp. Med., 107: 748- 750 (1961); and Rudt, S. and Muller, R. H., J. Contr. Rel, 22: 263-272 (1992). By administering particles with an aerodynamic volume greater than 3 microns, phagocytic engulfment by alveolar macrophages and clearance from the lungs can be minimized.

[0140] In some embodiments, at least about 80%, more preferably at least about 90%, most preferably at least about 95% of the particles in dry powder formulation have aerodynamic diameter of less than about 10 microns, more preferably less than about 7 microns, most preferably about 5 microns. In some embodiments, at least about 80%, more preferably at least about 90%, most preferably at least about 95%, of the particles in dry powder formulation have aerodynamic diameter of greater than about 0.5 microns. In some embodiments, at least about 80%, more preferably at least about 90%, most preferably at least about 95%, of the particles in dry powder formulation have an aerodynamic diameter of greater than about 0.1 microns.

[0141] In some embodiments, at least about 80%, more preferably at least about 90%, most preferably at least about 95%, of the particles in dry powder formulation have aerodynamic diameter of greater than about 0.5 microns and less than about 10 microns, more preferably greater than about 0.5 microns and less than about 7 microns, most preferably greater than about 0.5 microns and less than about 5 microns. In some embodiments, at least about 80%, more preferably at least about 90%, most preferably at least about 95% of the particles in dry powder formulation have aerodynamic diameter of greater than about 0.5 microns and less than about 3 microns. In some embodiments, at least about 80%, more preferably at least about 90%, most preferably at least about 95% of the particles in dry powder formulation have aerodynamic diameter of greater than about 3 microns and less than about 5 microns. In some embodiments, at least about 80%, more preferably at least about 90%, most preferably at least about 95% of the particles in dry powder formulation have aerodynamic diameter of greater than about 5 microns and less than about 7 microns. In some embodiments, at least about 80%, more preferably at least about 90%, most preferably at least about 95% of the particles in dry powder formulation have aerodynamic diameter of greater than about 7 microns and less than about 9.5 microns.

[0142] In some embodiments, the particles have a tap density of less than about 0.4 g / cm3, more preferably less than about 0.25 g / cm3, most preferably less than about 0.1 g / cm3. Features which can contribute to low tap density include irregular surface texture and porous structure.

[0143] In some cases, the particles are spherical or ovoid in shape. The particles can have a smooth or rough surface texture. The particles may also be coated with a polymer or other suitable material to control release of one or more active agents in the lungs.

[0144] Dry powder formulations can be administered as dry powder using suitable methods known in the art. Alternatively, the dry powder formulations can be suspended in the liquid formulations described below, and administered to the lung using methods known in the art for the delivery of liquid formulations.

[0145] Liquid Formulations

[0146] Liquid formulations contain apo-transferrin, haptoglobin, hemopexin, or a combination thereof dissolved or suspended in a liquid pharmaceutical carrier.

[0147] Suitable liquid carriers include, but are not limited to distilled water, de-ionized water, pure or ultrapure water, saline, and other physiologically acceptable aqueous solutions containing salts and / or buffers, such as phosphate buffered saline (PBS), Ringer's solution, and isotonic sodium chloride, or any other aqueous solution acceptable for administration to an animal or human.

[0148] Preferably, liquid formulations are isotonic relative to physiological fluids and of approximately the same pH, ranging e.g., from about pH 4.0 to about pH 7.4, more preferably from about pH 6.0 to pH 7.0. The liquid pharmaceutical carrier can include one or more physiologically compatible buffers, such as a phosphate buffer. One skilled in the art can readily determine a suitable saline content and pH for an aqueous solution for pulmonary administration.

[0149] Liquid formulations may include one or more suspending agents, such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, gum tragacanth, or lecithin. Liquid formulations may also include one or more preservatives, such as ethyl or n-propyl p- hydroxybenzoate.

[0150] In some cases, the liquid formulation may contain one or more solvents that are low toxicity organic (i.e. nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, ethyl ether, and propanol. These solvents can be selected based on their ability to readily aerosolize the formulation. Any such solvent included in the liquid formulation should not detrimentally react with the one or more active agents present in the liquid formulation. The solvent should be sufficiently volatile to enable formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as a freon, alcohol, glycol, polyglycol, or fatty acid, can also be included in the liquid formulation as desired to increase the volatility and / or alter the aerosolizing behavior of the solution or suspension.

[0151] Liquid formulations may also contain minor amounts of polymers, surfactants, or other excipients well known to those of the art. In this context, “minor amounts” means no excipients are present that might adversely affect uptake of the one or more active agents in the lungs.

[0152] Aerosol Formulations

[0153] The dry powder and liquid formulations described above can be used to form aerosol formulations for pulmonary administration. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art. The term aerosol as used herein refers to any preparation of a fine mist of solid or liquid particles suspended in a gas. In some cases, the gas may be a propellant; however, this is not required. Aerosols may be produced using a number of standard techniques, including as ultrasonication or high pressure treatment.

[0154] Preferably, a dry powder or liquid formulation as described above is formulated into aerosol formulations using one or more propellants. Suitable propellants include air, hydrocarbons, such as pentane, isopentane, butane, isobutane, propane and ethane, carbon dioxide, chlorofluorocarbons, fluorocarbons, and combinations thereof. Suitable fluorocarbons include 1-6 hydrogen containing fluorocarbons, such as CHF2CHF2, CF3CH2F, CH2F2CH3, and CF3CHFCF3 as well as fluorinated ethers such as CF3 — O — CF3, CF2H — O — CHF2, and CF3 — CF2 — O — CF2 — CH3. Suitable fluorocarbons also include perfluorocarbons, such as 1-4 carbon perfluorocarbons including CF3CF3, CF3CF2CF3, and CF3CF2CF2CF3.

[0155] Preferably, the propellants include, but not limited to, one or more hydrofluoroalkanes (HF A). Suitable HFA propellants, include but are not limited to, 1,1,1,2,3,3,-heptafluoro-n- propane (HFA 227), 1,1,1,2-tetrafluoroethane (HFA 134) 1,1,1,2,25 3,3,3- heptafluoropropane (Propellant 227), or any mixture of these propellants.

[0156] Preferably, the one or more propellants have sufficient vapor pressure to render them effective as propellants. Preferably, the one or more propellants are selected so that the density of the mixture is matched to the density of the particles in the aerosol formulation in order to minimize settling or creaming of the particles in the aerosol formulation.

[0157] The propellant is preferably present in an amount sufficient to propel a plurality of the selected doses of the aerosol formulation from an aerosol canister.

[0158] Methods of Use

[0159] The compositions and formulations described above can be administered to a subject in need thereof to treat hemolysis in pulmonary tissue in a subject.

[0160] In some embodiments, the hemolysis in the pulmonary tissue can be characterized by elevated levels of hemoglobin in the pulmonary tissue, elevated levels of free iron in pulmonary tissue, elevated levels of heme in the pulmonary tissue, or a combination thereof. In these embodiments, administration of the composition or formulation can reduce a level of hemoglobin in the pulmonary tissue of the subject, reduce a level of free iron in the pulmonary tissue, reduce a level of heme in the pulmonary tissue, or a combination thereof.

[0161] In some embodiments, the hemolysis in the pulmonary tissue in the subject can be associated with pulmonary hypertension associated with sickle cell disease, acute respiratory distress syndrome (ARDS), and transfusion-related acute lung injury (TRALI), inhalation of a toxic or irritating agent that induces hemolysis (e.g., ozone, ionizing radiation, smoking, vapes, asbestos, silica, etc.), mechanical ventilation, lung transplantation, lung surgery, or a combination thereof. In these embodiments, administration of the composition or formulation can comprise a method of treating pulmonary hypertension associated with sickle cell disease, a method of treating acute respiratory distress syndrome (ARDS), a method of treating transfusion-related acute lung injury (TRALI), a method of treating inhalation of a toxic or irritating agent that induces hemolysis (e.g., ozone, ionizing radiation, smoking, vapes, asbestos, silica, etc.), a method of mechanical ventilation, a method of lung transplantation, a method of lung surgery, or a combination thereof.

[0162] In some embodiments of the above methods, the administration comprises pulmonary administration.

[0163] In some embodiments of the above methods, the composition is administered to the subject using a nebulizer, a dry powder inhaler, or a pressurized metered dose inhaler, as described in more detail below.

[0164] In some embodiments of the above methods, the composition comprises a dry powder formulation and the administration comprises inhalation of the dry powder formulation. In some embodiments of the above methods, the dry powder formulation comprises particles having a tap density of less than 0.4 g / cm, a mass median aerodynamic diameter (MMAD) of between 0.5 micron and 5.0 microns, or a combination thereof.

[0165] In some embodiments of the above methods, the composition comprises an aqueous solution or suspension and the administration comprises inhalation of an aerosol formed by nebulization of the composition. In certain embodiments of the above methods, the nebulization of the composition creates: (a) a mass median aerodynamic diameter (MMAD) of droplet size of the composition emitted from a nebulizer of from 0.5 microns to 5 microns; (b) a volumetric mean diameter (VMD) droplet size of the composition emitted from a nebulizer of from 0.5 microns to 5 microns; (c) a Geometric Standard Deviation (GSD) of emitted droplet size distribution of the composition emitted from a nebulizer of from 1.0 micron to 3.4 microns; (d) a fine particle fraction (FPF=% of aerosol particles less than or equal to 5 microns) of droplets emitted from a nebulizer of at least 30%; (e) an output rate of at least 0.1 mL / min from a nebulizer; or a combination thereof.

[0166] In some embodiments of the above methods, the administration comprises pulmonary administration, and wherein pulmonary administration locally administers the apo-transferrin, the haptoglobin, the hemopexin, the apo-hemoglobin, the PEGylated apo-hemoglobin, the apo-hemoglobin-haptoglobin complex, or the combination thereof to the pulmonary tissue of the subject. In certain embodiments of the above methods, the administration comprises pulmonary administration, and wherein pulmonary administration of apo-transferrin, the haptoglobin, the hemopexin, the apo-hemoglobin, the PEGylated apo-hemoglobin, the apohemoglobin-haptoglobin complex, or the combination thereof does not substantially increase levels of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apohemoglobin, apo-hemoglobin-haptoglobin complex, or a combination thereof circulation in a blood stream of the subject.

[0167] Devices for Pulmonary Administration

[0168] To minimize the undesirable side effects associated with the systemic administration of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof is preferably administered locally (i.e., directly to the lungs).

[0169] In some cases, the one or more active agents are delivered into the lungs by inhalation of an aerosolized pharmaceutical formulation. Inhalation can occur through the nose and / or the mouth of the patient. Administration can occur by self-administration of the formulation while inhaling, or by administration of the formulation via a respirator to a patient on a respirator.

[0170] In some cases, a device is used to administer the formulations to the lungs. Suitable devices include, but are not limited to, dry powder inhalers, pressurized metered dose inhalers, nebulizers, and electrohydrodynamic aerosol devices.

[0171] Dry Powder Inhalers

[0172] The dry powder formulations described above can be administered to the lungs of a patient using a dry powder inhaler (DPI). DPI devices typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which can then be inhaled by the patient.

[0173] In a dry powder inhaler, the dose to be administered is stored in the form of a nonpressurized dry powder and, on actuation of the inhaler, the particles of the powder are inhaled by the subject. In some cases, a compressed gas (i.e., propellant) may be used to dispense the powder, similar to pressurized metered dose inhalers (pMDIs). In some cases, the DPI may be breath actuated, meaning that an aerosol is created in precise response to inspiration. Typically, dry powder inhalers administer a dose of less than a few tens of milligrams per inhalation to avoid provocation of cough.

[0174] DPIs function via a variety of mechanical means to administer formulations to the lungs. In some DPIs, a doctor blade or shutter slides across the dry powder formulation contained in a reservoir, culling the formulation into a flowpath whereby the patient can inhale the powder in a single breath. In other DPIs, the dry powder formulation is packaged in a preformed dosage form, such as a blister, tabule, tablet, or gelcap, which is pierced, crushed, or otherwise unsealed to release the dry powder formulation into a flowpath for subsequent inhalation. Still others DPIs release the dry powder formulation into a chamber or capsule and use mechanical or electrical agitators to keep the dry powder formulation suspended in the air until the patient inhales.

[0175] Dry powder formulations may be packaged in various forms, such as a loose powder, cake, or pressed shape for insertion in to the reservoir of a DPI.

[0176] Examples suitable DPIs for the administration of the formulations described above include the Turbohaler® inhaler (Astrazeneca, Wilmington, Del.), the Clickhaler® inhaler (Innovata, Ruddington, Nottingham, UK), the Diskus® inhaler (Glaxo, Greenford,

[0177] Middlesex, UK), the EasyHaler® (Orion, Expoo, FI), the Exubera® inhaler (Pfizer, New York, N.Y.), the Qdose® inhaler (Microdose, Monmouth Junction, N.J.), and the Spiros® inhaler (Dura, San Diego, Calif.).

[0178] Pressurized Metered Dose Inhalers

[0179] The liquid formulations described above can be administered to the lungs of a patient using a pressurized metered dose inhaler (pMDI).

[0180] Pressurized Metered Dose Inhalers (pMDIs) generally include at least two components: a canister in which the liquid formulation is held under pressure in combination with one or more propellants, and a receptacle used to hold and actuate the canister. The canister may contain a single or multiple doses of the formulation. The canister may include a valve, typically a metering valve, from which the contents of the canister may be discharged. Aerosolized drug is dispensed from the pMDI by applying a force on the canister to push it into the receptacle, thereby opening the valve and causing the drug particles to be conveyed from the valve through the receptacle outlet. Upon discharge from the canister, the liquid formulation is atomized, forming an aerosol. pMDIs typically employ one or more propellants to pressurize the contents of the canister and to propel the liquid formulation out of the receptacle outlet, forming an aerosol. Any suitable propellants, including those discussed above, may be utilized. The propellant may take a variety of forms. For example, the propellant may be a compressed gas or a liquefied gas. Chlorofluorocarbons (CFC) were once commonly used as liquid propellants, but have now been banned. They have been replaced by the now widely accepted hydrofluororalkane (HF A) propellants. pMDIs are available from a number of suppliers, incuding 3M Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome, Schering Plough and Vectura. In some cases, the patient administers an aerosolized formulation by manually discharging the aerosolized formulation from the pMDI in coordination with inspiration. In this way, the aerosolized formulation is entrained within the inspiratory air flow and conveyed to the lungs.

