Patent Application
20250169491
In the United States in 2020, there were almost 108,000 patients on the organ transplant waiting list, yet only 39,000 transplants were performed leading to 11,000 patients being removed from the waiting list due to death or sickness. A primary reason for the disparity between the number of patients on the waiting list and the number of transplants performed is an overall limited number of available organs and additionally some of the available supply of organs are deemed of poor quality and are not viable for transplantation. For organs such as the lungs, more than 80% of donor allografts offered by organ donors are declined due to poor organ quality. It is obvious that the best way to decrease the number of patients on the organ waiting list is to make more organs available for transplantation. In an effort to expand the donor pool, organs from marginal or extended criteria donors (ECD) may be evaluated. However, ECD grafts are associated with a significantly higher risk of ischemia-reperfusion injury (IRI) which leads to primary graft dysfunction and subsequently reduces organ viability. Due to a rising demand in the need for organ transplantation and a critical donor organ shortage, the need to fill this gap has increased the use of ECD and donation after cardiac death (DCD) organs viable for transplantation to lower the mortality of patients on the organ waiting list.
In accordance with the purposes of the disclosed materials and methods, as embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to a perfusion solution for tissue recovery. perfusion solution can comprise polymerized hemoglobin, wherein the perfusion solution comprises less than 5% by weight low molecular weight hemoglobin species, based on the total weight of the perfusion solution.
Additional advantages will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain examples of the present disclosure and together with the description, serve to explain, without limitation, the principles of the disclosure. The application includes reference to the accompanying figures, in which:
By the end of the NEVLP, the pH of the RBC perfusate was significantly higher than the other two perfusates. Given that lungs are supposed to be mildly acidotic, the increase in pH towards 7.4 corroborates the decline in lung function seen by other metrics at this time.
Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, formulations, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.
The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiments. Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.
Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
As can be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.
Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.
It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It can be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.
Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.
As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.”
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a disorder”, includes, but is not limited to, two or more such compounds, compositions, or disorders, and the like.
It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.
When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less' and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “substantially free,” when used in the context of a composition or component of a composition that is substantially absent, is intended to refer to an amount that is then about 1% by weight or less, e.g., less than about 0.5% by weight, less than about 0.1% by weight, less than about 0.05% by weight, or less than about 0.01% by weight of the stated material, based on the total weight of the composition.
As used herein, “molecular weight” refers to the sum of the atomic masses of all atoms in a molecule, based on a scale in which the atomic masses of hydrogen, carbon, nitrogen, and oxygen are 1, 12, 14, and 16, respectively.
As used herein, “albumin” means a small globular protein with a molecular weight of 66.5 kilodaltons (kDa). It consists of 585 amino acids which are organized into three repeated homologous domains and are made up of two separate sub-domains, A and B.
As used herein, “osmolarity” refers to the concentration of a solution expressed as the total number of solute particles per liter.
As used herein, “viscosity” refers to a quantity expressing the magnitude of internal friction, as measured by the force per unit area resisting a flow in which parallel layers unit distance apart have unit speed relative to one another.
As used herein, “colloid osmotic pressure” refers to the physiochemical phenomenon that occurs when two solutions with different colloid concentrations are separated by a semipermeable membrane. It is a type of osmotic pressure induced by colloids, which can include protein and more specifically albumin, in a blood vessel's plasma that cause a pull on fluid back into the capillary.
As used herein, “glutaraldehyde” refers to C5H8O2 or OCH(CH2)3CHO, is a transparent oily, liquid with a pungent odor. It is a dialdehyde comprised of pentane with aldehyde functions at C-1 and C-5. Alternatives to glutaraldehyde can include carboiimide, diisocyanates and polyepoxy compounds, as well as Genipin (Challenge Bioproducts Co., Ltd., Taiwan), epigallocatechin gallate (Sigma, St. Louis, MO), and grape seed proanthocyanidin (PureBulk, Inc., Roseburg, OR).
As used herein, “normothermic conditions” refers to a condition of normal body temperature. In some embodiments, normothermic conditions can include a temperature from 36° C. to 38° C.
The present disclosure provides for a perfusion solution for tissue recovery. The perfusion solution can comprise polymerized hemoglobin and less than 5% by weight low molecular weight hemoglobin species, based on the total weight of the perfusion solution.
As used herein, “polymerized hemoglobin”, also referred to as “polymerized Hb” or “PolyhHb”, refers to a class of hemoglobin (Hb) based O2 carrier (HBOC) that can be synthesized and purified at large scale such that it can transport and offload O2 to support cellular metabolism, while not demonstrating cytotoxic side-effects. In some embodiments, hemoglobin is polymerized with glutaraldehyde. As used herein, “hemoglobin species, or “hemoglobin (Hb)”, refers to the protein inside red blood cells that carries oxygen from the lungs to tissues and organs in the body and carries carbon dioxide back to the lungs. It can include four protein chains, two alpha chains and two beta chains, each with a ring-like heme group containing an iron atom. Oxygen can bind reversibly to these iron atoms and can be transported through blood.
As used herein, “perfusion solution”, also referred to as “perfusate”, refers to the solution used to perfuse a tissue sample during perfusion. In some embodiments, a perfusion solution can include PolyhHb diluted with William's cell culture media. In further embodiments, the perfusion solution can include albumin (e.g., human serum albumin). In some embodiments, the perfusion solution can include from 1 to 15 g/dL polymerized hemoglobin, 25 to 85 mM NaCl, 1 to 3 mM KCl, 6 to 20 mM KH2PO4, 20 to 70 mM sodium gluconate, 5 to 21 mM sodium lactate, 1 to 4 mM magnesium gluconate, 0.6 to 1.2 mM CaCl2 dihydrate, 11 to 16 mM NaOH, 1 to 4 mM adenine, 2 to 8 mM dextrose, 0.5 to 3 mM glutathione, 2 to 8 mM HEPES, 1 to 4 mM ribose, 7 to 30 mM mannitol, 10 to 40 g/L hydroxyethyl starch, and 40 to 160 mg/dL N-acetyl-L-cysteine. In further embodiments, the perfusion solution can include from 3 to 4 g/dL polymerized hemoglobin, 25 to 85 mM NaCl, 1 to 3 mM KCl, 6 to 20 mM KH2PO4, 20 to 70 mM sodium gluconate, 5 to 21 mM sodium lactate, 1 to 4 mM magnesium gluconate, 0.6 to 1.2 mM CaCl2) dihydrate, 11 to 16 mM NaOH, 1 to 4 mM adenine, 2 to 8 mM dextrose, 0.5 to 3 mM glutathione, 2 to 8 mM HEPES, 1 to 4 mM ribose, 7 to 30 mM mannitol, 10 to 40 g/L hydroxyethyl starch, and 40 to 160 mg/dL N-acetyl-L-cysteine.
As used herein, “hemoglobin species, or “hemoglobin (Hb)”, refers to the protein inside red blood cells that carries oxygen from the lungs to tissues and organs in the body and carries carbon dioxide back to the lungs. It can include four protein chains, two alpha chains and two beta chains, each with a ring-like heme group containing an iron atom. Oxygen can bind reversibly to these iron atoms and can be transported through blood.
In some embodiments, the low molecular weight hemoglobin species can have a molecular weight below 500 kDa (e.g., a molecular weight below 300 kDa). In some embodiments, the low molecular weight hemoglobin species can have a molecular weight from below 500 kDa to 400 kDa, from 400 kDa to 300 kDa, from 300 kDa to 200 kDa, from 200 kDa to 100 kDa, from 100 kDa to 50 kDa, or from 50 kDa to above 0 kDa.
In some embodiments, the low molecular weight hemoglobin species includes unreacted hemoglobin. As used herein, “unreacted hemoglobin” refers to hemoglobin that has not reacted or bound to another molecule and therefore has a low molecular weight. Unreacted hemoglobin, like cell-free hemoglobin, can extravasate out of circulation into tissue space, thereby causing nitric oxide scavenging and subsequently vasoconstriction, systemic hypertension, and/or oxidative tissue injury.
In some embodiments, the low molecular weight hemoglobin species can include cell-free hemoglobin. As used herein, “cell-free hemoglobin” refers to hemoglobin molecules that have been separated from the red blood cells within which they originally occurred, often through the process of hemolysis.
In some embodiments, the perfusion solution comprises less than 5% by weight (e.g., less than 4% by weight, less than 3% by weight, less than 2% by weight, less than 1% by weight, or less than 0.5% by weight) hemoglobin species having a molecular weight below 500 kDa, based on the total weight of the perfusion solution.
In some embodiments, the perfusion solution comprises less than 5% by weight (e.g., less than 4% by weight, less than 3% by weight, less than 2% by weight, less than 1% by weight, or less than 0.5% by weight) hemoglobin species having a molecular weight below 300 kDa, based on the total weight of the perfusion solution.
As used herein, “filter” can include tangential-flow filtration. 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 (e.g., filter). 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. As used herein, “filtration membrane” refers to microporous barriers of, for example, polymeric, ceramic, or metallic materials which are used to separate dissolved materials (solutes), colloids, or find particular from solutions. A filtration membrane can be rated for retaining solutes have a specific molecular weight range from the molecular weight of one component in the solution to another component in the solution. By way of example, a filtration membrane can be rated for retaining polymerized hemoglobin with a molecular weight above that of a low molecular weight hemoglobin species (e.g., such as a membrane rated for retaining solutes have a molecular weight above 300 kDa).
In some embodiments, the polymerized hemoglobin can have an average molecular weight from 300 kDa to 500 kDa and can be filtered using a filtration membrane with a cutoff value from 300 kDa to 500 kDa. In further embodiments, the polymerized hemoglobin can have an average molecular weight from 300 kDa to 350 kDa, 350 kDa to 400 kDa, 400 kDa to 450 kDa, or 450 kDa to 500 kDa and the cutoff value can be from 300 kDa to 350 kDa, 350 kDa to 400 kDa, 400 kDa to 450 kDa, or 450 kDa to 500 kDa.
In some embodiments, the polymerized hemoglobin can have an average molecular weight from 500 kDa to 750 kDa and can be filtered using a filtration membrane with a cutoff value from 500 kDa to 750 kDa. In further embodiments, the polymerized hemoglobin can have an average molecular weight from 500 kDa to 550 kDa, 550 kDa to 600 kDa, 600 kDa to 650 kDa, 650 kDa to 700 kDa, or 700 kDa to 750 kDa and the cutoff value can be from 500 kDa to 550 kDa, 550 kDa to 600 kDa, 600 kDa to 650 kDa, 650 kDa to 700 kDa, or 700 kDa to 750 kDa.
In some embodiments, the polymerized hemoglobin can have an average molecular weight from 750 kDa to 50 nm and can be filtered using a filtration membrane with a cutoff value from 750 kDa to 50 nm. In further embodiments, the polymerized hemoglobin can have an average molecular weight from 750 kDa to 800 kDa, 800 kDa to 850 kDa, 850 kDa to 900 kDa, 900 kDa to 950 kDa, 950 kDa to 1000 kDa, 1000 kDa to 25 nm, or 25 nm to 50 nm and the cutoff value can be from 750 kDa to 800 kDa, 800 kDa to 850 kDa, 850 kDa to 900 kDa, 900 kDa to 950 kDa, 950 kDa to 1000 kDa, 1000 kDa to 25 nm, or 25 nm to 50 nm.
In some embodiments, the polymerized hemoglobin can have an average molecular weight from 50 nm to 0.2 μm and can be filtered using a filtration membrane with a cutoff value from 50 nm to 0.2 μm. In further embodiments, the polymerized hemoglobin can have an average molecular weight from 50 nm to 100 nm, 100 nm to 150 nm, or 150 nm to 0.2 μm and the cutoff value can be from 50 nm to 100 nm, 100 nm to 150 nm, or 150 nm to 0.2 μm.
In some embodiments, the polymerized hemoglobin can be prepared by a process that includes polymerizing the hemoglobin and filtering the perfusion solution by ultrafiltration against a filtration membrane having a pore size that separates the low molecular weight hemoglobin species from the polymerized hemoglobin. As used herein, “filtration membrane” refers to microporous barriers of, for example, polymeric, ceramic, or metallic materials which are used to separate dissolved materials (solutes), colloids, or find particular from solutions. A filtration membrane can be rated for retaining solutes have a specific molecular weight range from the molecular weight of one component in the solution to another component in the solution. By way of example, a filtration membrane can be rated for retaining polymerized hemoglobin with a molecular weight above that of a low molecular weight hemoglobin species (e.g., such as a membrane rated for retaining solutes have a molecular weight above 300 kDa).
In further embodiments, polymerizing hemoglobin includes adding to a Hb solution a glutaraldehyde solution over a specified period of time. In certain embodiments, polymerizing hemoglobin includes adding to a Hb solution a 0.75 wt. % glutaraldehyde solution in phosphate buffered saline (0.1 M, pH 7.4) in a 30:1 molar ratio, glutaraldehyde to Hb, over a period of 3 hours and mixing the solution for an additional hour after the glutaraldehyde addition is completed.
In some embodiments, the filtration membrane can be rated for retaining solutes having a molecular weight greater than the molecular weight of the low molecular weight hemoglobin species, thereby forming a retentate fraction including the polymerized hemoglobin and a permeate fraction including the low molecular weight hemoglobin species. As used herein, a “retentate fraction” refers to the fraction of solution that is unable to pass through the filtration membrane. In some embodiments, the retentate fraction can include polymerized hemoglobin. As used herein, a “permeate fraction” refers to the fraction of solution that permeates the filtration membrane. In some embodiments, the permeate fraction can include low molecular weight hemoglobin species. In other embodiments, the permeate fraction can include polymerized hemoglobin and low molecular weight hemoglobin species.
In some embodiments, ultrafiltration can include tangential-flow filtration. 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 (e.g., filter).
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.
In some embodiments, the retentate fraction including polymerized hemoglobin can have a molecular weight of greater than 300 kDa and the permeate fraction including the low molecular weight hemoglobin species can have a molecular weight of less than 300 kDa. In certain embodiments, the polymerized hemoglobin can have a molecular weight from greater than 300 kDa to 500 kDa, 300 kDa to 750 kDa, 300 kDa to 50 nm, 300 kDa to 100 nm, 300 kDa to 0.2 μm, 500 kDa to 750 kDa, 500 kDa to 50 nm, 500 kDa to 100 nm, 500 kDa to 0.2 μm, 750 kDa to 50 nm, 750 kDa to 100 nm, 750 kDa to 0.2 μm, or 50 nm to 0.2 μm. In some embodiments, the low molecular weight hemoglobin species can have a molecular weight from below 300 kDa to 200 kDa, from 200 kDa to 100 kDa, from 100 kDa to 50 kDa, or from 50 kDa to above 0 kDa.
In some embodiments, the polymerized hemoglobin can be prepared by a process that can further include filtering the retentate fraction comprising the polymerized hemoglobin by ultrafiltration against a second filtration membrane, thereby forming a second retentate fraction comprising the polymerized hemoglobin with a molecular weight above a cutoff value and a second permeate fraction comprising species having a molecular weight below the cutoff value and above 300 kDa.
In some embodiments, the cutoff value can be from 300 kDa to 0.2 μm.
In some embodiments, the cutoff value can be from 300 kDa to 500 kDa. In further embodiments, the cutoff value can be from 300 kDa to 350 kDa, 350 kDa to 400 kDa, 400 kDa to 450 kDa, or 450 kDa to 500 kDa.
In some embodiments, the cutoff value can be from 500 kDa to 750 kDa. In further embodiments, the cutoff value can be from 500 kDa to 550 kDa, 550 kDa to 600 kDa, 600 kDa to 650 kDa, 650 kDa to 700 kDa, or 700 kDa to 750 kDa.
In some embodiments, the cutoff value can be from 750 kDa to 50 nm. In further embodiments, the cutoff value can be from 750 kDa to 800 kDa, 800 kDa to 850 kDa, 850 kDa to 900 kDa, 900 kDa to 950 kDa, 950 kDa to 1000 kDa, 1000 kDa to 25 nm, or 25 nm to 50 nm.
In some embodiments, the cutoff value can from 50 nm to 0.2 μm. In some embodiments, the cutoff value can be from 50 nm to 100 nm, 100 nm to 150 nm, or 150 nm to 0.2 μm.
In some embodiments, the polymerized hemoglobin can be prepared by a process that can further include filtering the second retentate fraction including the polymerized hemoglobin by ultrafiltration against a third filtration membrane, thereby forming a third retentate fraction comprising the polymerized hemoglobin with a molecular weight above a second cutoff value and a third permeate fraction including species having a molecular weight below the second cutoff value and above the cutoff value.
In some embodiments, the second cutoff value can be from the cutoff value to 0.2 μm.
In some embodiments, the second cutoff value can be from the cutoff value to 500 kDa. In some embodiments, the second cutoff value can be from the cutoff value to 350 kDa, 350 kDa to 400 kDa, 400 kDa to 450 kDa, or 450 kDa to 500 kDa.
In some embodiments, the second cutoff value can be from 500 kDa to 750 kDa. In some embodiments, the second cutoff value can be from 500 kDa to 550 kDa, 550 kDa to 600 kDa, 600 kDa to 650 kDa, 650 kDa to 700 kDa, or 700 kDa to 750 kDa.
In some embodiments, the second cutoff value can be from 750 kDa to 50 nm. In some embodiments, the second cutoff value can be from 750 kDa to 800 kDa, 800 kDa to 850 kDa, 850 kDa to 900 kDa, 900 kDa to 950 kDa, 950 kDa to 1000 kDa, 1000 kDa to 25 nm, or 25 nm to 50 nm.
In some embodiments, the second cutoff value can be from 50 nm to 0.2 μm. In some embodiments, the second cutoff value can be from 50 nm to 100 nm, 100 nm to 150 nm, or 150 nm to 0.2 μm.
In some embodiments, the polymerized hemoglobin can be prepared by a process that can further include filtering the third retentate fraction including the polymerized hemoglobin by ultrafiltration against a fourth filtration membrane, thereby forming a fourth retentate fraction comprising the polymerized hemoglobin with a molecular weight above a third cutoff value and a fourth permeate fraction including low molecular weight hemoglobin species having a molecular weight below the third cutoff value and above the second cutoff value.