[0181] In other cases, a breath- actuated trigger, such as that included in the Tempo® inhaler (MAP Pharmaceuticals, Mountain View, Calif.) may be employed that simultaneously discharges a dose of the formulation upon sensing inhalation. These devices, which discharge the aerosol formulation when the user begins to inhale, are known as breath- actuated pressurized metered dose inhalers (baMDIs).

[0182] Nebulizers

[0183] The liquid formulations described above can also be administered using a nebulizer. Nebulizers are liquid aerosol generators that convert the liquid formulation described able, usually aqueous-based compositions, into mists or clouds of small droplets, preferably having diameters less than 5 microns mass median aerodynamic diameter, which can be inhaled into the lower respiratory tract. This process is called atomization. The droplets carry the one or more active agents into the nose, upper airways or deep lungs when the aerosol cloud is inhaled. Any type of nebulizer may be used to administer the formulation to a patient, including, but not limited to pneumatic (jet) nebulizers and electromechanical nebulizers.

[0184] Pneumatic (jet) nebulizers use a pressurized gas supply as a driving force for atomization of the liquid formulation. Compressed gas is delivered through a nozzle or jet to create a low pressure field which entrains a surrounding liquid formulation and shears it into a thin film or filaments. The film or filaments are unstable and break up into small droplets that are carried by the compressed gas flow into the inspiratory breath. Baffles inserted into the droplet plume screen out the larger droplets and return them to the bulk liquid reservoir. Examples of pneumatic nebulizers include, but are not limited to, PARI LC Plus®, PARI LC Sprint®, Devilbiss PulmoAide®, and Boehringer Ingelheim Respima®.

[0185] Electromechanical nebulizers use electrically generated mechanical force to atomize liquid formulations. The electromechanical driving force can be applied, for example, by vibrating the liquid formulation at ultrasonic frequencies, or by forcing the bulk liquid through small holes in a thin film. The forces generate thin liquid films or filament streams which break up into small droplets to form a slow moving aerosol stream which can be entrained in an inspiratory flow.

[0186] In some cases, the electromechanical nebulizer is an ultrasonic nebulizer, in which the liquid formulation is coupled to a vibrator oscillating at frequencies in the ultrasonic range. The coupling is achieved by placing the liquid in direct contact with the vibrator such as a plate or ring in a holding cup, or by placing large droplets on a solid vibrating projector (a horn). The vibrations generate circular standing films which break up into droplets at their edges to atomize the liquid formulation. Examples of ultrasonic nebulizers include

[0187] DuroMist®, Drive Medical Beetle Neb®, Octive Tech Densylogic®, and John Bunn Nano- Sonic®.

[0188] In some cases, the electromechanical nebulizer is a mesh nebulizer, in which the liquid formulation is driven through a mesh or membrane with small holes ranging from 2 to 8 microns in diameter, to generate thin filaments which break up into small droplets. In certain designs, the liquid formulation is forced through the mesh by applying pressure with a solenoid piston driver (for example, the AERx® nebulizer), or by sandwiching the liquid between a piezoelectrically vibrated plate and the mesh, which results in a oscillatory pumping action (for example EFlow®, AerovectRx®, or TouchSpray® nebulizer). In other cases, the mesh vibrates back and forth through a standing column of the liquid to pump it through the holes. Examples of such nebilzers include the AeroNeb Go®, AeroNeb Pro®. PARI EFlow®, Omron 22UE®; and Aradigm AERx®.

[0189] Electrohydrodynamic Aerosol Devices

[0190] The liquid formulations described above can also be administered using an electrohydrodynamic (EHD) aerosol device. EHD aerosol devices use electrical energy to aerosolize liquid drug solutions or suspensions. Examples of EHD aerosol devices are known in the art. See, for example, U.S. Pat. No. 4,765,539 to Noakes et al. and U.S. Pat. No. 4,962,885 to Coffee, R.A. The electrochemical properties of the formulation may be important parameters to optimize when delivering the liquid formulation to the lung with an EHD aerosol device and such optimization is routinely performed by one of skill in the art.

[0191] Dosages

[0192] As discussed above, formulations containing a therapeutically effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof are administered to a patient in need thereof to treat or prevent hemolysis in pulmonary tissue in a subject.

[0193] A therapeutically effective treatment is one that results in alleviation of one or more symptoms of hemolysis.

[0194] Beneficial or desired clinical results include, but are not limited to, a reduction in a level of hemoglobin in the pulmonary tissue of the subject, a reduction in a level of free iron in the pulmonary tissue, a reduction a level of heme in the pulmonary tissue, or a combination thereof.

[0195] Beneficial or desired clinical results include, but are not limited to, a reduction in symptoms, alleviation of symptoms, and / or decreased progression of pulmonary hypertension associated with sickle cell disease, acute respiratory distress syndrome (ARDS), transfusion- related acute lung injury (TRALI), adverse effects associated with the inhalation of a toxic or irritating agent that induces hemolysis (e.g., ozone, ionizing radiation, smoking, vapes, asbestos, silica, etc.), adverse effects of mechanical ventilation, complications associated with lung transplantation, improved outcome of a lung transplantation, complications associated with lung surgery, improved outcome of a lung surgery, or a combination thereof.

[0196] Preferably, the compositions and formulations are administered non-systemically (i.e., locally within the lung and lung vasculature) using a device as described above. By administering the formulation locally, therapeutic efficacy can be achieved with a lower dosage of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apohemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof than is generally required for systemic administration.

[0197] In one embodiment, the dosage is from about 0.0001 mg / kg to 600 mg / kg, more preferably from 1 mg / kg to 600 mg / kg, more preferably from 10 mg / kg to 600 mg / kg, more preferably from 100 mg / kg to 600 mg / kg, more preferably from 200 mg / kg to 600 mg / kg, more preferably from 300 mg to 600 mg / kg.

[0198] The pharmaceutical compositions and formulations may be administered, for example, in a single dosage, as a continuous dosage, one or more times daily, once daily, or less frequently, such as every two days, every three days, twice per week, every four days, every five days, every six days, once a week, or every two weeks. In any of these administration regimens, the pharmaceutical formulation is preferably for pulmonary administration and may be in an immediate release form, or a sustained release form. In some cases, the pharmaceutical formulation is administered over a prolonged period of time to treat or prevent one or more symptoms of pulmonary arterial hypertension. The pharmaceutical formulations can be administered once a day or more than once a day, such as twice a day, three times a day, four times a day or more. The compositions can be administered for a period of at least one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer. In other embodiments, the pharmaceutical formulation is administered on a one-time basis (i.e., acute administration), for example, to a patient experiencing a surgery or receiving a transplant. The effective dosage can be decreased by using targeted or selective delivery to the pulmonary system, for example, using a targeting peptide as described by Urakami,et al. Am. J. Pathol. 178(6):2489- 2495 (2011).

[0199] In some cases, dosage forms useful for the administration a pharmaceutical formulation containing a therapeutically effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof will be distributed in an administration kit. The kit may include one or a plurality of doses of a pharmaceutical formulation containing a therapeutically effective amount of apo-transferrin, haptoglobin, hemopexin, apohemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof in combination with one or more devices for pulmonary administration of the formulation to a patient. In some cases, the kit can additionally contain a carrier or diluent (which is used, for example, to dissolve or suspend a solid formulation prior to delivery), a case, and instructions for employing the appropriate administration device.

[0200] In some cases, a pharmaceutical formulation containing apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof is administered to a patient in need thereof in a therapeutically effective amount to reduce a level of hemoglobin in the pulmonary tissue of the subject by at least 10%, more preferably at least 15%, most preferably at least 20%. In some embodiments, a pharmaceutical formulation containing apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof is administered to a patient in need thereof in a therapeutically effective amount to reduce a level of hemoglobin in the pulmonary tissue of the subject by greater than 20%, more preferably greater than 25%, most preferably greater than 30%.

[0201] In some cases, a pharmaceutical formulation containing apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof is administered to a patient in need thereof in a therapeutically effective amount to reduce a level of free iron in the pulmonary tissue of the subject by at least 10%, more preferably at least 15%, most preferably at least 20%. In some embodiments, a pharmaceutical formulation containing apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof is administered to a patient in need thereof in a therapeutically effective amount to reduce a level of free iron in the pulmonary tissue of the subject by greater than 20%, more preferably greater than 25%, most preferably greater than 30%.

[0202] In some cases, a pharmaceutical formulation containing apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof is administered to a patient in need thereof in a therapeutically effective amount to reduce a level of heme in the pulmonary tissue of the subject by at least 10%, more preferably at least 15%, most preferably at least 20%. In some embodiments, a pharmaceutical formulation containing apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof is administered to a patient in need thereof in a therapeutically effective amount to reduce a level of heme in the pulmonary tissue of the subject by greater than 20%, more preferably greater than 25%, most preferably greater than 30%.

[0203] Methods of Making

[0204] Also provided herein are methods of purifying apo-transferrin comprising obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50-100 kDa; isolating transferrin from the protein cocktail using chromatography; and contacting the transferrin with a chelating agent to remove transferrin-bound iron, thereby isolating the apo-transferrin. In certain embodiments, the chromatography comprises ion exchange chromatography, such as anion exchange chromatography.

[0205] Also provided herein are methods of purifying haptoglobin comprising obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50 kDa and 0.2 microns (e.g., 50-500 kDa, 50-100 kDa, 100-500 kDa, 200-500 kDa, 300-500 kDa, 100 kDa-0.2 microns); and isolating haptoglobin from the protein cocktail. In certain embodiments, isolating haptoglobin from the protein cocktail comprises chromatography, such as hydrophobic interaction chromatography (e.g., using an alkyl ligand or an aryl ligand such as a phenyl ligand). In certain embodiments, isolating haptoglobin from the protein cocktail comprises ammonium sulfate precipitation, polyethylene glycol (PEG) precipitation, or a combination thereof.

[0206] Also provided herein are methods of purifying hemopexin that comprise obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50-100 kDa; and isolating hemopexin from the protein cocktail using chromatography. In certain embodiments, the chromatography comprises immobilized metal affinity chromatography, such as nickel (Ni2+) affinity chromatography.

[0207] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0208] EXAMPLES

[0209] Example 1. Transferrin Purification, Biophysical Characterization, and Lung Biodistribution in Sickle Cell Disease Mice.

[0210] Summary

[0211] Plasma transferrin (Tf) is the transport protein central to the process of iron recycling and metabolism. Holo-Tf serves as the body’s pool of ferric iron, facilitating transport from tissues such as the intestine, liver, spleen and finally bone marrow, where iron is incorporated into erythropoiesis. In sickle cell disease (SCD), iron overload is primarily caused by chronic blood transfusions in patients at risk of stroke or frequent acute pain crisis. However, we have identified that pulmonary vascular iron accumulation, independent of transfusion, is a driver of pulmonary hypertension in SCD patients and murine models. Therefore, we hypothesize that intra-pulmonary administration of apo-Tf localizes the protein to sites of iron accumulation within the lung, where reactive iron driven pathology develops. This approach to therapeutic development focuses on optimizing administration using aerosol drug delivery, which can increase clinical compliance compared to subcutaneous or intravenous administration. The goal of this study was to purify apo-Tf, perform biochemical characterization on the material and test the proof of concept that apo-Tf protein can be delivered to lung regions where iron accumulation occurs in SCD pulmonary hypertension. We conclude that apo-Tf can be isolated from plasma Cohn fraction IV paste using a simple process and that characterization of the material identified a high purity apo-TF product with functional iron binding properties. Further, this material was administered to SCD mice to target pulmonary anatomical regions where pathology occurs. This data suggests an intriguing approach to iron chelation applicable to a relevant clinical population.

[0212] Introduction

[0213] Human transferrin (Tf) is a glycoprotein (MW = 79.5 kDa) present in human serum at concentrations between 2 and 4 mg / mL. Normal Tf iron saturations are typically 20 - 45% in healthy individuals, which accounts for a small portion of the total body iron pool. Nonetheless, Tf recycles all of the iron required for red blood cell production and does so in more than one hundred cycles of iron binding and delivery. The C- and N-lobe pockets of the Tf protein bind one ferric iron (Fe3+) atom each and Tf circulates in plasma as apo-Tf, mono- ferric-Tf, di-ferric (holo-Tf). Holo-Tf binds to cell surface Tf receptor 1 (TfRl) creating a receptor-ligand complex with TfRl and two holo-Tf proteins that initiates cellular iron utilization as well as recycling of apo-Tf. Further, Tf s function allows for the prevention of iron’s unwanted distribution and excess accumulation in tissues as well iron’s participation in unwanted oxidation reactions that contribute toward tissue damage.

[0214] We have identified a unique iron accumulation in adventitial macrophages surrounding pre- and post-capillary pulmonary vessels of patients and murine models of SCD pulmonary hypertension. Because iron accumulation is specific to the lung vasculature in SCD pulmonary hypertension, targeting the lungs with aerosolized Tf is a potential approach to therapeutic intervention. In this situation, labile or reactive iron in localized tissue parenchymal compartments facilitates protein and lipid peroxidation, senescence, fibrosis and contributes to cardiopulmonary dysfunction. We suggest that macrophage iron loading leads to excess transport of iron through ferroportin and that labile iron contributes toward pulmonary vascular remodeling and pulmonary hypertension in SCD. In support of this pathophysiological concept, Tf receptor 2 gene deficient and homeostatic iron regulator gene deficient mice show pulmonary iron accumulation, airway fibrosis and lung functional decline. In these preclinical models, iron chelators given by the intranasal route attenuate markers of fibrosis. In the distal lung, Tf functions as an antioxidant, but pulmonary Tf concentrations are decreased in the lung lavage fluid from patients with chronic obstructive pulmonary disease. SCD patients with low serum Tf concentrations (< 1.6 mg / mL, normal 2- 3 mg / mL) are reported to have higher tricuspid regurgitant j et velocity (an indicator of pulmonary hypertension) as well as increased endothelial dysfunction and mortality. Therefore, we have strategized therapeutic approaches to target pulmonary vascular sites, such as pre- and post-capillary vessels where iron loaded macrophages accumulate. Several methods to purify Tf have been developed, using plasma, and various Cohn fractions as the starting material. Tf purification techniques include ammonium sulfate precipitation, ethanol precipitation, rivanol precipitation, and ion exchange chromatography (IEX). Additionally, several methods have been developed to purify Tf using Cohn fractions I to IV. Recently, a purification method using a combination of cassette tangential flow filtration (TFF) and anion exchange chromatography to purify apo-Tf directly from Cohn fraction IV was reported. The abundance of Tf in plasma makes it a potential source to produce large quantities of Tf. The Cohn plasma fractionation process primarily purifies albumin, and generates various waste fractions. For example, Cohn fraction IV is one such waste product of the Cohn fractionation process that contains significant amounts of Tf. The preparation of apo-Tf by plasma fractionation may offer a material that can be delivered to vascularized lung regions to facilitate iron binding and enhance localized antioxidant activity.