In some embodiments, the third cutoff value can be from the second cutoff value to 0.2 μm.
In some embodiments, the third cutoff value can be from the second cutoff value to 500 kDa. In some embodiments, the third cutoff value can be from the second cutoff value to 350 kDa, 350 kDa to 400 kDa, 400 kDa to 450 kDa, or 450 kDa to 500 kDa.
In some embodiments, the third cutoff value can be from 500 kDa to 750 kDa. In some embodiments, the third cutoff value can be from 500 kDa to 550 kDa, 550 kDa to 600 kDa, 600 kDa to 650 kDa, 650 kDa to 700 kDa, or 700 kDa to 750 kDa.
In some embodiments, the third cutoff value can be from 750 kDa to 50 nm. In some embodiments, the third cutoff value can be from 750 kDa to 800 kDa, 800 kDa to 850 kDa, 850 kDa to 900 kDa, 900 kDa to 950 kDa, 950 kDa to 1000 kDa, 1000 kDa to 25 nm, or 25 nm to 50 nm.
In some embodiments, the third cutoff value can be from 50 nm to 0.2 μm. In some embodiments, the third cutoff value can be from 50 nm to 100 nm, 100 nm to 150 nm, or 150 nm to 0.2 μm.
In some embodiments, the polymerized hemoglobin can be prepared by a process that can further include filtering the fourth retentate fraction comprising the polymerized hemoglobin by ultrafiltration against a fifth filtration membrane, thereby forming a fifth retentate fraction comprising the polymerized hemoglobin with a molecular weight above a fourth cutoff value and a fifth permeate fraction comprising species having a molecular weight below the fourth cutoff value and above the third cutoff value.
In some embodiments, the fourth cutoff value can be from the third cutoff value to 0.2 μm.
In some embodiments, the fourth cutoff value can be from the third cutoff value to 500 kDa. In some embodiments, the fourth cutoff value can be from the third cutoff value to 350 kDa, 350 kDa to 400 kDa, 400 kDa to 450 kDa, or 450 kDa to 500 kDa.
In some embodiments, the fourth cutoff value can be from 500 kDa to 750 kDa. In some embodiments, the fourth cutoff value can be from 500 kDa to 550 kDa, 550 kDa to 600 kDa, 600 kDa to 650 kDa, 650 kDa to 700 kDa, or 700 kDa to 750 kDa.
In some embodiments, the fourth cutoff value can be from 750 kDa to 50 nm. In some embodiments, the fourth cutoff value can be from 750 kDa to 800 kDa, 800 kDa to 850 kDa, 850 kDa to 900 kDa, 900 kDa to 950 kDa, 950 kDa to 1000 kDa, 1000 kDa to 25 nm, or 25 nm to 50 nm.
In some embodiments, the fourth cutoff value can be from 50 nm to 0.2 μm. In some embodiments, the fourth cutoff value can be from 50 nm to 100 nm, 100 nm to 150 nm, or 150 nm to 0.2 μm.
In some embodiments, the filtration membrane can be rated for retaining solutes having a molecular weight greater than 0.2 μm, thereby forming a retentate fraction including species having a molecular weight of greater than 0.2 μm and a permeate fraction including the polymerized hemoglobin having a molecular weight of less than 0.2 μm and the low molecular weight hemoglobin species.
In some embodiments, the ultrafiltration includes tangential-flow filtration. Tangential-flow filtration has a meaning as described herein.
In some embodiments, the polymerized hemoglobin can be prepared by a process that can further comprise filtering the permeate fraction including the polymerized hemoglobin and the low molecular weight hemoglobin species by ultrafiltration against a second filtration membrane, thereby forming a second retentate fraction including the polymerized hemoglobin having a molecular weight below 0.2 μm and above a cutoff value and a second permeate fraction comprising the low molecular weight hemoglobin species. In some embodiments, the polymerized hemoglobin can have a molecular weight from 50 nm to below 0.2 μm, 750 kDa to 50 nm, 500 kDa to 750 kDa, or 300 kDa to 500 kDa.
In some embodiments, the cutoff value can be from 300 kDa to 0.2 μm.
In some embodiments, the cutoff value can be from 50 nm to 0.2 μm. In some embodiments, the cutoff value can be from 50 nm to 100 nm, 100 nm to 150 nm, or 150 nm to 0.2 μm.
In some embodiments, the cutoff value is from 750 kDa to 50 nm. In some embodiments, the cutoff value can be from 750 kDa to 800 kDa, 800 kDa to 850 kDa, 850 kDa to 900 kDa, 900 kDa to 950 kDa, 950 kDa to 1000 kDa, 1000 kDa to 25 nm, or 25 nm to 50 nm.
In some embodiments, the cutoff value is from 500 kDa to 750 kDa. In some embodiments, the cutoff value can be from 500 kDa to 550 kDa, 550 kDa to 600 kDa, 600 kDa to 650 kDa, 650 kDa to 700 kDa, or 700 kDa to 750 kDa.
In some embodiments, the cutoff value is from 300 kDa to 500 kDa. In some embodiments, the cutoff value can be from 300 kDa to 350 kDa, 350 kDa to 400 kDa, 400 kDa to 450 kDa, or 450 kDa to 500 kDa.
In some embodiments, the perfusion solution can comprise from 1% by weight to 5% by weight albumin, based on total weight of the perfusion solution.
In some embodiments, the perfusion solution can have an osmolarity from 270 to 370 mOsm. In further embodiments, the perfusion solution can have an osmolarity from 270 to 290 mOsm, 290 to 310 mOsm, 310 to 330 mOsm, 330 to 350 mOsm, or 350 to 370 mOsm.
In some embodiments, the perfusion solution can have a viscosity from 2 cP to 4.5 cP at normothermic conditions. In further embodiments, the perfusion solution can have a viscosity from 2 cP to 3 cP, 3 cP to 4 cP, or 4 cP to 4.5 cP.
In some embodiments, the perfusion solution can have a viscosity from 2.9 to 3.7 cP at normothermic conditions. In further embodiments, the perfusion solution can have a viscosity from 2.9 to 3.1 cP, 3.1 to 3.3 cP, 3.3 to 3.5 cP, or 3.5 to 3.7 cP.
In some embodiments, the perfusion solution can have a colloid osmotic pressure from 14 mm Hg to 20 mm Hg. In further embodiments, the perfusion solution can have a colloid osmotic pressure from 14 mm Hg to 16 mm Hg, 16 mm Hg to 18 mm Hg, or 18 mm Hg to 20 mm Hg.
In some embodiments, the perfusion solution can have a colloid osmotic pressure from 16.8 mm Hg to 17.6 mm Hg.
In some embodiments, the polymerized hemoglobin can be synthesized using a molar ratio from 20:1 to 40:1 of glutaraldehyde to hemoglobin. In some embodiments, the polymerized hemoglobin can be synthesized using a molar ratio from 25:1 to 35:1 of glutaraldehyde to hemoglobin. In further embodiments, the ratio can be at least 1:1 (e.g., at least 2:1, at least 5:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, or at least 90:1). In certain embodiments, the ratio can be 100:1 or less (e.g., 90:1 or less, 80:1 or less, 70:1 or less, 60:1 or less, 50:1 or less, 40:1 or less, 30:1 or less, 20:1 or less, or 10:1 or less).
The range can vary between any of the minimum values described above to any of the maximum values described above. For example, the polymerized hemoglobin can be synthesized at a molar ratio that can range from 1:1 to 100:1 (e.g., from 1:1 to 10:1, 10:1 to 20:1, 20:1 to 30:1, 30:1 to 40:1, 40:1 to 50:1, 50:1 to 60:1, 60:1 to 70:1, 70:1 to 80:1, 80:1 to 90:1, 90:1 to 100:1).
In some embodiments, the partial pressure of oxygen at which 50% of the polymerized hemoglobin is saturated with oxygen can be from 1 mm Hg to 50 mm Hg. In further embodiments, the partial pressure of oxygen at which 50% of the polymerized hemoglobin is saturated with oxygen can be from 10 mm Hg to 12 mm Hg, 12 mm Hg to 14 mm Hg, 14 mm Hg to 16 mm Hg, 16 mm Hg to 18 mm Hg, or 18 mm Hg to 20 mm Hg. In some embodiments, the partial pressure of oxygen at which 50% of the polymerized hemoglobin is saturated with oxygen can be from 14 mm Hg to 16 mm Hg. As used herein, saturation with oxygen refers to the binding of heme in hemoglobin with oxygen molecules. The degree of oxygen saturation of hemoglobin is dependent on the number of heme units that are bound to oxygen (e.g., 50% saturation with oxygen means that half of the heme units are bound to oxygen molecules).
In some embodiments, the polymerized hemoglobin can exhibit an auto-oxidation rate at 37° C. of from 0.0020 to 0.0085 h−1. In further embodiments, the polymerized hemoglobin can exhibit an oxidation rate of from 0.0020 to 0.0045 h−1, 0.0045 to 0.0065 h−1, or 0.0065 to 0.0085 h−1. In some embodiments, the polymerized hemoglobin can exhibit an oxidation rate of from 0.0045 to 0.0065 h−1.