[0215] The objectives of this study were to optimize a scalable Tf purification process using anion exchange chromatography on a protein cocktail obtained from TFF fractionation of Cohn fraction IV. Previous studies purifying Tf from Cohn fraction IV required multiple steps for purification such as precipitation and several filtration steps. In this study, we propose a simple and scalable process that only requires TFF for protein separation based on size, followed by IEX with a strong anion exchanger for the purification of apo-Tf. The purified Tf was then analyzed for structural and functional integrity by various biophysical techniques. Preliminary studies in Berkely SCD mice were then conducted using the novel Tf preparation to determine lung distribution and systemic absorption following intra-pulmonary delivery using a rodent micro-sprayer.

[0216] Materials and Methods

[0217] Materials. Trizma, trizma HC1, sodium chloride, sodium phosphate dibasic, sodium phosphate monobasic, iron chloride (97%), and nitrilotriacetate (99%) were purchased from Sigma Aldrich (St. Louis, MO), while 0.2 pm polyethersulfone syringe filters were purchased from ThermoFisher (Waltham, MA). Human Cohn fraction IV was purchased from Seraplex (Pasadena, CA). All protein purifications were performed using prepacked columns (Cytiva, Marlborough, MA) on an NGC Quest 100 chromatography system (BioRad, Hercules, CA).

[0218] Tf Purification. Human Cohn fraction IV (FIV) paste was suspended in 3.75 mM phosphate buffered saline (PBS) at 4°C and centrifuged along with fumed silica for lipid removal. The protein solution was then clarified using TFF to yield a protein cocktail bracketed between 50-100 kDa. The protein cocktail was then used as the starting material for Tf purification using a 20 mL HiPrep Q Sepharose FF column (Cytiva, Marlborough, MA). Two buffers, 25 mM Tris, pH 8.00 (buffer A), and 25 mM Tris pH 8.00 containing 0.5 M NaCl (buffer B) were used for binding and eluting protein from the column. The protein cocktail was diluted to a concentration of 25 mg / mL using buffer A and filtered using a 0.2 pm filter before it was loaded onto the column. After completion of protein binding onto the column, the column was then washed with buffer A for 5 column volumes (CVs). Proteins eluted with increasing concentrations of buffer B at 10%, and 20%. A clean in place run was performed between each purification using 2 M NaCl to remove any bound proteins, followed by washing with 5 CVs of buffer A. Pure Tf fractions were pooled and concentrated using a 50 kDa Amicon ultracentrifuge filter (Millipore Sigma, St. Louis, MO). The purified Tf was treated with 50 mM EDTA over night at 4°C to remove Tf bound iron to obtain apo- Tf, followed by buffer exchange into 50 mM phosphate buffer, pH 7.4 using a 50 kDa Amicon ultra centrifuge filter. The protein was concentrated post buffer exchange and stored at -20°C until further use.

[0219] Total Protein Concentration. Total protein concentration was determined using the Bradford assay.

[0220] SDS-PAGE Analysis. Pre-cast 4-20% Novex tris-glycine mini protein gels Invitrogen (Waltham, MA) were used with the Novex mini gel tank (Waltham, MA) for SDS-PAGE analysis. Sodium dodecyl sulfate (SDS) running buffer (10x) was purchased from Invitrogen (Waltham, MA). Samples were diluted to 1 mg / mL in 3.75 mM phosphate buffered saline (PBS) (pH: 7.4) before addition of SDS in a 1 : 1 ratio and heated at 85°C for 10 minutes. Twenty microliters of the sample were then loaded onto the gels and ran at 220 V for 40 minutes. The completed gel was then stained with Coomassie blue (Thermofisher, Waltham, MA) for 40 minutes and destained overnight using a destaining reagent (60% deionized water, 30% methanol, 10% acetic acid). Densitometric analysis was performed with GelQuantNet software provided by biochemlabsolutions.com to determine protein purity.

[0221] MALDI-TOF Mass Spectrometry. Samples were diluted to 1 mg / mL on a protein basis in 50 mM PB (pH 7.4). A saturated solution of a-cyano-4-hydroxy cinnamic acid (CHCA) was prepared for use as the matrix by adding 10 mg of CHCA to 1 mL of 50% v / v acetonitrile with 0.1% tri fluoroacetic acid. The sample was prepared by mixing protein, matrix and 1 M HC1 in a 1 :5: 1 ratio. A 1 pL sample was loaded onto a matrix assisted laser desorption ionization (MALDI) plate. The MALDI plate was then analyzed on a Bruker Microflex MALDI time of flight (MALDI-TOF) mass spectrometry system (Bruker, Billerica, MA). The data was analyzed using the Bruker Flex Analysis software (Bruker, Billerica, MA).

[0222] Trypsin Digest Mass Spectrometry. Samples were resuspended in 50 mM ammonium bicarbonate solution. Five microliters of dithiothreitol (5 pg / pL in 50 mM ammonium bicarbonate) was added and the sample was incubated at 65°C for 15 min. Post incubation, 5 pL of iodoacetamide (15 mg / mL in 50 mM ammonium bicarbonate) was added and the samples were kept in the dark at room temperature for 30 min. Sequencing grade- modified trypsin (Promega, Madison, WI) was prepared in 50 mM ammonium bicarbonate. The trypsin was added to the sample reaction in a 1 :50 (enzyme: sample) ratio at 37°C overnight. The reaction was quenched the following morning by adding formic acid (FA) (50 pL, 0.1%) for acidification. Samples were dried in a SpeedVac (Eppendorf, Enfield, CT) and resuspended in 0.1% FA. The concentration of the resultant peptides was measured by Nanodrop (Fisher, Pittsburgh, PA) before liquid chromatography tandem mass spectrometry (LC / MS-MS) (Fisher, Pittsburgh, PA) analysis. Data analysis was performed using Scaffold 5 (Proteome Software, Portland, OR).

[0223] UV-Visible Spectroscopy. The ferric Fe binding capacity of the purified Tf was determined by monitoring the reaction of Tf with iron-nitrilotriacetic acid (FeNTA). The reaction mixture was kept at room temperature overnight on a shaker plate. The absorbance spectra of Tf with and without FeNTA was measured between 350 - 700 nm at 25°C using a Hewlett-Packard 8452A spectrophotometer (HP, Palo Alto, CA). The holo-Tf extinction coefficient of E 465 nm = 4.86 rnNf'cm'1was used to determine the total amount of active protein (i.e. apo-Tf).

[0224] Circular Dichroism Spectroscopy. The circular dichroism (CD) spectra in the far ultraviolet region between 190-260 nm in a 0.1 cm path length quartz cuvette was measured on purified Tf samples using a JASCO J-815 CD spectrometer (Jasco, Easton, Maryland). Samples were diluted to 2 mg / mL (protein basis) in 50 mM phosphate buffer (pH 7.4). Baseline subtraction was performed using 50 mM phosphate buffer (PB), and the protein CD spectra consisted of an average of three scans. Data analysis was performed using Spectra Manager™ (Jasco, Easton, Maryland).

[0225] Hemoglobin Auto-Oxidation. Auto-oxidation of hemoglobin (Hb) was monitored on a Hewlett-Packard 8452A spectrophotometer (HP, Palo Alto, CA). Hb at a concentration of 100 pM was combined with 50 pM FeNTA and 50 pM apo-Tf. The absorbance spectra between 350-700 nm were measured at 37°C to determine the ability of apo-Tf to reduce auto-oxidation of Hb in the presence of Fe. The data was regressed to a linear fit in R Studio (Posit, Boston, MA) and the slope was taken as the first order rate constant for Hb autooxidation.

[0226] Fe Titration. Tf samples were diluted to ~ 2.9 mg / mL in 50 mM PB (pH 7.4). FeNTA was combined with Tf at concentrations of 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120 pM before incubating at 25°C for 1 hour. Post incubation, 350 pL of each aliquot were then transferred to a 96 well plate and absorbances at 450 nm were measured on a VersaMax tunable microplate reader (Molecular Devices, San Jose, CA). The data was regressed to a linear fit in R Studio (Posit, Boston, MA). The intersection of the two lines is indicative of apo-Tf saturation with iron.

[0227] Fe Binding Kinetics. The binding kinetics between apo-Tf and FeNTA was performed using a SX-20 stopped-flow spectrophotometer (Applied Photophysics, Leatherhead, U.K.). Purified Tf was diluted to 5 pM, with Fe ligand (i.e., FeNTA) concentrations of 10, 20, 40, 80 pM in 2 mM Tris buffer (pH 7.4) and fluorescence quenching was measured at a wavelength of 285 nm at room temperature. All experiments were performed in triplicate. The data was regressed to a double exponential fit in R Studio (Posit, Boston, MA).

[0228] Electron Paramagnetic Resonance Spectroscopy. Tf samples were incubated with FeNTA in excess at room temperature while shaking for 2 hours prior to experiments. Four mm medium wall Suprasil EPR sample tubes 250 mm L (Wilmad Life Sciences, Vineland, NJ) were used for loading the samples. The holo-Tf sample was then buffer exchanged with 50 mM PB (pH 7.4) twice on a 50 kDa Amicon centrifuge filter. EPR measurements were performed at 20 K using an X-Band (9.4 GHz) EMXPlus (Bruker, Billerica, MA). Temperature control was maintained using a Mercury ITC (Oxford Instruments, Oxfordshire, U.K.). The following settings were used for sample analysis: microwave power 2 mW, receiver gain 30 dB, modulation amplitude 10 G, modulation frequency 100 kHz. The sample was measured at center 2600 G with a sweep width of 4000 G. Five measurements were averaged to reduce noise in the spectra. Data analysis was performed using Xenon (Bruker, Billerica, MA) and EasySpin Documentation on the EasySpin.org website.

[0229] Human Tissue Preparation. Human lung tissue, deidentified at autopsy was obtained from deceased SCD patients with identified pulmonary hypertension from the University of Colorado Denver Anschutz Medical Center and Royal London Hospital. Use of deceased patient tissue was considered by the Colorado Multiple Institutional Review Board and deemed to be non-human subject research and given an exempt status.

[0230] Animal Studies. Berkeley SCD (Berk-SS) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed and bred at the University of Maryland Baltimore School of Medicine. Female heterozygous Berk-SS mice were bred with male homozygous Berk-SS mice to generate homozygous offspring. Berk-SS mice with genotype Tg(Hu-miniLCR al Gy Ay 8 Bs) Hba0 / 0 Hbb0 / 0 and the hemizygous with genotype Tg(Hu-miniLCR al Gy Ay 8 Bs) Hba0 / 0 HbbO Hbb+ were littermates. Genotyping of mice used for breeding and experiments was performed by Transnet YX (Cordova, TN, USA). All experimental procedures were conducted under the guidelines recommended by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committees at the University of Maryland Baltimore School of Medicine and the University of Colorado Anschutz Medical Campus.

[0231] Tf was dosed by aerosolized intrapulmonary delivery in N=28 mice (mixed male and female). Mice were lightly anesthetized with isoflurane (3-4%), and then placed in the prone position with the mouth open and the vocal cords visualized. The tip of the Penn-Century micro-sprayer was inserted through the middle of the two vocal cords into the upper trachea. Three hundred mg / kg of the protein was prepared in 0.9% sodium chloride (75 pL) and was aerosolized through the micro-sprayer tip. The rationale for this dose was based on work with human Tf in beta thalassemia Hbbth3 / +mice where 300 mg / kg was given through the intraperitoneal route daily for 30 days. This dose was used to determine Tf absorption from the lung and subcutaneous absorption. Further for this study, intraperitoneal administration was not considered to be a translationally relevant route of administration. The procedure lasted 3-5 seconds and animals were observed until fully awake and alert and moving normally about their cages. Dosing was performed daily for five days and after the final dose, animals were allocated to pre- and post-dosing groups at 1, 2, 4, 8, 12, 24 hours (N=4 / time point) for blood collection and lung tissue harvesting. Under isoflurane (4%) blood was collected from the right ventricle into 1 mL lithium heparin tubes (Greiner Bio-One GmbH, Austria). The aorta was severed, and the lungs were perfused of remaining blood with PBS (5 mL) through the right ventricle. Lung fixation was performed with 10% buffered formalin (3 mL) by airway inflation under constant pressure at 25 cm of H2O pressure, after which the lungs were removed. Blood was processed to plasma for analysis of endogenous murine and dosed human Tf. In a second set of mice, Tf was dosed by subcutaneous injection (N=28 mice, mixed male, and female). Three hundred mg / kg of protein in 0.2 mL of 0.9% sodium chloride was dosed for five days. After the final dose, animals were allocated to pre- and post-dosing groups at 1, 2, 4, 8, 12, 24 hours (N=4 / time point) for blood collection and processing as stated previously. Analysis of lung distribution was performed on lung samples prior to treatment (NT) and at 2, 4, 8, 12 and 24 hours post dosing. Plasma concentrations were analyzed for pre- and post-dosing groups at 1, 2, 4, 8, 12, 24 hours to determine if Tf was absorbed from lung following intrapulmonary dosing. AUCs of plasma concentrations versus time were used to determine bioavailability (%F) using the formula (%F = AUC intrapulmonary / AUC subcutaneous x 100). Here we evaluated Berk-SS mice rather than background wild type mice (C57BL / 6) with specific interest in assessing disease state biodistribution and pharmacokinetics. This is further rationalized by the development path for SCD therapeutics where Phase I trials to assess pharmacokinetics, biodistribution and safety are often on conducted patients rather than healthy volunteers).

[0232] Tissue and Plasma Analysis. Whole lung clearing and imaging: Berk-SS mice were dosed by aerosolized intra-pulmonary administration with 300 mg / kg Alexa Fluor 647 conjugated Tf from human serum (Alexa Fluor™ 647 Conjugate, Cat # T23366) for five days. Ten minutes after the final dose, blood was collected from the heart, and mice were euthanized. The lungs were perfused with 5 mL saline and 1 mL of 4% PFA through the trachea to maintain the lung structure. Lungs were removed and fixed into cold 4% PFA overnight at 4°C. Post-fixed lungs were washed in PBS 3 times and the PBS was changed every 2 hrs. Further, the lunges were transferred into Cubic-L at 37 °C and the solution was replaced with fresh Cubic-L (TCI, T3740) every two days until the lobes were uniformly opaque (total of 3-4 days). After Cubic clearance, the lungs were transferred to a small amount of Cubic-R+(M) (TCI, T3741) and gently shaken to remove most of the Cubic-L and move into fresh Cubic-R+(M). Lungs were incubated in Cubic-R+(M) at room temperature for two days or until the lobes were transparent. Bondic glue was used on the flat surface of the sample holder and lung lobes were fixed on the sample holder and UV light was used to dry the glue on the sample and sample holder. The sample chamber was filled with Cubic- R+(M) and the sample holder with the sample was placed into the chamber for imaging. A Lightsheet 7 microscope and ZEN 3.1 (Black edition) software was used to image and analyze the whole lungs. To identify autofluorescence, the samples were excited with a 488 nm laser, and to detect Alexa fluor 647, samples were exited with a 638 nm laser. Image analysis was performed using Arivis Vision4D 3.5.0 software. Lung Immunofluorescence. Formalin-fixed tissue sections from Berk-SS mice dosed with apo-Tf (300 mg / kg) purified from human Cohn fraction IV paste were processed for immunofluorescence. Briefly, sections were then incubated (overnight, 4°C). Tf immunofluorescence was performed using an anti-Tf primary antibody (Proteintech, # 17435-1-AP) with an anti -Rabbit IgG (H+L) followed by a secondary antibody with Alexa flour TM647 for 1 hr. The sections were then washed three times and mounted with DAPI (VECTASHIELD with DAPI, Vector Laboratories, Newark, California, USA) and stored in the dark at 4°C. Slides were imaged at 20* and 63* using a DM4 B microscopy system (Leica Biosystems, Wetzlar, Germany) with an LED 3 light source and a Cy5 filter set at 4*, 20*, and 63* magnifications. Image immunofluorescence intensity quantitation was determined for each of the four primary mouse lung lobes (N=4 mice / lobe). A minimum of 5 images per lobe per animal were used in the analysis. All tissue images were analyzed using Imaged software.

[0233] Plasma Concentrations. Mouse and Human Tf concentrations were determined in murine plasma after aerosol and subcutaneous dosing at 1, 2, 4, 8, 10 and 24 hours using a commercial ELISA kit (human Tf, ab 108911, abeam).

[0234] Results and Discussion

[0235] Anion Exchange Chromatography Purification and SDS-PAGE Analysis. A protein cocktail bracketed between (50-100 kDa) was purified from Cohn fraction IV via TFF. A single step anion exchange purification process was then used to purify Tf starting from the protein cocktail (Figure 1). Small scale Tf purifications were first performed using 5 mL HiTrap Q FF columns to determine optimal protein binding and elution conditions. After optimization on small scale columns, the purification process was scaled up to a 20 mL HiPrep Q FF column. A wash step of 5 CVs using 0% buffer B at 1 mL / min was applied to remove any unbound protein from the column. At 50 mM NaCl (10% buffer B), a single peak was observed (peak 1, Figure 2A), made up primarily of human serum albumin (HSA), along with other proteins (Figure 2B). The second peak contained primarily Tf (peak 2, Figure 2A, and Figure 2B). The third peak contained primarily HSA and most of the non-Tf proteins (wash peak, Figure 2A, and Figure 2B). The major impurity remaining in the Tf rich fractions appears to be ~ 60 kDa in MW on the SDS-PAGE (Figure 2B) and could either be HSA or hemopexin (Hpx). Post purification, EDTA was used to remove Tf bound iron to obtain apo- Tf. MALDI-TOF Analysis. The protein cocktail (starting material) and purified apo-Tf were subjected to MALDI-TOF mass analysis (Figures 3A-3B). Mass spectral analysis of the protein cocktail shows that it contains a mixture of several expected proteins and consists primarily of human serum albumin (HSA), Tf and hemopexin (Hpx) (Figure 3 A). In the case of mass spectral analysis of the purified Tf, only a single major peak corresponding to the apparent molecular mass of Tf was observed (Figure 3B). A faint band at ~ 60 kDa was observed in the SDS-PAGE gel (Figure 2B), which appears to be hemopexin. However, MALDI mass spectrometry analysis did not detect hemopexin, but this could be attributed to differences in protein ionization. The purified Tf molecular mass was determined to be 78.4 kDa (Figure 3B). The small difference between our measured value and previously reported Tf values can be attributed to variances in protein glycosylation. The final product appeared free of contamination from other proteins when comparing the final Tf product with the initial protein composition of the protein cocktail shown in Figure 3 A. The protein cocktail is made up of HSA, Tf, and other plasma proteins and Figure 3 A also shows the original protein cocktail, which has an intense signal at ~ 65 kDa, indicating the presence of HSA. The Tf peak is present at ~ 79 kDa but occurs at a much lower intensity in the protein cocktail.

[0236] Protein purity was first determined using a gel densitometric technique (Figure 2B) followed by trypsin digestion mass spectrometry for protein composition and MW was confirmed using MALDI mass spectrometry (Figures 4A-4B). Tf purity was determined to be -97% ± 1.5% from gel densitometry. However, trypsin digestion mass spectroscopy indicated that the purity of the final Tf product was closer to 95% ± 0.5% (Figure 4B). Although the Tf purity from densitometry is similar to what has been obtained in the past at 98%, the true purity we report is lower based on the accuracy of trypsin digestion mass spectrometry. Trypsin digestion mass spectroscopy was also performed on the protein cocktail starting material to obtain the initial composition of Tf in the protein cocktail. The total Tf yield was determined by comparing the final mass of purified Tf against the initial mass of Tf in the protein cocktail (Table 1). A significant amount of Tf may have been lost in the fractions that contained significant impurities. Additionally, some Tf was lost in the 10% buffer B wash step or remained bound to the column until the 2M NaCl wash step and this likely decreased the overall Tf yield. Table 1. Estimation of Tf purity and yield.

[0237] UV-Visible Spectroscopy. The activity of the purified Tf was assessed using UV- visible spectroscopy (Figures 5A-5B). Apo- and holo- Tf have an identical absorption band at ~ 280 nm, which is attributed to the aromatic amino acid residues in the protein. However, Fe-bound Tf has a unique characteristic absorption band at ~ 470 nm due to metal-protein interactions, which is attributed to the phenomena of ligand to metal charge transfer. This indicates that the purified protein is properly folded and has intact ligand binding properties. We further used this characteristic Fe specific absorption band of Tf for quantification of protein activity. For this assay, apo-Tf was incubated with FeNTA for 2 hours at room temperature. The difference in absorbance between Tf with bound ferric Fe, Tf by itself, and ferric Fe by itself was calculated to determine the total amount of holo-Tf produced in the reaction (Figure 5 A). This relative increase was used to determine the amount of active apo- Tf in the sample prior to incubation with FeNTA. From this study, we determined the amount of active protein (i.e. apo-Tf) available to bind to ferric Fe was -90.9 ± 14.8%. Titration of the purified Tf at a concentration of 36 pM was performed with increasing concentrations of FeNTA (Figure 5B). The saturation point was determined to be - 75 pM FeNTA, which very closely replicates the expected value for Tf at a protein: ligand binding ratio of 1 :2 (Tf:Fe3+) and provides additional evidence that the purified Tf is primarily in the apo-protein form.

[0238] Auto-oxidation of Hb occurs naturally when the environment surrounding Hb is devoid of antioxidants and generates superoxide, a reactive oxygen species (ROS) that can damage surrounding tissue. The auto-oxidation rate constant of Hb has been reported in the past as 4.5 x 1 O’7s’1, which is close to our experimental kauto-ox of 2 x 10’6s’1under similar experimental conditions. However, Hb exhibits a higher auto-oxidation rate constant when exposed to free ferric Fe (kauto-ox = 1x10’5s’1) but this is attenuated by the addition of Tf kauto-ox = 3 x 10’6s’1) (Figure 6). When only Tf is added to Hb, the auto-oxidation rate constant of Hb decreases to 7 x 10’7s’1. This shows that apo-Tf reduces auto-oxidation of Hb both in the presence and absence of free Fe, which agrees with the literature. Circular Dichroism Spectroscopy. The secondary structure of purified apo-Tf, was analyzed by circular dichroism (CD) spectroscopy (Figure 7). Tf shows a single positive peak at ~ 196 nm, and a major negative peak at ~ 208 nm, followed by a minor negative peak at ~ 218 nm. This shows that the protein primarily consists of antiparallel P sheets with a relatively smaller amount of a helices. CD analysis shows that the protein is properly folded and our results are in agreement with the literature.

[0239] Fe Binding Kinetics. The kinetics of Fe binding to Tf has been reported in the literature as a two-step process, in which ferric Fe bound to a chelator such as NTA binds to Tf, followed by the loss of NTA.

[0240] The binding kinetics of Fe to Tf was monitored by measuring the fluorescence change at 285 nm on a stopped flow spectrophotometer (Figure 8A). The bimolecular rate constant was found by fitting the apparent rate constants as a linear function of FeNTA concentration and found to be kfast= 1.9 x 105M’1, and ksiow= 3.0 x 104M^s'1(Figure 8B). This compares to the rate constant previously determined of k = 8.0 x io4, but differs because our pseudo-first-order data was fit to a biexponential function. We believe the data is better explained by a fast and a slow rate, based on the reaction mechanism of FeNTA binding to Tf. The interaction between FeNTA and Tf is described by the fast rate, where the Fe binds to Tf, while the slow rate captures stabilization of the Fe atom by a carbonate anion and removal of the NTA moiety.

[0241] EPR Spectroscopy. Post incubation with FeNTA, Tf was characterized for its ability to bind to Fe via EPR spectroscopy at 20 K and yielded three distinct peaks (Figure 9). The g- factor was assigned to the inflection point between a peak and a trough for a region with the highest amplitude and was used to characterize the magnetic moment and angular momentum of the Fe atom bound to Tf. The g-factors for Tf coordinated ferric Fe measured in the past are, in order of increasing magnetic induction, g ~ 4.36 - 4.39, g ~ 4.19 - 4.25, g ~ 4.07 - 4.12. In that study, the Fe iron was present in a high spin state of S = 5 / 2 and is bound in a distorted octahedral coordination with four amino acids and two oxygen atoms from the carbonate ion to synergistically bind Fe3+to Tf. Although carbonate ions have been used in the past for EPR studies on Fe bound Tf (i.e. holo-Tf), our present EPR study did not have experimentally added carbonate ions, but still exhibited the same coordination for Fe with g- factors of g = 4.38, g = 4.26, and g = 4.11 with increasing magnetic induction (Figure 9). This suggests ferric Fe is in the correct coordination state, and bicarbonate ions were found in solution under ambient conditions during sample preparation upon exposure to the atmosphere which contains carbon dioxide.

[0242] In summary, the approach to purification and characterization of apo-Tf suggested a highly pure and efficient iron binding protein that could be useful in diseases where reactive iron contributes toward pathophysiology. Targeting lung regions that have iron accumulation, such as that observed in SCD pulmonary hypertension offer a novel use for Tf using intra- pulmonary lung delivery.

[0243] Whole lung distribution of Tf in Berk-SS mice. Hemolytic severity is associated with increased pulmonary systolic pressure and is a risk factor for death in SCD. Data from humans and murine models supports that hemoglobin (Hb) and its degradation product, heme are important toxins that drive vasculopathy in SCD. Macrophages with iron accumulation are present in the lung vasculature of humans and murine models of pulmonary hypertension. The cell type is consistent with macrophages that accumulate in the vascular adventitia and export iron into the localized tissue environment. Based on this finding, we tested if the human apo-Tf preparation could be delivered to the distal lung in Berk-SS mice following aerosolized intra-pulmonary administration. Light sheet microscopy of lungs excised from Berk-SS mice after dosing with Alexa Fluor 647 conjugated to purified human apo-Tf are shown in Figure 10. Auto-fluorescence (AF) is shown in green after laser excitation at 488 nm (Figure 10, Panel A). A section of the right superior lung lobe (box 1 region, Figure 10, Panel A) shows distribution of Alexa fluor 647 conjugated Tf fluorescence after laser excitation at 638 nm (red) merged with auto-fluorescence (Figure 10, Panel B). Images merging AF and Alexa fluor 647 conjugated Tf fluorescence for the right middle lobe (box 2, Figure 10, Panel A) is shown in Figure 10, Panel C. Images merging AF and Alexa fluor 647 conjugated Tf fluorescence for the left lobe (box 3, Figure 10, Panel A) is shown in Figure 10, Panel D. The right superior and middle lobe sections as well as the left lobe section were also imaged. Typically, particles <1 pm distribute to distal airways, while particles > 1 pm deposit in the bronchi and bronchioles (collectively known as conducting airways). The present data shows our delivery method that aerosolizes particles indeed distributes them to the distal lung sites that are downstream of conducting airways.

[0244] Lung Vascular Accumulation of Tf After Intra-Pulmonary Dosing. We next sought to determine if the distribution of intra-pulmonary aerosolized apo-Tf could be localized lung vasculature, which is the specific anatomical region of interest for delivery. In Berk-SS mice, intrapulmonary dosing of Tf shows localization to the lung vasculature (Figure 11). Here the left lobe is shown as a representative for vascular tissue accumulation. Nonetheless, the distribution of Tf throughout the lung shows immunofluorescence of Tf protein in all major lung lobes (Figures 12A-12E). The major lobes of the left lung show Tf protein fluorescence intensity at 2, 4, 8, 12 and 24 hours after intrapulmonary dosing (Figure 12A). The major lobes of the right lung include the superior, middle, and inferior lobes and each show accumulation of protein at 2, 4, 8, 12 and 24 hours after intrapulmonary dosing (Figures 12B-12D). Except for the left lobe at 2 and 4 hours post administration, all lobes demonstrated similar retention of Tf after its intrapulmonary administration. The mean fluorescence intensity for Tf comparing fluorescence intensity between lung lobes over 24 hours post dosing is shown in Figure 12E, indicating the left lobe receives a significantly greater distribution of Tf compared to the right lung lobes.