In some embodiments, the perfusion solution can further include one or more of a metabolic suppressant agent. As used herein, “metabolic suppressant agent” refers to an agent used to suppress the metabolic demand that is required to keep organs and/or tissue at normothermic conditions during preservation for transplantation.
A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.
The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.
Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.
Normothermic ex vivo lung perfusion (NEVLP) is able to resuscitate marginal lung allografts to make more organs available for transplantation. During normothermic perfusion, cellular metabolism is active, creating a need for an oxygen (O2) carrier to adequately oxygenate the graft. As an O2 carrier, red blood cells (RBCs) are prone to hemolysis in perfusion circuits leading to cell-free hemoglobin (Hb) toxicity, oxidative tissue damage, and cell death. Polymerized human Hb (PolyhHb) is a class of Hb-based O2 carrier (HBOC) that can be synthesized and purified at a large scale such that it can transport and offload O2 to support cellular metabolism, while not demonstrating cytotoxic side-effects. In this example, a next-generation PolyhHb was synthesized and purified at the 30 L pilot scale with a suite of analytical techniques including size exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to validate that the majority of low molecular weight Hb polymers (<500 kDa) were removed from the solution. The PolyhHb was then added to an existing colloid solution and compared to both RBC and asanguinous perfusates in a rat NEVLP model. The pulmonary artery pressure and pulmonary vascular resistance were both higher in lungs perfused with RBCs, likely due to vasoconstriction from hemolysis and subsequent exposure to cell-free Hb. Lungs perfused with PolyhHb also demonstrated greater oxygenation than those perfused with RBCs and elicited less cellular damage and edema than both other perfusates.
Normothermic machine perfusion (NMP) has shown promise in the field of organ storage and preservation to improve the viability of transplanted organs. Studies have shown that NMP can resuscitate ECD organs to obtain pre-transplant quality comparable to nonmarginal organs for subsequent transplantation. There is a significant metabolic demand maintaining organs at normothermia and this metabolic demand is only exacerbated in DCD organs. There is a demand for an artificial RBC substitute that can store and transport oxygen (O2) to meet the metabolic demands of perfused organs while not being subject to hemolysis or Hb/heme/iron toxicity.
Of all the different types of HBOCs being studied to carry and offload O2 to surrounding tissue, polymerized Hb (PolyHb) has the ability to be synthesized and purified at large scale. Any adverse side-effects are the result of the presence of cell-free Hb and low molecular weight (MW) Hb polymers (<500 kDa) that extravasate out of the circulation into the tissue space, which leads to nitric oxide (NO) scavenging and subsequent vasoconstriction, systemic hypertension, and oxidative tissue injury. Elimination of these low MW Hb species from the NMP perfusate may mitigate these deleterious side-effects. In ex vivo lung perfusion (EVLP), there is heightened concern about the development of edema which further necessitates the need for development of a perfusate that will not damage the especially delicate tissues of the lung. Currently, the majority of EVLPs, such as those that follow the Toronto Protocol, contain no O2 carrying molecule in the perfusate. Without an O2 carrier in the perfusate, the metabolic demand during normothermic EVLP (NEVLP), especially when perfusing DCD lungs, is not fully met.
This example utilizes a clinically relevant, validated, lung DCD model and NEVLP platform to assess the ability of a polymerized human Hb (hHb) (PolyhHb) HBOC perfusate to meet DCD organ metabolic demands and evaluate organ quality as compared to an RBC and asanguinous perfusate.
Materials. Glutaraldehyde (70/6), sodium chloride (NaCl), potassium chloride (KCl), sodium hydroxide (NaOH), sodium dithionite (Na2S2O4), calcium chloride (CaCl2·2H2O), sodium lactate, Nacetyl-L-cysteine (NALC), sodium cyanoborohydride (NaCNBH3), sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic (NaH2PO4), trichloroacetic acid (C2HCl3O2), sodium acetate (C2H3NaO2), ascorbic acid (C6HO6), and citrate buffer were purchased from Sigma-Aldrich (St. Louis, MO). Hollow fiber tangential flow filtration (TFF) modules (polyethersulfone (PES) 0.2 μm and polysulfone (PS) 500 kDa) were purchased from Spectrum Laboratories (Rancho Dominguez, CA). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA). Expired human RBC units were generously donated by Canadian Blood Services, Ottawa, Canada.
William's E Medium (A12176-01) was purchased from Gibco, Thermo Fisher Scientific (Waltham, MA). Sprague-Dawley rat RBCs were purchased from Innovative Research Inc. (Novi, MT). Male Sprague-Dawley rats were purchased from Envigo (Indianapolis, IN) and housed under pathogen-free conditions at The Ohio State University Animal Facility. All procedures were humanely performed according to the NIH and the National Research Council's Guide for the Humane Care and Use of Laboratory Animals and with the approval of The Ohio State University Institutional Animal Care and Use Committee (IACUC Protocol #2012A00000135-R2)
PolyhHb Synthesis. The PolyhHb was produced from a single pilot batch. Twenty units of expired human RBCs were added to 7 L of 0.9 wt % saline to achieve a pooled hematocrit of 22%. RBCs were washed with 6 volume difiltrations of saline over a 0.65 μm modified polyethersulfone (mPES) TFF filter. The RBC solution was then concentrated down to 10 L before being lysed for 1 hour with an equal volume of phosphate buffer (PB) (3.75 mM, pH 7.4). Cell debris was eliminated by filtration of the RBC lysate through a 500 kDa PES filter with purified Hb and other intracellular proteins present in the permeate flowing into a 30 L bioreactor. 470 g of Hb was allowed to flow into the bioreactor before the permeate feed line was closed. The Hb in the bioreactor had deionized water, NaCl, and KCl added to it to bring the final solution to 18.2 gHb/L in phosphate buffered saline (PBS). The bioreactor was held under a nitrogen (N2) head at 14° C. overnight.
The next morning, the cooling coils were turned off and the bioreactor was brought to and maintained at 37° C. using a heating jacket. The Hb solution was deoxygenated using an external flow loop with a G420 X40 gas-liquid exchanger (3M, Maplewood, MN) and N2 as the sweep gas. Once the partial pressure of O2 (pO2) reached below 5 mm Hg on a Rapidlab 248 (Siemens, Malvern, PA) blood gas analyzer (BGA), a bolus of 6 g of Na2S2O4 was added to reduce the pO2 to below readable levels on the BGA. Upon full deoxygenation of the solution, a 0.75 wt %/o glutaraldehyde solution in PBS (0.1 M, pH 7.4) was added in a 30:1 molar ratio glutaraldehyde to hemoglobin over a period of 3 hours. The solution was allowed to mix for an additional hour after glutaraldehyde addition was completed. After polymerization was completed, the heating jacket was removed and the cooling coils were turned back on. NaCNBH3 was used to quench the reaction at a 7:1 molar ratio of NaCNBH3 to glutaraldehyde through rapid delivery of a 3.5 wt % solution of NaCNBH3 in PBS (0.1 M, pH 7.4). The solution continued to quench and cool overnight.
PolyhHb purification followed methods described previously in the literature. The sole deviation was that the number of diafiltrations was increased from 8 to 16 to ensure the final PolyhHb product contained <5% low MW Hb species (<500 kDa). The final product was concentrated to 11.5 g/dL and stored at −80° C.
Protein Quantification. The total Hb concentration and percentage of methemoglobin (metHb) were determined using the cyanomethemoglobin method. This assay was used to quantify PolyhHb during production, after purification, and during NEVLP. Cell-free Hb in RBC perfusates was quantified by centrifuging the perfusate until the RBCs formed a pellet. The resulting supernatant was then analyzed via the cyanomethemoglobin method. To determine the total Hb concentration in RBCs, RBCs were lysed through freeze thaw cycles followed by dilution in PB (3.75 mM, pH 7.4). The lysed RBCs were again centrifuged down to a pellet and the supernatant assayed for the total Hb concentration.
Biophysical Parameters. The oxygen equilibrium curve, MW distribution, and auto-oxidation kinetics for PolyhHb and Hb were all measured as previously described in the literature. Briefly, oxygen equilibrium curves were measured using a Hemox Analyzer (TCS Scientific, New Hope, PA) at 37° C. and pH 7.4. The MW distribution was estimated by performing size exclusion high pressure liquid chromatography (SEC-HPLC) using an Acclaim SEC-1000 column (Thermo Scientific, Waltham, MA) on a Thermo Scientific UHPLC System using MW standards. Auto-oxidation kinetics were measured via UV-visible spectrometry over 24 hours at 37° C. The solution viscosity was measured using a DV3T-CP cone and plate viscometer (Brookfield AMETEK, Middleboro, MA), and osmolarity was measured using a Gonotech 010 freezing point osmometer (Gonotech GmbH, Berlin, Germany). Colloid osmotic pressure (COP) was measured using a Wescor 4420 Colloid Osmometer (Wescor, Logan, UT).