[0245] It is worth noting that the anti-Tf antibody used in Figure 11 does have cross reactivity with murine and human Tf. Therefore, the absence of a signal from the NT mice reflects the comparatively low concentration of endogenous Tf in the lung vasculature compared with the human Tf dosed mice.

[0246] Tf plasma concentrations were determined to understand if the dosed protein was retained in the lungs or if pulmonary vascular absorption led to accumulation of Tf in the systemic circulation. Plasma concentration versus time data was compared after subcutaneous and intra-pulmonary injection (Figures 13A-13B). Subcutaneous injection (300 mg / kg) of Tf for five days resulted in a steady state plasma concentration of 0.35 ± 0.022 mg / mL and an area under the plasma concentration time curve (AUC) of 8.66 ± 1.10 mg*hour / mL in Berk- SS mice. After intra-pulmonary Tf injection (300 mg / kg) plasma concentrations did not exceed 3 pg / mL (approximately 100 times lower than subcutaneous injection) over a 24-hour period after the last administration. Based on our calculation pulmonary bioavailability (%F) using the formula (%F = AUC intra-puimonaiy / AUC subcutaneous) x 100), less than 1% of Tf administered to lung was detectable in the plasma. These data suggest that intra-pulmonary delivery of Tf would have only localized effects at the lung vasculature, which may be effective for further evaluation in SCD pulmonary hypertension mouse models.

[0247] Conclusion

[0248] Our observations of lung vascular pathology in SCD patients with PH shows a clear iron and oxidative role within the lung vasculature. This observation is unique and not observed in idiopathic PH or in SCD patients without PH. Therefore, we developed the current study to purify apo-Tf from Cohn fraction IV paste at a pilot scale with a priori knowledge of pulmonary vascular iron deposition in SCD PH. Further, this work for the first time demonstrates potential for the use of an aerosolized large protein (80 kDa) that directly reaches the peripheral lung and sites of pathology. We expect that this purification process will allow for apo-Tf production at a scale needed for pre-clinical proof-of-concept effectiveness studies. Indeed, aerosol intra-pulmonary therapeutic delivery may prevent the need for intravenous or subcutaneous administration of novel therapeutic proteins. Translational aspects of this work offer insight into a disease specific application (i.e., SCD PH), which currently has no options for treatment and significantly increases morbidity and mortality.

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[0290] 44. Tang, S., MacColl, R. & Parsons, P. J. Spectroscopic study of the interaction of aluminum ions with human transferrin. J. Inorg. Biochem. 60, 175-185 (1995).

[0291] 45. Bates, G. W. & Wernicke, J. The Kinetics and Mechanism of Iron (III) Exchange between Chelates and Transferrin. J. Biol. Chem. 246, 3679-3685 (1971).

[0292] 46. Pakdaman, R. & El Hage Chahine, J. M. A mechanism for iron uptake by transferrin. Eur. J. Biochem. 236, 922-931 (1996).

[0293] 47. Kubiak, T., Krzyminiewski, R. & Dobosz, B. EPR Study of Paramagnetic Centers in Human Blood. Curr. Top. Biophys. 36, 7-13 (2013).

[0294] 48. Rottman, G. A., Doi, K., Zak, O., Aisen, P. & Aasa, R. Hyperfine Interactions of 57Fe in Human Transferrin: An ENDOR Spectroscopic Study. J. Am. Chem. Soc. Ill, 8613-8618 (1989).

[0295] 49. Adams, T. E. et al. The position of arginine 124 controls the rate of iron release from the N-lobe of human serum transferrin: A structural study. J. Biol. Chem. 278, 6027-6033 (2003).

[0296] 50. Faber, H. R., Baker, C. J., Day, C. L., Tweedie, J. W. & Baker, E. N. Mutation of arginine 121 in lactoferrin destabilizes iron binding by disruption of anion binding: Crystal structures of R121S and R121E mutants. Biochemistry 35, 14473-14479 (1996). 51. Nouraie, M. et al. The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe. Haematologica 98, 464-472 (2013).

[0297] 52. Reiter, C. D. et al. Cell-free hemoglobin limits nitric oxide bioavailability in sicklecell disease. Nat. Med. 8, 1383-1389 (2002).

[0298] 53. Belcher, J. D. et al. Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease. Blood 123, 377-390 (2014).

[0299] 54. Ferguson, S. K. et al. Effects of living at moderate altitude on pulmonary vascular function and exercise capacity in mice with sickle cell anaemia. J. Physiol. 597, 1073-1085 (2019).

[0300] 55. Gordeuk, V. R., Castro, O. L. & Machado, R. F. Pathophysiology and treatment of pulmonary hypertension in sickle cell disease. Blood 127, 820-828 (2016).

[0301] Example 2. Purification, Biophysical Characterization and Response of Hemopexin Derived from Human Cohn Fraction IV.

[0302] Summary

[0303] Hemopexin (Hpx) is an acute phase plasma protein that is responsible for sequestration and removal of cell-free heme with very high affinity (Kd < 1 pM). Hpx expression in liver cells is induced following an inflammatory event such as severe hemolysis in patients with sickle cell disease (SCD). Therefore, plasma Hpx has potential clinical relevance due to its’ ability to bind free heme, thus reducing lipid peroxidation and activation of inflammatory pathways in patients with SCD. This provides the rationale for purifying Hpx at high purity and at large scale. Starting from human Cohn fraction IV, we purified a protein cocktail using tangential flow filtration for use in this study as the starting material for purification of Hpx. Hpx was purified from the protein cocktail using immobilized metal ion affinity chromatography. SDS-PAGE and densitometric analysis showed that Hpx was purified to homogeneity and was approximately 99% pure. To gain further insight into Hpx activity and its’ ligand binding properties, we employed comparative biophysical techniques such as UV-visible, circular dichroism, electron paramagnetic resonance and stopped flow spectroscopy.

[0304] Introduction

[0305] Hemopexin (Hpx) is an acute phase plasma protein (~60 kDa), whose primary function is the binding and removal of cell-free heme in an equimolar ratio (1 : 1 Hpx:heme). Hpx is produced in the liver and consists of a single polypeptide chain with 439 amino acid residues. Hpx binds heme with the highest affinity of any protein (Ka < 1 pM) to form a low spin bis-histidyl heme complex inside the heme pocket surrounded by 8 hydrophobic residues. Once heme is bound to the heme pocket of Hpx, removal of heme from the plasma is mediated by liver macrophages through receptor-mediated endocytosis of the Hpx-heme complex (holo-Hpx) to the LDL receptor-related protein 1 (LRP1), which facilitates recycling of Hpx. Binding and clearance of heme prevents toxicological responses such as lipid peroxidation, and activation of inflammatory pathways. Hpx is present in plasma at a concentration of 0.4 - 1.5 mg / mL. Under clinical conditions characterized by severe hemolysis such as sickle cell disease (SCD), heme is first sequestered by human serum albumin (HSA) in the plasma before binding to Hpx. Consistently high levels of heme leads to a systemic reduction in the plasma levels of Hpx, which directly relates to increased levels of apo-lipoprotein and tissue damage in SCD patients.

[0306] Previously reported Hpx purification methods that start with a basis of plasma or plasma fractions include heme-agarose affinity chromatography, lectin chromatography, anion exchange chromatography and utilizing various forms of chemical treatment such as ammonium sulfate and rivanol precipitation. In addition to these purification techniques, purification based on the strong interactions between Hpx and metal ions is the newest and perhaps the most cost effective and efficient approach for removing contaminants. Metal affinity chromatography together with size exclusion chromatography is often used in tandem to purify Hpx.

[0307] The goal of this study is to develop a single step nickel (Ni2+) affinity chromatography purification process to obtain highly pure Hpx from a protein cocktail obtained from tangential flow filtration (TFF) purification of human Cohn fraction IV. Purified Hpx will be assessed for its’ purity, structural and functional integrity by various analytical techniques.

[0308] Materials and Methods

[0309] Materials. Sodium phosphate monobasic anhydrous (Na^PCU), sodium phosphate dibasic anhydrous (ISfeHPCU), sodium chloride (NaCl), nickel sulfate heptahydrate (NiSO4’7H2O), and imidazole (C3H2N4) were purchased from Sigma Aldrich (St. Louis, MO), and 0.2 pm polyethersulfone syringe filters were purchased from ThermoFisher (Waltham, MA). Human Cohn fraction IV paste was purchased from Seraplex (Pasadena, CA). Protein purifications were performed using prepacked columns purchased from Cytiva (Marlborough, MA) on an NGC Quest 100 chromatography system (BioRad, Hercules, CA). Hpx Purification. Human Cohn fraction IV (FIV) paste was suspended in 3.75 mM phosphate buffered saline (PBS) at 4 °C, and fractionated using tangential flow filtration to yield a protein cocktail with proteins bracketed between 50 - 100 kDa in molecular weight (MW). The protein cocktail was used as the starting material for subsequent purification of Hpx using Ni affinity chromatography on a 20 mL HisPrep FF prepacked column (Cytiva, Marlborough, MA). The binding and elution buffers used were 20 mM phosphate buffer (PB) with 250 mM NaCl pH 7.40 (buffer A), and 20 mM PB with 250 mM NaCl and 100 mM imidazole pH 7.4 (buffer B) respectively. The protein cocktail was first diluted to a concentration of 20 mg / mL using buffer A and filtered through A 0.2 pm dead end filter prior to column loading at 0.5 mL / min. Post column loading of the protein cocktail, the column was washed with 5% buffer B for 5 column volumes (CVs) at 5 mL / min. Protein elution was performed with increasing amounts of buffer B in isocratic steps at 25% for 10 CVs and 50% for 10 CVs at 5 mL / min. Columns were regenerated after each run by first cleaving the Ni2+ions with 5 CVs of 50 mM EDTA and finally loaded with 3 CVs of 100 mM NiSCL heptahydrate. Five CVs of deionized (DI) water was used to wash the column between each regeneration step and post Ni2+loading. Fractions collected with high Hpx purity were pooled and buffer exchanged to 50 mM PB pH 7.4, using 10 kDa Amicon ultracentrifuge filters (Millipore Sigma, St. Louis, MO) and stored at -20 °C until further use.

[0310] SDS-PAGE. Pre-cast 4-20% Novex tris-glycine mini protein gels (Invitrogen, Waltham, MA) were used with the Novex mini gel tank (Waltham, MA) for SDS-PAGE analysis. Sodium dodecyl sulfate (SDS) running buffer (10x) was purchased from Invitrogen (Waltham, MA). Samples were diluted to 1 mg / mL before addition of SDS in a 1 : 1 ratio and heated at 85 °C for 10 minutes. Twenty microliters of the sample were then loaded onto the gels and ran at 220 V for 40 minutes. The gel was stained with Coomassie blue (Thermofisher, Waltham, MA) for 40 minutes and destained overnight using a destaining solution (60% deionized water, 30% methanol, 10% acetic acid). Densitometric analysis was performed with GelQuantNet software provided by biochemlabsolutions.com to determine protein purity.

[0311] Total Protein Concentration. The total protein concentration was determined using the Bradford assay.

[0312] MALDI-TOF Mass Spectrometry. Samples were diluted to 1 mg / mL on a protein basis in 50 mM phosphate buffer (PB) (pH 7.4). A saturated solution of sinapic acid was prepared for use as the matrix by adding 10 mg of sinapic acid to 1 mL of 50% v / v acetonitrile with 0.1% tri fluoroacetic acid. The sample was prepared by mixing protein with the matrix in a 1 : 1 ratio, and 1 pL of sample was loaded onto a matrix assisted laser desorption (MALDI) plate. The MALDI plate was then analyzed on a Bruker Microflex MALDI time of flight (MALDI-TOF) mass spectrometry system (Bruker, Billerica, MA). The data was analyzed using the Bruker Flex Analysis software (Bruker, Billerica, MA).

[0313] Circular Dichroism. The circular dichroism (CD) spectra in the far ultraviolet region between 190-260 nm in a 0.1 cm path length quartz cuvette was measured on purified Hpx samples and Hpx samples treated with heme using a JASCO J-815 CD spectrometer (Jasco, Easton, Maryland). Samples were diluted to 9 pM (protein basis) in phosphate buffered saline (PBS) (pH 7.4). Heme solutions were prepared with 5 mg hemin chloride (Milipore Sigma, Burlington, MA) in 1 mL 100% DMSO. Concentrations were measured spectrophotometrically using an extinction coefficient of 120,000 M'1s'1at 280 nm. Samples treated with heme were diluted to 9 pM (protein basis) along with 9 pM heme. Baseline subtraction was performed using PBS, and the protein CD spectra consisted of an average of three scans. Data analysis was performed using Spectra Manager™ (Jasco, Easton, Maryland).

[0314] Thermal stability was analyzed by monitoring the change in molar ellipticity at 231 nm. Purified apo-Hpx and holo-Hpx samples were subjected to heating at a ramp of 2 °C / min in the temperature range between 20-90 °C in a 0.1 cm path length quartz cuvette. Heme was prepared as described previously in DMSO. Samples were diluted to 9 pM (protein basis), with 9 pM heme added. Data analysis was performed using Spectra Manager™.

[0315] Heme Titration. Titration of Hpx with heme was measured spectrophotometrically using a plate reader at 414 nm. Samples were prepared at a 9 pM protein basis. Heme was prepared in DMSO as described above and concentrations were determined spectroscopically. Ten ligand (L) : protein (P) ratios between 0.1 L / P to 2.0 L / P were measured in triplicate and the data was regressed to a linear fit in R Studio (Posit, Boston, MA) to determine Hpx saturation with heme.

[0316] Stopped Flow Kinetics. The kinetics of apo-Hpx binding to free heme was performed using a SX-20 stopped flow spectrophotometer (Applied Photophysics, Leatherhead, U.K.). Five milligrams of hemin chloride (Sigma- Aldrich, St. Louis, MO) was solubilized in 1 ml 0.1 M NaOH and the concentration was determined using UV-visible spectrometry with an extinction coefficient of 120,000 M^cm’1. Purified apo-Hpx samples were diluted to 5 pM, with heme concentrations of 10, 20, 30, and 40 pM in 2 mM Tris buffer (pH 7.4). Stopped flow readings were measured in absorbance mode at 414 nm at room temperature. All experiments were performed in triplicate and the data was regressed to a double exponential in R Studio (Posit, Boston, MA).

[0317] Hydrogen Peroxide Mediated Oxidation. Hydrogen peroxide (H2O2) mediated oxidation of holo-Hpx was monitored in a photodiode array spectrophotometer following procedures outlined in the literature. Briefly, a sample was prepared with 19 pM heme:holo- Hpx at a L / P molar ratio of 0.8 to yield 8-fold molar excess (H2O2) over the Hpx-heme complex and a reference sample with free heme (19 pM) in PBS with 8-fold molar excess H2O2. The absorbance change at 414 nm versus time was monitored immediately after addition of H2O2 for 10 min with measurements every 30 seconds at room temperature in a 10 mm pathlength quartz cuvette.

[0318] Electron Paramagnetic Resonance Spectroscopy. Hpx samples at a concentration of 200 pM were incubated with heme at a 1 :0.9 molar ratio at room temperature in 50 mM PB while shaking for 1 hr prior to experiments. Four millimeter medium wall Suprasil EPR sample tubes 250 mm L (Wilmad Life Sciences, Vineland, NJ) were used for loading the samples. EPR measurements were performed at 10 K using an X-Band (9.37 GHz) EMXPlus (Bruker, Billerica, MA). Temperature control was maintained using a Mercury ITC (Oxford Instruments, Oxfordshire, U.K.). The following settings were used for sample analysis: microwave power 0.2 mW, power attenuation 30 dB, modulation amplitude 10 G, modulation frequency 100 kHz. The sample was measured at center 3300 G with a sweep width of 2800 G. Thirty measurements were averaged to reduce noise in the spectra. Data analysis was performed using Xenon (Bruker, Billerica, MA) and EasySpin Documentation on the EasySpin.org website.

[0319] Results and Discussion

[0320] Ni-NTA Affinity Chromatography Purification of Hpx and SDS-PAGE Analysis. The protein cocktail containing proteins bracketed between (50 - 100 kDa) that was purified from Cohn Fraction IV via TFF was used as the basis for Hpx purification in this study. Preliminary studies were performed using immobilized metal ion affinity chromatography (IMAC) with 1 mL HiTrap columns, saturated with different metal ions (Ni2+, Zn2+and Cu2+) (data not shown). Purification using immobilized Ni2+showed promising results for separation of Hpx. For initial column screening studies, the column volume was scaled up to 5 mL HiTrap columns before ultimately using 20 mL HisPrep columns. A single step Hpx purification profile using immobilized metal ion (Ni2+) affinity chromatography is shown in Figure 14A. The protein cocktail was diluted 10*, and loaded onto the Ni-NTA column, and unbound proteins, primarily HSA, are observed in the flow through (Figure 14B, lane 3). A column wash with 5% buffer B removed any unbound contaminants (Figure 14B, lane 4). Additional isocratic steps at increasing % B removed various contaminants until only Hpx eluted at 50 % B at peak 3 (Figure 14A), as shown in the SDS-PAGE (Figure 14B, lanes 7-12). Gel densitometry was performed on the purified Hpx fractions, which yielded a purity of - 99%. Based on SDS-PAGE analysis and given that Hpx and HSA have similar apparent MW, the purified Hpx MW was measured by MALDI-TOF spectrometry.

[0321] MALDI-TOF Analysis. Mass spectral analysis was performed to determine the protein species present in the fractions. Both the protein cocktail and the purified Hpx were subject to MALDI-TOF analysis (Figures 15A-15B). It is important to note that the intensity of a certain protein relative to another does not indicate its relative concentration in a sample, as each protein has a unique degree of ionization. MALDI-TOF analysis shows that the protein cocktail is primarily made up of HSA at 66.5 kDa and transferrin (Tf) at - 79 kDa with a negligible reading corresponding to the MW of Hpx (~ 60 kDa) (Figure 15 A). This is in agreement with what we have previously observed from prior analysis of the protein cocktail and is consistent in all of the stock material used for Hpx purification.

[0322] In the purified Hpx samples, a major intensity peak at 59,800 m / z was observed, with a minor intensity peak at -89,000 m / z, which is potentially a low MW or monomeric form of haptoglobin, Hp 1-1 (aiP)2 (Figure 15B). This confirms that peak 3 shown in Figure 15B corresponds to Hpx with a MW of - 60 kDa, and does not have any impurities consisting of HSA at 66.5 kDa or Tf at- 79 kDa. The relatively broad peak of Hpx is due to the fact that Hpx is highly glycosylated, and a range of MW values from 57 kDa to 63 kDa have been reported in the literature. Our values for the mass of Hpx is very similar to previously reported values of Hpx at - 60 kDa.

[0323] Circular Dichroism. To determine if the purification process had negative effects on the secondary structure of the purified Hpx, the far-UV CD spectra of apo-Hpx and holo- Hpx was measured. Our findings match previously reported data for apo-Hpx which exhibited a negative minima at 206 nm and a positive maxima at 231 nm (Figure 16 A) and suggests that the purified Hpx has an a-helical content of -7% and P-sheet content of Holo-Hpx has a similar CD spectra compared to apo-Hpx with a slight change in intensity at 206 nm and 231 nm. This is likely due to conformational changes in the protein upon binding to heme. It has previously been reported in the literature that this conformational change has physiological importance in the binding of holo-Hpx to the membrane receptor protein LRP1. It is difficult to determine if this conformational shift in the secondary structure is reflected in the far UV CD spectra, as even a slight change in the very negative trough at 206 nm would cause the 231 nm peak to shift. However, it is very likely that the change in ellipticity at 231 nm is caused by the tryptophan residues on domain II of Hpx during heme binding.

[0324] Temperature induced unfolding of the purified Hpx was measured at 231 nm from 20 °C to 90 °C with holo-Hpx at a L / P molar ratio of 1.0). For purified Hpx, Tm= 62 ± 0.3, and for holo-Hpx, Tm= 73 ± 0.5 (Figure 16B). The increase in melting temperature is expected, as heme is known to stabilize apo-Hpx, and our values are corroborated by the literature.

[0325] Hpx Activity. The activity of purified Hpx was determined via heme binding to apo- Hpx in increasing L / P molar ratios. Titration of heme to Hpx showed a significant break in the slope at a L / P ~ 1.0, which corresponds to equimolar binding of heme by Hpx, as previously reported in the literature. This suggests that the majority of the protein in the purified sample correspond to apo-Hpx.

[0326] Heme Binding Kinetics. The kinetics of heme (in 0.1 M NaOH) binding to Hpx was measured using the stopped flow spectroscopy. Heme was combined with apo-Hpx in increasing concentrations from 10 - 40 pM and the change in absorbance at 414 nm was measured (Figure 17A). The plots were fit to a double exponential to obtain the kapparent values for both the fast and the slow rates. The heme to Hpx binding rate constant was determined by Pasternack et. al., with heme solubilized in 40% dimethyl sulfoxide (DMSO) in DI water to be a single rate constant of 1.8 x io6M^s'1. From these kinetics studies, we found that a double exponential regression fit the data significant better than a single exponential fit. Therefore, the kapparent values were assigned to the fast step and the slow step in the reaction and were plotted against the concentration in Figure 17B to obtain kFast = 4.4 x 105M^s'1and ksiow= 5.6 x 104M’1. Our rate constants are slightly lower than what Pasternack et. al., obtained, and this can be attributed to the difference in solutions used to solubilize the heme. DMSO may be a better alternative to NaOH to keep heme in a monomeric form, whereas NaOH may have more dimer heme molecules, thus slowing heme binding to Hpx. Hydrogen Peroxide Mediated Oxidation. The effect of H2O2 mediated oxidation on holo-Hpx and free heme shown is shown in Figure 17D. In the case of holo-Hpx, the change in the absorbance of the Soret peak at 414 nm was significantly reduced compared to free heme in PBS, and is similar to previous observations in the literature. This indicates that free heme is easily oxidized, while the heme in the heme-Hpx complex remains stable and is not as readily oxidized by excess chemical oxidant (H2O2). Additionally, this observation is corroborated with heme-Hpx coordination chemistry, where heme binds to the Hpx heme binding pocket in a bis-histidyl coordination, where such hexa coordinated species with low spin chemistry is inert and not accessible for H2O2 binding.

[0327] Electron Paramagnetic Resonance Spectroscopy. The EPR spectra of Hpx with heme at a ~ L / P = 0.9 molar ratio was measured to determine the coordination of heme within the Hpx heme binding pocket (Figure 18). Hpx was combined in excess with heme to prevent any unintended binding of heme to the protein. The observed signal was significantly lower compared to the adventitious or otherwise “junk” iron observed at ~ 1100 G (g = 5.87).35This is expected since high spin iron at S = 3 / 2 is known to have a more intense signal. The 10 K measurement temperature along with an attenuation of 30 dB was chosen to maximize the signal to noise ratio of the protein coordination to the low spin iron of heme as well as to obtain readings below the saturation point.

[0328] The g tensor is used to determine the coordination of Hpx to heme. The following g values were observed, g = 2.89, g = 2.27, and g = 1.55, and is characteristic of low spin iron of S = / i in heme bound to Hpx. Additionally, these g values suggest that the low spin iron in heme is coordinated by 2 histidine residues in the heme pocket of Hpx.

[0329] Conclusion

[0330] In this study, we developed a single step process for purifying Hpx to high purity from a protein cocktail solution using Ni-NTA immobilized metal affinity chromatography. The purified protein was characterized to have a MW of 60 kDa and a purity of - 99%. Proper protein folding was determined by CD and matches previously reported values. Protein activity was assessed using titration with heme, which was observed to saturate at a L / P - 1.0 and with proper heme coordination.

[0331] References

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[0368] Example 3. Purification, Biophysical Characterization, and Response of Haptoglobin

[0369] Derived from Human Cohn Fraction IV. Summary

[0370] Haptoglobin (Hp) is a polymorphic acute phase a-2 glycoprotein found in plasma that has the critical role of binding, neutralizing, and removing cell-free hemoglobin (Hb) from the circulation. Under clinical conditions characterized by high levels of hemolysis, such as patients with sickle cell disease (SCD), large quantities of cell-free Hb are released from lysed red blood cells (RBCs) into the extracellular space and bind to Hp, which decreases the plasma concentration of Hp below basal levels and lowers its’ Hb binding capacity. Therefore, plasma-derived Hp has the potential to be used as a therapeutic to scavenge, neutralize, and remove excess cell-free Hb from the blood, thus preventing Hb-mediated toxicity. Hence, this provides the motivation for purifying Hp at high purity and at large scale from sustainable sources such as waste plasma fractions from the Cohn plasma fractionation process which is used to produce human serum albumin from pooled human plasma. Starting from human Cohn fraction IV, we first purified a Hp rich fraction using tangential flow filtration (TFF) that was then used as the starting material for Hp purification in this study. Hp was then purified from this Hp rich fraction using hydrophobic interaction chromatography to homogeneity, and the final Hp purity was determined to be 98% via SDS- PAGE gel densitometry and 87% via trypsin digest LC-MS / MS analysis. The purified Hp was further characterized to determine its’ molecular weight, secondary structure, Hb binding capacity, and its’ Hb binding kinetics by applying various biophysical techniques such as MALDI-TOF, circular dichroism, size exclusion HPLC, and UV-visible stopped flow spectroscopy.

[0371] Introduction

[0372] Haptoglobin (Hp) is a highly polymorphic acute phase a-2 glycoprotein found in plasma with a wide molecular weight (MW) range (90 kDa - 900 kDa). Hp is a polymeric protein made up of a and P chains connected by intermolecular disulfide bonds, the extent of which depends on the Hp phenotype. The molecular polymorphism of Hp occurs because of genetic differences in the a- chains, which are coded in humans by a pair of co-dominant alleles Hp 1 and Hp 2 for the a-1- subunit (9 kDa) and the a-2 subunit (18 kDa) , each sharing the same P chain (36 kDa). The difference in the structures of Hp phenotypes are regulated by the different a chains, where Hp 1-1 is formed by two aiP dimers linked through a disulfide bond. Higher degree Hp polymers arise from the Hp 2 allele, which codes for the a?P dimer, which has an additional free thiol group (Cys-15) on the a-chain which can interact with an additional aP dimer. This leads to either homozygous Hp 1, Hp 2, or a heterozygous polymer of Hp 1 with Hp 2, leading to the three major phenotypes: Hp 1-1 (89 kDa), Hp 2-1 (200 - 500 kDa), and Hp 2-2 (200 - 900 kDa). Hp is primarily synthesized in the liver, and is responsible for binding, neutralization, and removal of cell-free Hb, with concentrations ranging from 1.0 to 1.5 mg / mL in human plasma. The Hp P- subunit binds to the Hb aP dimer stoichiometrically with very high affinity in plasma to form the Hp-Hb complex that is subsequently recognized by the surface receptor CD 163 on macrophages and removed through endocytosis, thus preventing heme release from Hb which reduces oxidative damage as well as inflammation to surrounding tissues.

[0373] Hp is the primary scavenger protein for the removal of cell-free Hb. Therefore, Hp has the potential to serve as a therapeutic treatment for diseases or clinical conditions characterized by acute and systemic hemolysis. Hemolysis is characterized by the release of Hb from damaged RBCs into the plasma. Individuals with sickle cell disease (SCD) suffer from systemic hemolysis, which results in low plasma levels of Hp, which can lead to systemic tissue and organ damage due to the excess cell-free Hb in circulation. In the long term, tissue accumulation of cell-free Hb and its’ resultant oxidative damage is a contributing factor to vaso-occlusive crisis and acute chest syndrome.

[0374] There are many options for the purification of Hp from either human plasma, human serum, or Cohn fraction IV (FIV). These methods range from Hb affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), size exclusion chromatography, ammonium sulfate precipitation, polyethylene glycol (PEG) precipitation, and a combination of the techniques listed above. Therefore, in this study, we propose to purify Hp using a single chromatographic purification step on a pooled Hp rich protein bracket (100 - 500 kDa) obtained from FIV using tangential flow filtration (TFF).