Perfusate Formulation. Perfusates were formulated using William's cell culture media as the primary fluid at a final volume of 165 mL. For the HBOC perfusate, PolyhHb was diluted with William's media to a final concentration of 3.7±0.1 g/dL. Twenty-five percent human serum albumin (HSA) was added such that the final perfusate consisted of 3% HSA by weight. Rat RBCs were diluted to a final hematocrit of 15% to be in line with the Lund protocol. The asanguinous control consisted of 4% HSA in William's media. Each of these perfusates was brought to 37° C. and pH 7.4 using THAM buffer before beginning NEVLP.
NEVLP. Briefly, rats were fully anesthetized by a ketamine and xylazine injection via intraperitoneal injection then shaved and positioned for lung procurement. The incision was started by cutting the abdomen to open the peritoneum. Heparin (100 IU/kg) was injected via the inferior vena cava (IVC) and allowed to circulate for 10 minutes. Rats were then connected to the ventilator via tracheostomy and exsanguinated by cutting the IVC. The thoracic cavity was opened, and the pulmonary vein and left atrium were cannulated via transapical approach. The heart-lung bloc was removed from the chest cavity with the lung-no-touch technique before being connected to the ex vivo perfusion system as shown in
Lungs were ventilated for 2 hours or until pulmonary artery pressure (PA pressure) exceeded 100 mmHg. Ventilation was performed with ambient air at 60 bpm and tidal volume of 4 mL/kg of rat with a positive end expiratory pressure (PEEP) of 2 cmH2O. The perfusion flow rate was set at 20% of estimated cardiac output (75 mL/kg of rat). Perfusate samples were collected at each timepoint of NEVLP and snap frozen until needed for analysis.
Post-NEVLP Analysis. Lactate dehydrogenase (LDH) released into the perfusate was measured using a LDH cytotoxicity detection kit (Clontech Laboratories, Mountain View, CA) and following the manufacturer's instructions. The right inferior lobe was used for wet to dry ratio determination. The lobe was weighed immediately upon perfusion termination for wet weight, dried at 60° C. for 48 hr., and then weighed again for the dry weight.
Lung Histology. Snap frozen superior lung lobe samples were homogenized in 10 volume equivalents of deionized water. 1 mL of the homogentate was incubated (60 min, 50° C.) with 500 μL of a solution containing 1 M hydrochloric acid (HCl) and 10% trichloratic acid. Samples were then centrifuged at 8,000 g for 15 minutes and 375 μL of supernatant was mixed with 125 μL of a 2% ascorbic acid solution to reduce ferric iron. To quantify, ferrous iron 100 μL of a solution containing 1 g/L of FerroZine™ (ACROS Orgnaics, Geel, Belgium) and 1.5 M sodium acetate was added to the aliquot. Samples were allowed to develop for 30 minutes, after which the absorbance at 562 nm was measured using a BioTek Synergy HTX plate reader.
Paraffin embedded tissue blocks from the lung middle lobe were prepared, and tissue was sectioned (5-micron thickness) by the University of Maryland Baltimore Histology Core. Sections were dewaxed and hydrated in graded ethanol percentages. Heat-mediated antigen retrieval was performed using pH 6.0 citrate buffer. The solution was brought to a boil, and then allowed to cool for 30 minutes. Once cool, sections were incubated at room temperature for 30 minutes with 3% horse serum. Sections were then incubated overnight at 4° C. with an antibody against the Hb α-chain (1:300, Abcam, Ab92492). A biotinylated secondary antibody was used for Hb α-chain staining with sections incubated for 30 minutes (1:300, Invitrogen, 31820). Sections were then incubated for 30 minutes with avidin and biotinylated horseradish peroxidase (VECTASTAIN Elite® ABC Kit, Vector Laboratories), and incubated for 3 minutes with 3,3′-diaminobenzidine and H2O2 (SIGMAFAST™, Sigma). Sections were then counterstained in hematoxylin (Gil no. 2, Fisher). All images were obtained using a Leica DM4-B TL bright field microscope (Leica, Wetzlar, Germany) and captured with LAS X software at 630 times total magnification using a 63 times objective with oil immersion at 22.02 milliseconds of exposure.
Statistical Analysis. All statistical analysis was done using RStudio (Version 1.4.1106, RStudio, Inc., Boston, MA) using a one-way ANOVA to determine significance. Significance was reported to an a value of 0.05. In all figures, an asterisk is used to indicate significance between groups. A pound sign is used to indicate significance within a group compared to its initial value.
PolyhHb Biophysical Properties. The potential ability of the PolyhHb synthesized in this study to function as a perfusate can be assessed by a variety of in vitro parameters. The metHb level (%), average MW, pO2 at which 50% of the Hb or PolyhHb is saturated with O2 (P50), cooperativity coefficient (n), percentage of low MW species in solution (<500 kDa), and auto-oxidation rate constant (kox) are listed in
The percentage of metHb in the PolyhHb produced in this study is marginally lower than other high-MW PolyhHbs. This can likely be explained by the significantly lower kox compared to previous generations of commercial HBOCs. Hemolink (Hemosol Inc.), Hemopure (Biopure Corp.), Oxyglobin (Biopure Corp.), and PolyHeme (Northfield Laboratories) are all commercial polymerized Hbs that failed Phase III clinical trials and each of these HBOCs were reported to have a kox between 0.13-0.26 h−1 which represents an auto-oxidation rate 20-40 times faster than the PolyhHb produced in this example. The lower rate of auto-oxidation means that in an NEVLP circuit, the PolyhHb HBOC formulation will retain its ability to load and offload O2 as intended over a longer period of time, since most of the Hb will exist in the ferrous form (HbFe2+) instead of the oxidized ferric form (HbFe3+, metHb) which cannot bind O2. This is invaluable in NEVLP experiments because prior HBOCs can lose a third of their O2-carrying capacity in as little as an hour during NMP.
The average MW of the pilot scale PolyhHb preparation is 5 times larger than prior generations of commercial HBOCs. Additionally, the PolyhHb produced in this example only contains a small fraction of the low MW species (<500 kDa) compared to previous generation of commercial HBOCs. As mentioned above, the high concentrations of Hb and low MW Hb polymers in prior generations of commercial HBOCs have created a host of disqualifying problems preventing them from being successful in transfusion medicine despite their ability to carry and offload O2 as intended.
The P50 and n are lower for this PolyhHb compared to previous generations of commercial HBOCs, which were synthesized in the low O2-affinity tense quaternary state (T-state). These prior HBOCs readily offloaded O2 to surrounding tissue to support metabolic activity. The indiscriminate offloading of O2 can actually be detrimental to the organ due to a phenomenon called autoregulation, whereby in the presence of high O2 levels, the organism limits O2 consumption in an attempt to keep tissue O2 levels constant. Additionally, too much O2 offloading during perfusion has been shown to generate reactive oxygen species (ROS) which are highly detrimental to graft survivability. Therefore, it is more beneficial to design an HBOC with an O2 affinity similar to or less than that of RBCs in order to support metabolic activity without inducing autoregulation or ROS formation. Hence, the P50 of the PolyhHb described in this study being half that of previous generations of commercial HBOCs may actually prove to be beneficial given that the P50 of human RBCs is about 26 mmHg.
NEVLP. In order for the full potential of NEVLP to be realized, an Oz carrier needs to be present in the perfusate in order to support the metabolic activity of the lung during NMP. The PolyhHb perfusate concentration remains very stable throughout the perfusion (
As shown in
Another shortcoming of previous generations of commercial HBOCs is their propensity for low molecular weight Hb species (<500 kDa) extravasation out of circulation into the tissue space, which has detrimental side-effects that were previously elaborated on. To validate that the PolyhHb is not extravasating out of the NEVLP circuit into the lung, SEC-HPLC was run on the PolyhHb perfusate both before and after NEVLPs. These results are shown in
The organ metrics measured during NEVLP demonstrated equal or superior graft performance in lungs perfused with PolyhHb compared to both the asanguinous control as well as the RBC perfusate. The pO2 of the post-bloc perfusate demonstrates the graft's ability to supply O2 to the system and eventually potentially to circulating blood in a transplant recipient. As such, maintaining an adequate post-bloc pO2 is an important indicator of graft health.
The O2-carrying ability of PolyhHb was likely the driving factor for this behavior. The increase in post-bloc pO2 for the asanguinous control compared to PolyhHb can partially be explained by the increase in oxygenation potential of the perfusate. Having an O2 carrier in solution will inherently facilitate more O2 to be stored and transported in the perfusate compared to only using a colloid. Given that grafts perfused with RBCs also had an O2 carrier in the perfusate, the improvement in PolyhHb lungs over RBC lungs can be attributed to an increase in graft health. Not only did lungs perfused with PolyhHb have a higher post-bloc pO2, but they also did not experience a significant decrease in pO2 over time. This also can only be attributed to an increase in graft health. Taken together, the PolyhHb perfusate sustained the ability of the lungs to provide O2 to the perfusate better than either RBCs or an asanguinous solution.
In addition to facilitating O2 delivery to the perfusate, the other primary function of the lungs is carbon dioxide (CO2) clearance.
Beyond the ability to exchange gases, organ health on the circuit can be evaluated by measuring various physiological parameters during NEVLP. PA pressure is one of the most critical metrics of proper lung function, and a PA pressure elevated above 100 cm H2O is a disqualifying event for continuation of NEVLP. All three perfusates led to an increase in PA pressure by 60 minutes as shown in
The trends displayed by the PA pressure over the course of NEVLP were mimicked by the results of tracking the pulmonary vascular resistance (PVR).
Along with vasoconstriction and systemic hypertension, the cytotoxicity caused by cell-free Hb and low MW Hb polymers (<500 kDa) is another serious side-effect associated with previous generations of PolyhHbs. On the NEVLP circuit, this manifests in real time in the change in lung weight. There are significant increases in lung weight when tissue is damaged leading to edema. The change in lung weight over time for the three perfusates is shown in
Lung tissue iron was quantified as a parameter of Hb degradation following perfusion of explanted tissue. RBC perfused tissue retained visually greater quantities of Hb compared to the control or PolyhHb perfused lungs prior to tissue homogenization and ferrozine assay analysis. Ferrozine functions as a water-soluble Fe2+ chelator with an absorbance at 562 nm and is therefore specific to reaction with unconjugated iron. Based on this data, RBC lung perfusion resulted in an average iron concentration equal to 1.63 μg/g of lung tissue. RBC perfused tissue contained residual iron concentrations that were 74% greater than after control perfusion and 123% greater than after PolyhHb perfusion as shown in
Traditionally, NEVLP has been performed using either a colloid based asanguinous solution or an RBC-based perfusate. Both of these options have shortcomings in preserving graft health and viability during NEVLP. Earlier HBOCs including previous generations of PolyhHbs have caused detrimental side-effects due to the presence of cytotoxic cell-free Hb and other low MW Hb polymers in solution. Improvements to the synthesis and purification of PolyhHb described in this example yield a product that is significantly less likely to elicit the negative side effects observed in previous generations of PolyhHbs. Our PolyhHb demonstrates improved lung oxygenation as well as overall graft health by eliciting less edema, Hb extravasation, iron deposition, and cellular damage. This improved HBOC is a perfusate for NEVLP, which delivers O2 while simultaneously not damaging the lungs.
Polymerized human hemoglobin (PolyhHb) is being studied as a possible red blood cell (RBC) substitute for use in scenarios where blood is not available. While the O2 carrying capacity of PolyhHb makes it appealing as an O2 therapeutic, the commercial PolyhHb PolyHeme® (Northfield Laboratories Inc., Evanston, IL) was never approved for clinical use due to the presence of large quantities of low molecular weight polymeric (LMW) Hb species (<500 kDa), which have been shown to elicit vasoconstriction, systemic hypertension, and oxidative tissue injury in vivo. PolyhHb can by synthesized and purified using a two-stage tangential flow filtration (TFF) purification process to remove almost all undesirable Hb species (>0.2 μm and <500 kDa) in the material, in order to create a product that should be safer for transfusion. To enable future large animal studies and eventual human clinical trials, PolyhHb synthesis and purification processes need to be scaled up to the pilot scale. In this example, we describe pilot scale synthesis and purification of PolyhHb. Characterization of pilot scale PolyhHb showed that PolyhHb could be successfully produced to yield biophysical properties conducive for its use as an RBC substitute. Size exclusion chromatography showed that pilot scale PolyhHb yielded a high MW Hb polymer containing a small percentage of LMW Hb species (<500 kDa). Additionally, the auto-oxidation rate of pilot scale PolyhHb was even lower than that of previous generations of PolyhHb. Taken together, these results demonstrate that PolyhHb has the ability to be seamlessly manufactured at the pilot scale to enable future large animal studies
Allogenic blood transfusion is the most definitive treatment for blood loss. Unfortunately, the American Red Cross is experiencing one of the worst blood shortages in over a decade because of the COVID-19 pandemic. This has caused deferment of life-saving procedures such as organ transplants for some patients. Unfortunately, blood shortages are not limited to pandemics, and can occur seasonally and during other crises such as natural disasters or wars. In these situations, blood availability may not meet the demand for blood, or blood may not be available near to the trauma site. To address these challenges, there needs to be alternatives to blood that can keep an individual alive to bridge the gap between blood loss and definitive blood transfusion.
Given the scarcity of blood, the current standard of care administered by emergency personnel involves transfusion of crystalloid or colloid solutions to maintain mean arterial pressure and blood volume. These emergency transfusions are only meant to alleviate the immediate effects of hypovolemic shock and do not provide oxygen (O2) to tissues, unlike blood. Therefore, hemoglobin (Hb)-based O2 carriers (HBOCs) are actively being developed as an alternative to crystalloid or colloid solutions as a red blood cell (RBC) substitute to replace lost blood volume. HBOCs are able to resuscitate hypoxic tissues, which is facilitated by Hb's innate ability to bind and release oxygen (O2). A solution of acellular (cell-free) Hb cannot be used in transfusion medicine as a suitable HBOC, since the protein is small enough (64 kDa) to extravasate through the pores lining the blood vessel wall into the tissue space. As a result, cell-free Hb scavenges nitric oxide (NO), eliciting vasoconstriction and systemic hypertension. In addition, Hb deposition into the tissue space leads to oxidative tissue injury.
To mitigate the cytotoxic effects of cell-free Hb, HBOCs require modification of Hb via processes such as chemical crosslinking or particle encapsulation to generate a molecule/particle large enough to prevent tissue extravasation, while still maintaining Hb's native O2 transport properties. Polymerized Hb (PolyHb) is the most well-studied HBOC and typically uses glutaraldehyde as a non-specific Hb crosslinker. PolyHeme® (Northfield Laboratories Inc., Evanston, IL), Hemopure® (HbO2 Therapeutics, Souderton, PA), and Hemolink® (Hemosol, Toronto, ON, Canada) are all commercial PolyHbs that failed phase III clinical trials. These HBOCs are mostly composed of low molecular weight (LMW) Hb species (<500 kDa) that pose similar side-effects to cell-free Hb, thus resulting in their failure in clinical trials. Thus, despite their clinical potential, there are still no FDA approved HBOCs for use in transfusion medicine.
To minimize the adverse side-effects observed in PolyHb clinical trials derived by the presence of LMW polymer species, LMW polymer species can be removed from the PolyHb product via tangential flow filtration (TFF). Building on the success of the bench-top scale polymerized human Hb (PolyhHb) synthesis protocol that can produce 15 g of PolyhHb in one batch, this example focuses on scaling up PolyhHb production to the 200-300 g scale that brackets PolyhHb between 500 kDa and 0.2 μm, therefore removing the majority of LMW polymeric species and cell-free Hb that could elicit vasoconstriction, systemic hypertension, and oxidative tissue injury; as well as, any high molecular weight (HMW) species over 0.2 μm in size that could signal the reticuloendothelial system (RES). Therefore, PolyhHb scaleup is essential to enable safety and efficacy studies in large animals before subsequent evaluation in humans
In this example, PolyhHb scaleup followed a protocol mirroring the PolyhHb bench-top synthesis parameters. The scale of production was increased from the 1.5 L bench-top scale reactor system to a 30 L pilot-scale reactor system. The use of TFF modules enabled scalable purification of PolyhHb. Therefore, this example describes the synthesis and characterization of pilot scale PolyhHb produced in both the low and moderate O2 affinity state and compares the pilot scale PolyhHb product to previously published bench-top scale PolyhHb product.
Materials. Sodium dithionite (Na2S2O4), glutaraldehyde (C5H8O2) (70 wt %), sodium cyanoborohydride (NaCNBH3), sodium lactate (NaC3HO3), N-acetyl-L-cysteine (NALC, C5H9NO3S) and calcium chloride dihydrate (CaCl2)·2H2O) were purchased from Sigma Aldrich (St. Louis, MO). Sodium chloride (NaCl), potassium chloride (KCl), sodium phosphate monobasic (NaH2PO4), sodium phosphate dibasic (Na2HPO4), sodium hydroxide (NaOH), and 0.2 μm Titan3 sterile filters were purchased from Fisher Scientific (Pittsburgh, PA). Hollow fiber tangential flow filter (TFF) modules N02-P500-05-N (polysulfone (PS), 500 kDa pore size), N02-S20U-05-N (polyethersulfone (PES), 0.2 μM pore size), and N02-E65U-07-N (polyethersulfone (PES), 0.65 μM pore size) were purchased from Repligen (Rancho Dominguez, CA). The Liqui-Cel™ EXF Series G420 Membrane Contactor was purchased from 3M (St. Paul, MN). A minicentrifuge (50-090-100, with a working speed of 6,000 rpm and maximum speed of 6,600 rpm) was obtained from Fisher Scientific. Expired human RBC units were acquired from Transfusion Services at the Wexner Medical Center (Columbus, OH), Canadian Blood Services (Ottawa, Canada), and Zen-Bio Inc. (Durham, NC).