[0375] Materials and Methods

[0376] Materials. Sodium phosphate monobasic anhydrous (Na^POQ, sodium phosphate dibasic anhydrous (ISfeHPOQ, sodium chloride (NaCl), and ammonium sulfate ((NH^SCU, [AS]) were purchased from Sigma Aldrich (St. Louis, MO), 0.2 pm polyethersulfone syringe filters were purchased from ThermoFisher (Waltham, MA), and TFF filters were purchased from Repligen (Boston, MA). Human Cohn fraction IV paste was purchased from Seraplex (Pasadena, CA). Protein purifications utilized prepacked columns purchased from Cytiva (Marlborough, MA) on an NGC Quest 100 chromatography system (BioRad, Hercules, CA).

[0377] Hp Purification. Human Cohn fraction IV (FIV) paste was suspended in 3.75 mM phosphate buffered saline (PBS) at 4 °C, and fractionated using TFF to yield a Hp rich fraction with proteins bracketed between 100 - 500 kDa in MW. The Hp rich fraction will be used as the starting material for the purification of Hp using HIC with a prepacked 1 mL PhenylHP column (Cytiva, Marlborough, MA). The binding and elution buffers used in this study consisted of 20 mM PBS with 1.5 M AS pH 7.00 (buffer A), and 20 mM PBS pH 7.00 (buffer B) respectively. The Hp rich fraction (bracketed between 100 - 500 kDa using TFF) was first diluted to a concentration of 1 mg / mL using buffer A and filtered through a 0.2 pm dead end polyethylenesulfone (PES) filter prior to column loading at a flow rate 0.5 mL / min. Post column loading of the Hp rich fraction (bracketed between 100 - 500 kDa using TFF), the column was washed with 100% buffer A for 5 column volumes (CVs) at 1 mL / min. Protein elution was performed with increasing amounts of buffer B in isocratic steps at 20% for 10 CVs and 50% for 10 CVs and finally 100% for 5 CVs at 1 mL / min. Columns were cleaned after each run by flushing 20 CVs of deionized water (DI) through the column at 1 mL / min. High purity Hp fractions were collected, pooled, and buffer exchanged into 50 mM PB pH 7.4, using 50 kDa Amicon ultracentrifuge filters (Millipore Sigma, St. Louis, MO) and stored at -20 °C until further use.

[0378] SDS-PAGE. Pre-cast 4-20% Novex tris-glycine mini protein gels (Invitrogen, Waltham, MA) were used with the Novex mini gel tank (Waltham, MA) for SDS-PAGE analysis. Sodium dodecyl sulfate (SDS) running buffer (10x) was purchased from Invitrogen (Waltham, MA). Samples were diluted to 1 mg / mL before addition of SDS sample loading buffer (Invitrogen, Waltham, MA) in a 1 : 1 ratio and heated at 85 °C for 10 minutes. For samples under reducing conditions, 4 pL of 1 M dithiothreitol (DTT) was added before heating the samples. 20 pL of the sample were then loaded onto the gel and run at 220 V for 40 minutes. The gel was stained with Coomassie blue (Thermofisher, Waltham, MA) for 40 minutes and destained overnight using a destaining solution (60% deionized water, 30% methanol, 10% acetic acid). Densitometric analysis was performed with GelQuantNet software provided by biochemlabsolutions.com to determine protein purity.

[0379] Total Protein Concentration. The total protein concentration was determined using the Bradford assay.

[0380] MALDI-TOF Mass Spectrometry. Samples were diluted to 1 mg / mL on a protein basis in PBS (pH 7.4). A saturated solution of sinapic acid was prepared for use as the matrix by adding 10 mg of sinapic acid to 1 mL of 70% v / v acetonitrile with 0.1% trifluoroacetic acid. The sample was prepared by mixing protein with the matrix in a 1 : 1 ratio, and 1 pL of sample was loaded onto a matrix assisted laser desorption (MALDI) plate. The MALDI plate was then analyzed on a Bruker Microflex MALDI time of flight (TOF) mass spectrometry system (Bruker, Billerica, MA). The data was analyzed using the Bruker Flex Analysis software (Bruker, Billerica, MA).

[0381] Hp and Hb Binding Capacity. The Hb binding capacity (HbBC) of Hp was determined using size exclusion high performance liquid chromatography (SEC-HPLC) (ThermoFisher, Waltham, MA). Hb was first diluted to 1 mg / mL in PBS and used as the stock solution for dilution of Hp samples. Hp samples were diluted 20 x to approximately 0.5 mg / mL. Elution of the Hp-Hb complex was monitored at 413 nm (Soret peak of Hb), and the difference in area of the Hb elution peak was used to determine the HbBC of Hp.

[0382] Titration of Hp with Hb was performed to determine the Hb saturation point of the purified Hp using SEC-HPLC monitored at 413 nm (Soret peak of Hb). The Hp protein concentration was determined by using the HbBC of Hp determined via SEC-HPLC as determined above. A stock solution of 5 pM Hp in PBS (pH 7.4) was diluted for titration against an increasing concentration of Hb from 0.25 pM to 20 pM Hb on a Hb tetramer basis.

[0383] Circular Dichroism Spectroscopy. The circular dichroism (CD) spectra in the far ultraviolet region between 190 - 260 nm was measured on purified Hp and Hp-Hb samples in a 0.1 cm path length quartz cuvette on a JASCO J-815 CD spectrometer (Jasco, Easton, Maryland) at 25 °C. Hb samples were prepared to 2.5 pM on a Hb tetramer basis and bound in a 1 :2 Hb:Hp molar ratio in PBS (pH: 7.4). Baseline subtraction was performed using PBS (pH: 7.4), and each protein CD spectra was recorded as an average of three scans. Molar ellipticity was recorded on a protein basis. Data analysis was performed using Spectra Manager™ (Jasco, Easton, Maryland).

[0384] Trypsin Digest LC / MS-MS analysis. Samples were resuspended in 50 mM ammonium bicarbonate solution. Five microliters of dithiothreitol (5 pg / pL in 50 mM ammonium bicarbonate) was added, and the sample was incubated at 65 °C for 15 min. Post incubation, 5 pL of iodoacetamide (15 mg / mL in 50 mM ammonium bicarbonate) was added and the samples were kept in the dark at room temperature for 30 min. Sequencing grade- modified trypsin (Promega, Madison, WI) was prepared in 50 mM ammonium bicarbonate. The trypsin was added to the sample reaction in a 1 :50 (enzyme: sample) ratio at 37 °C overnight. The reaction was quenched the following morning by adding formic acid (FA) (50 pL, 0.1%) for acidification. Samples were dried in a SpeedVac (Eppendorf, Enfield, CT) and resuspended in 0.1% FA. The concentration of the resultant peptides was measured by Nanodrop (Fisher, Pittsburgh, PA) before liquid chromatography tandem mass spectrometry (LC / MS-MS) (Fisher, Pittsburgh, PA) analysis. Data analysis was performed using Scaffold 5 (Proteome Software, Portland, OR).

[0385] Stopped Flow Kinetics. The kinetics of Hp binding to Hb was performed using a SX- 20 stopped flow spectrophotometer (Applied Photophysics, Leatherhead, U.K.). Purified Hp samples were diluted to 0.25 pM in 3.75 mM PBS (pH 7.4) based on the HbBC in mg / mL. Hb samples were prepared in increasing concentrations from 5 pM, 10 pM, 15 pM and 20 pM on a Hb tetramer basis. Stopped flow kinetics were measured in fluorescence mode using an excitation at 285 nm and monitoring the emission at 285 nm at room temperature. All experiments were performed in triplicate, and the data was fit to a single exponential function in R Studio (Posit, Boston, MA).

[0386] Oxygen Equilibrium and Offloading kinetics. The oxygen (O2) equilibrium curve of Hb and the Hb-Hp complex was measured using a Hemox Analyzer (TCS Scientific, New Hope, PA). Samples were prepared in 3 mL Hemox buffer (pH 7.4) at a concentration of 30 pM Hb (tetramer basis) and 60 pM Hp along with 20 pL bovine serum albumin and 20 pL anti-foaming agent. The oxygen affinity (P50, partial pressure of O2 [PO2], where half of the Hb O2 binding sites are saturated with O2) and cooperativity coefficient were obtained from regressing the data to the Hill equation in Igor (Wavemetrics, Portland, OR).

[0387] The O2 offloading kinetics of Hb and the Hp-Hb complex was monitored using a SX- 20 stopped flow spectrophotometer. The purified Hp samples were reacted with Hb in molar excess of 2: 1 with 25 pM Hp and 12.5 pM Hb (Hb tetramer basis). A separate Hb sample was prepared at a concentration of 12.5 pM Hb (Hb tetramer basis). Both samples were oxygenated and combined with 1 M dithionite in the stopped flow spectrophotometer, and the change in absorbance at 437.5 nm was monitored. Data was collected, normalized in R Studio, and fit to a single exponential function.

[0388] Results and Discussion

[0389] HIC Purification of Hp and SDS-PAGE and trypsin digest LC-MS / MS Analysis. A Hp rich solution with MW bracketed between 100 - 500 kDa was purified from FIV paste via TFF as described in the literature, and used as the starting material for the purification of Hp in the MW range 100 - 500 kDa, which primarily consists of Hp 2-1. Because the starting material used in this study already contains a high concentration of Hp, we chose to use hydrophobicity to further purify the Hp from the contaminants in order to increase the purity above 90%. A 500 pL sample was diluted by 10x in 20 mM PBS + 1.5 M AS and loaded onto a 1 mL PhenylHP column, with no protein flow through observed in the loading step (Figure 19). However, unbound proteins were removed in the wash step with 0% B. Three isocratic steps were employed to separate contaminants from Hp in the protein sample, but these stages also removed some of the bound Hp as well as shown in Figure 20A, where Hp dimers were observed in both the 20% and 100% B fractions (Figure 20B). The second peak at 50% B elution had the highest concentration of Hp with the lowest number of contaminants determined from SDS-PAGE. Gel densitometry indicated the overall Hp purity to be ~ 98% ± 0.6%. The Hp purity as determined by trypsin digest mass spectrometry was slightly lower, at around 87.4 % ± 4.6 when including both the Hp and Hp related protein (Hpr). The most likely reason for the discrepancy between densitometry and trypsin digest is due to the higher sensitivity of the trypsin digest method. Additionally, the yield was determined to be 40%, but the purity increased from 67% to 87% on total Hp + Hpr in the product (Table 2).

[0390] Table 2. Hp purification summary. The Hp purity was assessed using tryptic digest mass spectrometry.

[0391] To determine the activity and available sites on Hp capable of binding to Hb (i.e.

[0392] HbBC), Hp was mixed with Hb on a mass basis of 0.5 mg / mL Hp to 1 mg / mL Hb. The change in the area under the curve for the Hb absorbance peak at 413 nm was then used to calculate the HbBC. A representative plot of this characterization is shown in Figure 21, where a decrease in the peak area at for Hb at an elution time of 9.2 min directly correlates to an increase in the peak area for the Hp-Hb complex at an elution time of 8.2 min. From this experiment, we were able to determine the HbBC of the purified Hp samples (Table 2), which has half of the HbBC of the starting material, but this makes sense due to the decrease in concentration of protein. A titration experiment was then performed to further confirm the activity of the purified Hp and determine the Hb saturation point for the polymeric Hp. This was accomplished by increasing the Hb concentration from 0.25 pM to 20 pM (Hb tetramer basis) in the presence of 5 pM Hp and measuring the Hp / Hb mixture via SEC-HPLC (Figures 22A-22B). Interestingly, we can see in Figure 22A that the purified Hp has virtually no 413 nm absorbance at an elution time of 8.2 min, where the Hp-Hb complex appears, suggesting that all the purified Hp is in the apo protein form with no bound Hb. The Hp-Hb complex peak at 8.2 min increases with increasing concentration of Hb starting at 0.25 pM and shortly reaches the maxima at approximately 3 pM Hb (Figure 22B), with the remaining unbound Hb shown as an increase in the 413 nm absorbance at an elution time of 9.2 min as the Hp is saturated with Hb. The approximately 1 :2 Hb:Hp molar binding ratio occurs because Hb at low concentrations is present as aP dimers, which results in an underestimation of the tetrameric HbBC. Additionally, because the Hp concentration is based off the HbBC, the actual molar concentration of Hp may be much lower. If we take these into consideration, the actual HbBC of the purified Hp is slightly above a 1 : 1 Hb:Hp molar ratio, which is comparable to previously reported values in the literature. This can also be explained because the Hp we purified is primarily Hp 2-1 with small amounts of Hp 2-2, which has more than 2 sites for binding Hb aP dimers.

[0393] MALDI-TOF Mass Spectrometry. Mass spectral analysis was performed on reduced protein samples to determine the presence of Hp a- and P-chains as well as potential protein contaminants. Both the initial protein sample (Hp rich fraction purified via TFF) and the purified Hp were subjected to MALDI-TOF and monitored between 3,000 - 70,000 m / z, with an emphasis on the 3,000 - 20,000 m / z region to detect the a-chains (Figures 23A-23B). It is important to note that the mass spectra is influenced significantly by the ability of the target protein to ionize, which affects the total intensity of the different species observed in the spectrum, as different proteins have varying degrees of ionization. MALDI-TOF is however, an excellent tool to determine the exact MW of protein species.

[0394] The MALDI-TOF spectra shows that the starting material (Hp rich fraction purified via TFF) contains a peak corresponding to the Hp 012 subunit at 15,949 m / z and the P- subunit at 32,007 m / z, with a noticeable peak at 66,455 m / z corresponding to human serum albumin (HSA) (Figures 23 A-23B). There is no noticeable ai peak for the starting material, but one is clearly present for the purified Hp sample, which was observed at 9,000 m / z. This may be due to the much higher relative intensity of the a.2 subunit and HSA as compared to the ai subunit, which would render it too low to detect via MALDI-TOF. Compared to the initial sample, the purified Hp sample has clear ai, 012, and P peaks along with no peak at 66,500 m / z which corresponds to HSA. The observed peaks at 9,000 m / z, 18,000 m / z, and 34,000 m / z match closely with values previously reported in the literature for Hp, and we can confirm that the purified sample is indeed Hp. Circular Dichroism Spectroscopy. The secondary structure of Hp and the Hp-Hb complex was measured via CD in the far UV region 190 - 260 nm (Figure 24). Data was presented in units of molar ellipticity on the basis of the total protein in solution. The Hb sample displayed two troughs at 209 nm and 222 nm, which indicates that Hb mainly consists of a-helices. In comparison, the purified Hp sample showed a significant negative molar ellipticity, in the range of 203 - 205 nm and a small trough at ~ 219 nm which indicates that Hp mainly consists of P- sheets. When Hp is combined with Hb in an excess molar ratio, the positive peak due Hb at 198 nm disappears and the overall spectra is shifted such that there’s only a continuous minima ranging from 210 - 222 nm. This is very close to previously reported CD spectra of the Hp-Hb complex, and suggests that the proteins are bound properly, with the correct secondary structure consisting of a mixture of a- helices and P- sheets.