RBC Washing Process. 180 L of 0.9 wt % saline was prepared the night prior to the start of the RBC washing process. To prepare the saline, a vessel was first filled with 176 L of deionized (DI) water followed by the addition of 4 L of 40.5 wt % saline. The vessel was then stirred for 30 minutes and stored at 4° C. For each batch of pooled RBCs, a total of 18 RBC units were used in the washing process. Initially, the system vessel was primed with 0.9 wt % saline before addition of the RBC units. All RBC units were gently inverted and massaged to properly mix blood bag contents and then transferred into a 20 L Nalgene container in a biosafety cabinet and an initial pooled sample of RBCs was taken for analysis. The vessel was then transferred into a chromatography refrigerator, where it was maintained at 4° C. for the entirety of the RBC washing process. The vessel containing pooled RBCs was diluted with 0.9 wt % saline to obtain a 22±2% hematocrit (HCT) to reduce RBC solution viscosity and standardize the HCT between replicates. Once the HCT was confirmed, the permeate line in the TFF RBC washing system (
HCT Quantification. RBC solution HCT was determined by loading 65 μL of the retentate sample into a 75 mm mylar wrapped capillary tube (Drummond, Broomall, PA) followed by centrifugation in a Sorvall Legend micro 17 microcentrifuge (Fisher Scientific, Waltham, MA) at 17 g for 5 minutes. The HCT was determined using a standardized HCT calibration curve.
RBC Cell Concentration Quantification. The cell concentration for retentate samples taken during the RBC washing process was measured using a Multisizer 4e Coulter Counter (Beckman Life Sciences, Indianapolis, IN). RBC samples were diluted 100-followed by the addition of 100 μL of the diluted sample into 20 mL of filtered Isoton solution (Beckman Life Sciences) for cell concentration analysis in the Coulter Counter.
Hb Quantification. The cell-free Hb concentration in both permeate and retentate samples from the RBC washing process was measured using UV-visible absorbance spectrometry on a diode array spectrophotometer HP 8452A (Olis, Bogart, GA). Retentate samples were first centrifuged on a minicentrifuge (Fisher Scientific, Waltham, MA) at 6,000 RPM for 2 minutes, followed by removal of the supernatant for spectrometry measurements. The supernatant Hb concentration was analyzed by the cyanomethemoglobin method.
hHb Purification. Post wash, RBCs were lysed with phosphate buffer (PB) (3.75 mM, pH 7.4) for 1 hour at 4° C. with constant stirring. Lysed RBC membrane fragments and large aggregates were removed using a 500 kDa TFF module (
hHb Polymerization. The next day, the hHb solution in the reactor was brought up to physiological temperature (37° C.) using a thermal jacket wrapped around the reactor. The pO2 of the system was checked periodically using a RAPIDLab 248 blood gas analyzer (Siemens, Munich, Germany) with the goal of achieving a pO2 value of 0.0±2.0 mmHg before hHb polymerization in order to achieve 100% tense quaternary state (T-state) PolyhHb. To ensure complete hHb deoxygenation, sodium dithionite was added in 1 g charges until a pO2 of 0 mm Hg was achieved.
Once the hHb in the reactor was fully deoxygenated, the gas contactor was temporarily bypassed to begin hHb polymerization as shown in
PolyhHb Separation and Purification. Following the hHb polymerization process, the PolyhHb solution was pumped from the reactor to a stage 1 PolyhHb purification vessel into a chromatography refrigerator as shown in
Oxygen Equilibrium Curve. A Hemox Analyzer (TCS Scientific Corp., New Hope, PA) was used to measure the oxygen equilibrium curve (OEC) of hHb and PolyhHb. The OEC was fit to the Hill equation to regress the cooperativity coefficient (nH) and partial pressure of O2 at which 50% of the hHb or PolyhHb was saturated with O2 (P50).
Polymer MW and Size. The MW distribution of hHb and PolyhHb was measured using size exclusion chromatography (SEC) on a high-pressure liquid chromatography (HPLC) system. The separation was performed using an Ultimate 3000 system with an SEC-1000 column (ThermoFisher Scientific, Waltham, MA). The absorbance was measured at 412 nm to monitor the Soret peak characteristic of hHb. The hydrodynamic diameter of hHb and PolyhHb was measured using a Zetasizer Nano Dynamic Light Scattering (DLS) Spectrometer (Malvern Instruments, Worcestershire, UK).
Auto-Oxidation Kinetics. UV-visible absorbance spectrometry was used to measure the auto-oxidation kinetics of hHb and PolyhHb. The hHb/PolyhHb solution was diluted in PB to 12.5 g/L to simulate the average concentration of product in the systemic circulation following transfusion. The solution was monitored for 24 hours, while maintaining a physiological temperature (37° C.) and pH (7.40). The absorbance at 630 nm was monitored over a 24-hour period. Analyzing the kinetics for hHb and PolyhHb, the fraction of heme in the ferrous state as a function of time was analyzed assuming first-order rate kinetics to regress the auto-oxidation rate constant (kox) in h−1.
Stopped-Flow Kinetics. A SX-20 micro-volume stopped-flow apparatus (Applied Photophysics, Leatherhead, UK) was used to measure the O2 offloading kinetics and the haptoglobin (Hp) binding kinetics of hHb/PolyhHb. For the O2 offloading kinetics, the absorbance at 437.5 nm was measured when a 12.5 μM (heme-basis) solution of oxygenated hHb/PolyhHb in PBS was rapidly mixed with a 1.5 M solution of sodium dithionite in degassed PBS. The average of the kinetic traces was fit to an exponential function using the Applied Photophysics software to regress the first-order Oz dissociation rate constant (kO2,off) in s−1.
Hp binding kinetics was monitored by measuring the fluorescence change at 285 nm when Hp binds to hHb/PolyhHb. A 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM solution of hHb/PolyhHb sample in PBS was rapidly mixed with a 0.25 μM solution of Hp. The average of the kinetic traces was fit to an exponential function and a pseudo-first order rate constant was regressed at each concentration. The pseudo-first order rate constants were plotted against hHb/PolyhHb concentration and fit to a linear function to regress the second-order Hp binding rate constant (kHp-Hb) in μM−1s−1.
Computational Methods. The fluid dynamics and mixing performance in the PolyhHb reactor vessel was modeled as a continuous stirred tank reactor (CSTR) and evaluated via Comsol Multiphysics (Version 5.3a, Comsol, Inc., Burlington, MA). In this example, the Rotating Machinery module was used to solve for the turbulent flow properties in the reactor. The simulated results were presented at steady state. Constant circulation of PolyhHb solution was maintained in between the inlet and outlet as shown in
Statistical Analysis. All data is presented as the mean±standard deviation. Statistical analysis for data collected in the study was performed on RStudio using t-tests. For all tests, p<0.05 was considered statistically significant.
RBC Washing Process. The RBC washing process successfully removed the majority of cell-free Hb from the expired pooled RBC units, while maintaining the more mechanically resilient RBCs for use in the subsequent hHb purification step of the pilot scale hHb polymerization process.
The HCT is defined as the volume fraction of packed RBCs present in the RBC solution and is directly related to the concentration of RBCs in a well-mixed solution. In
The concentration of intact RBCs was then used to determine whether washing RBCs using TFF and subjecting RBCs to shear forces during the washing process is potentially detrimental to cell integrity and causes more unintentional hemolysis. No significant drop in RBC concentration was observed (p=0.999) between the first and sixth diacycles as shown in
PolyhHb Quantification.
All pilot scale PolyhHb batches produced in this example were concentrated to at least 100 g/L. This is in line with the protocol for PolyhHb production used in our lab for bench-top scale PolyhHb production. Additionally, the average yield for pilot scale PolyhHb batches was 42%, which is identical to the yield of previous bench-top scale 30:1 T-state PolyhHb batches produced in our lab. In summary, the scaled-up PolyhHb production process does not result in a loss in yield compared to the bench-top scale process. On average, the pilot scale process produced 201 g of PolyhHb—16.3× more compared to the benchtop scale—with moderate P50 batches producing 18.6× as more compared to the bench-top scale. This exponential increase in PolyhHb production in a single batch helps to both validate the success of the scaled up process, and gives promise to the use of the next-generation PolyhHb in future large animal studies.
The most significant difference in protein quantification between pilot scale and bench-top scale PolyhHb process is the percentage of metHb (i.e., metHb level) in the PolyhHb product. The PolyhHb produced in this example included on average 3.2% metHb, which is significantly lower compared to the 30:1 T-state PolyhHb produced previously at the bench-top scale (p=0.0014). Upon analysis of the two subgroups, this trend appears to be due to the lower metHb level of the moderate P50 pilot scale batches. The five moderate P50 pilot scale batches have a significantly lower metHb level than the three fully T-state batches produced at the pilot scale (p=0.015). This is likely due to two separate factors. First, the pilot-scale PolyhHb synthesis protocol had the purified hHb added directly to the reactor after RBC lysis and filtration through a 500 kDa TFF module. In the bench-top scale synthesis of PolyhHb, the hHb was first filtered through a 500 kDa TFF module and then further concentrated over a 50 kDa TFF module for subsequent storage at −80° C. before use. The final concentration step on the 50 kDa TFF module removes excess buffer as well as any proteins<50 kDa, including superoxide dismutase (SOD) which is critical in preventing Hb oxidation. Co-polymerizing SOD with PolyhHb can limit the oxidation of PolyhHb, and a similar mechanism is likely at play here to reduce the metHb level of all pilot-scale batches compared to bench-top scale PolyhHb. Second, the low O2 affinity (i.e., high P50) of T-state PolyhHb leads to a higher rate of metHb formation compared to species with lower P50s. This is discussed more in later sections, but for now it would help explain the difference in metHb level between moderate P50 and fully T-state PolyhHb pilot scale batches.