[0395] Hp Binding Kinetics. The Hb binding kinetics of the purified Hp was determined via UV-visible stopped flow spectrometry to measure the Hp-Hb binding rate constant (Figures 25A-25B). Hb samples were prepared in increasing concentrations (5 pM, 10 pM, 15 pM and 20 pM, Hb tetramer basis) relative to 0.25 pM of Hp (Hb tetramer basis). Representative time courses for the change in the fluorescence signal of Hp binding to Hb are shown in Figure 25 A, and the data was fit to a single exponential function to calculate the pseudo-first order rate constant (kob . The slope from the plot of the pseudo first order rate constant as function of Hb concentration was used to determine the second order rate constant of Hp binding to Hb (kHp-Hb). The rate of association constant (kHp-Hb) was determined to be (0.2013 ± 0.001709 pM^s'1) which is comparable to previous reports in the literature.

[0396] Hb Oxygen Equilibrium and Offloading Kinetics. The Hb-O? equilibrium is altered when Hb is bound to Hp. Under normal physiological conditions inside the erythrocyte, Hb at ~ 3 mM exists as tetramer, and binds O2 cooperatively. However, the cooperative binding of O2 to Hb is decreased when it is bound to Hp, because in the Hb-Hp complex, Hb exist as a aP dimer, which reduces the cooperativity from n = 2.54 ± 0.034 to n = 0.95 ± 0.02 as shown in Figure 26A. Interestingly, the P50 (partial pressure at which 50 % of Hb is saturated with O2) of the Hb-Hp complex decreased from 15.53 ± 1.00 mmHg to 5.59 ± 0.19 mmHg, shifting the oxygen equilibrium curve to the left, which suggests that O2 binds with high affinity to Hb-Hp complex (Figure 26A).

[0397] The O2 offloading rate constant of Hb was fit to a single exponential function and the measured O2 dissociation rate constant was determined to be 45.6 ± 0.9 s'1(Figure 26B). When bound to Hp, O2 dissociation from the Hp-Hb complex displayed biphasic kinetics and was regressed to a double exponential function to calculate kfast (59.55 ± 0.7 s'1) and ksiow(10.11 ± 0.07 s'1). The rate of O2 dissociation from the Hp-Hb complex increases compared to Hb as Hp binding leads to Hb dimerization. The rate constant for O2 dissociation from the Hp-Hb complex is within the expected range, and is close to what has been observed in the literature.

[0398] Conclusion

[0399] In this example, we presented a simple method for the purification of Hp, primarily in the 100 - 500 kDa MW range. The single step purification process used HIC to purify Hp starting from a Hp rich fraction derived from TFF bracketing of FIV (100 - 500 kDa). The purified Hp is functionally active as well and has high HbBC, with an overall purity of > 98% or 87% based on densitometric and trypsin digest LC-MS / MS analysis, respectively.

[0400] The compositions, systems, kits, and methods of the appended claims are not limited in scope by the specific compositions, systems, kits, and methods described herein, which are intended as illustrations of a few aspects of the claims. Any compositions, systems, kits, and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions, systems, kits, and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions, systems, kits, and method steps disclosed herein are specifically described, other combinations of the compositions, systems, kits, and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

[0401] The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.

[0402] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a therapeutically effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobinhaptoglobin complex, or a combination thereof to treat hemolysis in pulmonary tissue in a subject.

2. The composition of claim 1, wherein the hemolysis in pulmonary tissue is characterized by elevated levels of hemoglobin in the pulmonary tissue, elevated levels of free iron in pulmonary tissue, elevated levels of heme in the pulmonary tissue, or a combination thereof.

3. The composition of any one of claims 1-2, wherein the composition comprises an effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apohemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof to reduce a level of hemoglobin in the pulmonary tissue of the subject, reduce a level of free iron in the pulmonary tissue, reduce a level of heme in the pulmonary tissue, or a combination thereof.

4. The composition of any one of claims 1-3, wherein the hemolysis in the pulmonary tissue in the subject is associated with pulmonary hypertension associated with sickle cell disease, acute respiratory distress syndrome (ARDS), and transfusion-related acute lung injury (TRALI), inhalation of a toxic or irritating agent that induces hemolysis (e.g., ozone, ionizing radiation, smoking, vapes, asbestos, silica, etc.), mechanical ventilation, lung transplantation, lung surgery, or a combination thereof.

5. The composition of any one of claims 1-4, wherein the composition is formulated for pulmonary administration to the subject.

6. The composition of any one of claims 1-5, wherein the composition comprises an aqueous solution or suspension.

7. The composition of any one of claims 1-5, wherein the composition comprises a dry powder formulation.

8. The composition of claim 7, wherein the dry powder formulation comprises particles having a tap density of less than 0.4 g / cm, a mass median aerodynamic diameter (MMAD) of between 0.5 micron and 5.0 microns, or a combination thereof.

9. The composition of any one of claims 1-5, wherein the composition comprises an aerosol comprising liquid droplets or solid particles suspended in a gas.

10. The composition of claim 9, wherein the aerosol comprises liquid droplets having a droplet diameter of less than 5 microns MMAD suspended in the gas.

11. The composition of claim 9, wherein the aerosol comprises solid particles having a tap density of less than 0.4 g / cm, a mass median aerodynamic diameter (MMAD) of between 0.5 micron and 5.0 microns, or a combination thereof suspended in the gas.

12. The composition of any one of claims 1-11, wherein the composition comprises apotransferrin.

13. The composition of claim 12, wherein the apo-transferrin comprises recombinant apotransferrin.

14. The composition of any one of claims 12-13, wherein the apo-transferrin is purified by ultrafiltration, such as tangential flow filtration (TFF).

15. The composition of any one of claims 12-14, wherein the apo-transferrin is purified by a process that comprises obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50-100 kDa; isolating transferrin from the protein cocktail using chromatography; and contacting the transferrin with a chelating agent to remove transferrin-bound iron, thereby isolating the apo-transferrin.

16. The composition of claim 15, wherein the chromatography comprises ion exchange chromatography, such as anion exchange chromatography.

17. The composition of any one of claims 1-16, wherein the composition comprises haptoglobin.

18. The composition of claim 17, wherein the haptoglobin comprises recombinant haptoglobin.

19. The composition of any one of claims 17-18, wherein the haptoglobin is purified by ultrafiltration, such as tangential flow filtration (TFF).

20. The composition of any one of claims 17-19, wherein the haptoglobin is purified by a process that comprises obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50 kDa and 0.2 microns (e.g., 50-500 kDa, 50-100 kDa, 100-500 kDa, 200-500 kDa, 300-500 kDa, 100 kDa- 0.2 microns); and isolating haptoglobin from the protein cocktail.

21. The composition of claim 20, wherein isolating haptoglobin from the protein cocktail comprises chromatography, such as hydrophobic interaction chromatography (e.g., using an alkyl ligand or an aryl ligand such as a phenyl ligand).

22. The composition of claim 20, wherein isolating haptoglobin from the protein cocktail comprises ammonium sulfate precipitation, polyethylene glycol (PEG) precipitation, or a combination thereof.

23. The composition of any one of claims 1-22, wherein the composition comprises hemopexin.

24. The composition of claim 23, wherein the hemopexin comprises recombinant hemopexin.

25. The composition of any one of claims 23-24, wherein the hemopexin is purified by ultrafiltration, such as tangential flow filtration (TFF).

26. The composition of any one of claims 23-25, wherein the hemopexin is purified by a process that comprises obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50-100 kDa; and isolating hemopexin from the protein cocktail using chromatography.

27. The composition of claim 26, wherein the chromatography comprises immobilized metal affinity chromatography, such as nickel (Ni2+) affinity chromatography.

28. The composition of any one of claims 1-27, wherein the composition comprises at least two of apo-transferrin, haptoglobin, and hemopexin.

29. The composition of any one of claims 1-28, wherein the composition comprises apotransferrin, haptoglobin, and hemopexin.

30. A method of treating hemolysis in pulmonary tissue in a subject comprising administering a composition comprising a therapeutically effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobinhaptoglobin complex, or a combination thereof to the subject.

31. The method of claim 30, wherein the hemolysis in pulmonary tissue is characterized by elevated levels of hemoglobin in the pulmonary tissue, elevated levels of free iron in pulmonary tissue, elevated levels of heme in the pulmonary tissue, or a combination thereof.

32. The method of any one of claims 30-31, wherein the composition comprises an effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apohemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof to reduce a level of hemoglobin in the pulmonary tissue of the subject, reduce a level of free iron in the pulmonary tissue, reduce a level of heme in the pulmonary tissue, or a combination thereof.

33. The method of any one of claims 30-32, wherein the hemolysis in the pulmonary tissue in the subject is associated with pulmonary hypertension associated with sickle cell disease,acute respiratory distress syndrome (ARDS), and transfusion-related acute lung injury (TRALI), inhalation of a toxic or irritating agent that induces hemolysis (e.g., ozone, ionizing radiation, smoking, vapes, asbestos, silica, etc.), mechanical ventilation, lung transplantation, lung surgery, or a combination thereof.

34. A method of reducing a level of hemoglobin in the pulmonary tissue of a subject, reducing a level of free iron in the pulmonary tissue of the subject, reduce a level of heme in the pulmonary tissue of the subject, or a combination thereof, the method comprising administering a composition comprising a therapeutically effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobinhaptoglobin complex, or a combination thereof to the subject.

35. A method of treating pulmonary hypertension associated with sickle cell disease, a method of treating acute respiratory distress syndrome (ARDS), a method of treating transfusion-related acute lung injury (TRALI), a method of treating inhalation of a toxic or irritating agent that induces hemolysis (e.g., ozone, ionizing radiation, smoking, vapes, asbestos, silica, etc.), a method of mechanical ventilation, a method of lung transplantation, a method of lung surgery, or a combination thereof, the method comprising administering a composition comprising a therapeutically effective amount of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apo-hemoglobin, an apo-hemoglobin-haptoglobin complex, or a combination thereof to the subject.

36. The method of any one of claims 30-35, wherein the administration comprises pulmonary administration.

37. The method of any one of claims 30-36, wherein the composition comprises a composition defined by any one of claims 1-29.

38. The method of any one of claims 30-37, wherein the composition is administered to the subject using a nebulizer, a dry powder inhaler, or a pressurized metered dose inhaler.

39. The method of any one of claims 30-38, wherein the composition comprises a dry powder formulation and the administration comprises inhalation of the dry powder formulation.

40. The method of claim 39, wherein the dry powder formulation comprises particles having a tap density of less than 0.4 g / cm, a mass median aerodynamic diameter (MMAD) of between 0.5 micron and 5.0 microns, or a combination thereof.

41. The method of any one of claims 30-38, wherein the composition comprises an aqueous solution or suspension and the administration comprises inhalation of an aerosol formed by nebulization of the composition.

42. The method of claim 41, wherein nebulization of the composition creates:(a) a mass median aerodynamic diameter (MMAD) of droplet size of the composition emitted from a nebulizer of from 0.5 microns to 5 microns;(b) a volumetric mean diameter (VMD) droplet size of the composition emitted from a nebulizer of from 0.5 microns to 5 microns(c) a Geometric Standard Deviation (GSD) of emitted droplet size distribution of the composition emitted from a nebulizer of from 1.0 micron to 3.4 microns;(d) a fine particle fraction (FPF=% of aerosol particles less than or equal to 5 microns) of droplets emitted from a nebulizer of at least 30%;(e) an output rate of at least 0.1 mL / min from a nebulizer; or a combination thereof.

43. The method of any one of claims 30-42, wherein the administration comprises pulmonary administration, and wherein pulmonary administration locally administers the apotransferrin, the haptoglobin, the hemopexin, the apo-hemoglobin, the PEGylated apohemoglobin, the apo-hemoglobin-haptoglobin complex, or the combination thereof to the pulmonary tissue of the subject.

44. The method of any one of claims 30-43, wherein the administration comprises pulmonary administration, and wherein pulmonary administration of apo-transferrin, the haptoglobin, the hemopexin, the apo-hemoglobin, the PEGylated apo-hemoglobin, the apo-hemoglobin-haptoglobin complex, or the combination thereof does not substantially increase levels of apo-transferrin, haptoglobin, hemopexin, apo-hemoglobin, PEGylated apohemoglobin, apo-hemoglobin-haptoglobin complex, or a combination thereof circulation in a blood stream of the subject.

45. A method of purifying apo-transferrin comprising obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50-100 kDa; isolating transferrin from the protein cocktail using chromatography; and contacting the transferrin with a chelating agent to remove transferrin-bound iron, thereby isolating the apo-transferrin.

46. The method of claim 45, wherein the chromatography comprises ion exchange chromatography, such as anion exchange chromatography.

47. A method of purifying haptoglobin comprising obtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50 kDa and 0.2 microns (e.g., 50-500 kDa, 50-100 kDa, 100-500 kDa, 200-500 kDa, 300-500 kDa, 100 kDa-0.2 microns); and isolating haptoglobin from the protein cocktail.

48. The method of claim 47, wherein isolating haptoglobin from the protein cocktail comprises chromatography, such as hydrophobic interaction chromatography (e.g., using an alkyl ligand or an aryl ligand such as a phenyl ligand).

49. The method of claim 47, wherein isolating haptoglobin from the protein cocktail comprises ammonium sulfate precipitation, polyethylene glycol (PEG) precipitation, or a combination thereof.

50. A method of purifying hemopexin comprisingobtaining a protein cocktail from a plasma fraction, such as Human Cohn plasma fraction IV, by ultrafiltration, wherein the protein cocktail is bracketed between 50-100 kDa; and isolating hemopexin from the protein cocktail using chromatography.

51. The method of claim 50, wherein the chromatography comprises immobilized metal affinity chromatography, such as nickel (Ni2+) affinity chromatography.

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