O2 Equilibria. The PolyhHb produced in this study fell into two distinct groups: T-state PolyhHb and a moderate O2 affinity PolyhHb, as shown in
T-state PolyhHb is traditionally used for hemorrhagic shock resuscitation and other therapeutic applications. The low O2 affinity of T-state PolyhHb results in the ability of O2 to be readily offloaded to surrounding tissue to reduce tissue hypoxia. More recent studies have shown that the indiscriminate offloading of O2 by T-state PolyhHb may not be as beneficial as originally thought. For instance, there is an increase in reactive oxygen species (ROS) generation under hyperoxic conditions. This triggers a cascade of inflammatory response pathways, leading to increased cellular damage. Additionally, the principle of autoregulation—whereby in the presence of an O2-rich environment, cells will automatically reduce O2 uptake—has been shown to limit the efficacy of Oz carriers with P50s significantly higher than RBCs (26 mm Hg). It is for these reasons that moderate P50 PolyhHbs show promise as viable RBC substitutes with reduced potential for ROS generation and autoregulatory response.
Size Distribution. The size of the PolyhHb synthesized in this example was measured using both DLS and SEC-HPLC and the SEC-HPLC results are shown in
The size difference between pilot scale and bench-top scale batches is likely due to mixing differences between the pilot-scale reactor system compared to the bench-top scale reactor system. Bench-top PolyHb syntheses used a stir bar as the agitator for mixing and the reactor has no baffles, while the pilotscale reactor is a baffled CSTR with an impeller, so it inherently generates more uniform mixing. Ostwald ripening is a well-described process whereby particles in solution are more likely to interact with larger particles over smaller ones. In a reactor set-up where mixing is not as uniform as in a CSTR, it is to be expected that Ostwald ripening will occur, resulting in a more bimodal distribution of Hb polymers. This is opposed to a perfectly mixed system, where there should be a more even distribution of polymer sizes, which would explain the smaller MW of the PolyhHb produced at the pilot-scale compared to bench-top scale. The optimal mixing of the pilot scale reactor is confirmed in the computational fluid modeling described in a later section.
Auto-Oxidation. Similar to the discussion regarding PolyhHb oxygen affinity, the auto-oxidation rate constant of pilot scale PolyhHbs produced in this study fell into two distinct groups. The auto-oxidation rate constant and oxygen affinity appear to be mildly correlated in
The kox for both pilot scale groups in this study were lower compared to other HBOCs in the literature. Unmodified hHb has been shown to have a kox of 0.043 h−1, which is 2× higher than the pilot scale T-state PolyhHb group and ˜9× higher than the moderate P50 pilot scale PolyhHb group. Furthermore, the commercial PolyhHb PolyHeme® (Northfield Labs, Evanston, IL) was shown to have a kox of 0.26 h−1, which is 10-fold and 45-fold higher than the pilot scale PolyhHbs synthesized in this example. The low concentration of LMW Hb polymers (<500 kDa) present in the pilot scale materials is thought to be the reason for the lower auto-oxidation rate constant compared to prior generations of HBOCs. A positive correlation between the concentration exists between low MW species in the PolyhHb solution and the auto-oxidation rate constant. All PolyhHbs synthesized in this example had less than one tenth of the LMW species (<500 kDa) that were present in previous commercial HBOCs, so it makes sense for the auto-oxidation rate constant to also be an order of magnitude lower as well.
The lower auto-oxidation rate constant is important for two major reasons. The first is that a lower rate of auto-oxidation will correspond to a greater amount of ferrous PolyhHb being capable of carrying and offloading O2 while in circulation. When ferrous PolyhHb oxidizes, it converts into the ferric state (i.e., metHb), and is no longer able to bind and release O2. For HBOCs with high kox values, the O2-carrying capacity of the solution drops off rapidly as the ferrous HBOC converts to metHb and therefore loses functionality over a very short time period. In contrast, low kox HBOCs, such as the pilot scale PolyhHb produced in this example, do not have this problem and remain functional for a much longer period of time, making them significantly more viable as an O2 therapeutic. Additionally, the Fe3+ in metHb is a strong oxidizer, which gives it the ability to produce ROS that are cytotoxic. This further supports the advantages of the low kox HBOCs produced in this study as these molecules will be less likely to catalyze ROS production and elicit cellular toxicity.
Deoxygenation Kinetics.
Hp Binding Kinetics. The plot of the pseudo-first order rate constant as a function of Hb concentration for Hp binding (kHp-Hb) to the PolyhHbs produced in this study is shown in
Because Hp is the native hHb clearance protein, a high level of Hp-binding would imply the presence of large quantities of the cytotoxic species. The elimination of such species would therefore lead to a decrease in kHp-Hb. This can be taken to mean that kHp-Hb is inversely proportional to the safety of the PolyhHb if it were to be transfused, since a low Hp binding constant would imply less cytotoxic potential. The kHp-Hb of the PolyhHb produced in this example is trivial and 100-fold less than that of unmodified hHb, alluding to its safety as a potential RBC substitute.
Mixing Modeling.
This example establishes that a high MW next-generation PolyhHb with limited LMW species can be produced at the pilot scale (200-300 g of PolyhHb product). Bench-top and pilot-scale PolyhHb processes exhibited nearly identical protein yields. Additionally, pilot-scale PolyhHb produced in this example was shown to have a lower metHb level and auto-oxidation rate constant compared to bench-top scale PolyhHb. We also demonstrated that PolyhHb could be produced in either the low O2 affinity T-state or at a moderate P50 making it possible to produce an HBOC that can readily offload O2 without possible concerns over autoregulation. Optimized mixing parameters most likely lead to the pilot-scale PolyhHb being smaller compared to previous bench-top scale PolyhHbs. Furthermore, the pilot scale PolyhHbs produced in this exhibited a low Hp binding rate constant, further indicating the potential the safety of the material.
In this example, we produced and characterized 12 batches of tense (T) quaternary state polymerized human hemoglobin (PolyhHb) of varying size. The PolyhHbs were then separated into 4 molecular weight (MW) brackets using tangential flow filtration (TFF): 50-300 kDa (PolyB1), 100-500 kDa (PolyB2), 500-750 kDa (PolyB3), and 750 kDa-0.2 μm (PolyB4). Each PolyhHb batch was synthesized using the chemical cross-linker glutaraldehyde (GA) at various cross-link densities to optimize product yield within the designated MW bracket. Specifically, PolyB1 was synthesized at a 10:1 molar ratio of glutaraldehyde to human hemoglobin (hHb) (GA:hHb) and bracketed using a 50 kDa TFF filter and a 300 kDa modified polyethersulfone (mPES) TFF filter. PolyB2 was synthesized at a 25:1 molar ratio of GA:hHb and bracketed using a 100 kDa TFF filter and a 500 kDa mPES TFF filter. Similarly, PolyB3 was produced at a 26.5:1 molar ratio of GA:hHb and bracketed using a 500 kDa polysulfone (PS) TFF filter and a 750 kDa mPES TFF filter. Finally, PolyB4 was synthesized at a 30:1 molar ratio of GA:hHb and the resultant material was bracketed using a 750 kDa filter and a 0.2 μm mPES TFF filter. The bracketed materials were subject to diafiltration with a modified lactated Ringer's solution (pH=7.40) and concentrated to ≥10 g/dL for subsequent use in animal models. The biophysical properties such as the O2 affinity, O2 offloading kinetics, haptoglobin (Hp) binding kinetics, total heme concentration, methemoglobin concentration, total protein concentration, effective diameter, and size/MW distribution of each batch were analyzed after preparation.
Our primary goal during the synthesis and production of PolyhHb was to determine the optimal cross-linking density which yielded the target MW bracket with low polydispersity index (PDI).
The P50 (partial pressure of O2 at which 50% of the hHb/PolyhHb is saturated with O2) was measured using a Blood Oxygen Binding System. The P50 for the fractions PolyB1, B2, B3, and B4 were 40.53 mm Hg, 34.78 mm Hg, 45.06 mm Hg, and 45.11 mm Hg, respectively. The P50 for the PolyhHb was greater than that of unmodified hHb (12 mm Hg). This increase in P50 is consistent with locking the heme in the tense (T) quaternary state conformation prior to synthesis. The resulting oxygen equilibrium curves for each fraction bracket (B1-B4) and free hHb are shown in
The elution time of bracketed PolyhHb fractions was measured using size exclusion chromatography (SEC). This data is shown in
Other advantages which are obvious, and which are inherent to the invention will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
This application claims benefit of U.S. Provisional Application No. 63/314,843, filed Feb. 28, 2022, and U.S. Provisional Application No. 63/327,979, filed Apr. 6, 2022, each of which is hereby incorporated herein by reference in its entirety.
This invention was made with government support under Grant No. W81XWH-18-1-0059 awarded by the Department of Defense, and Grant Nos. R01HL126945, R01HL138116, R01HL156526, and R01EB021926 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/041743 | 8/26/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63327979 | Apr 2022 | US | |
| 63314843 | Feb 2022 | US |
