Method for producing high molecular weight heparin compounds

Abstract

SOLUTION: A method for producing a high molecular weight heparin (HMWH) compound is disclosed. The method includes dissolving heparin to form a heparin solution and fractionating the heparin solution by tangential flow filtration (TFF) using a membrane having a molecular weight cut-off (MWCO) between about 8 kDa and about 12 kDa. TFF produces a retentate that contains fractionated heparin, i.e., high molecular weight heparin compounds, having a weight average molecular weight of about 20 kDa or more. A significant proportion of the heparin chains in the fractionated heparin have a high molecular weight, e.g., 50% or more of the heparin chains have a molecular weight of 20 kDa or more.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 187,624, entitled "Methods of Manufacturing a High Molecular Weight Heparin Compound," filed May 12, 2021, which is incorporated by reference in its entirety. Summary of the Invention

[0002] The present disclosure generally relates to a method for producing high molecular weight heparin compounds.The disclosed subject matter is applied to the production of compounds and / or compositions having the same for imaging, diagnosis, monitoring, and / or treatment of various conditions.For example, the compounds produced by the methods disclosed herein are useful for imaging, diagnosis, monitoring, and / or treatment of eosinophil-associated inflammation and eosinophil-associated diseases, such as eosinophilic esophagitis and eosinophil-associated eye diseases.

[0003] Historically, high molecular weight heparin has been avoided in the medical field in favor of low molecular weight heparin. Heparin is a polysaccharide that is inherently heterogeneous in polymer chain length to contain heparin chains of various molecular weights. Typically, heparin is depolymerized and fractionated to reduce the molecular weight, and low molecular weight heparin is administered to patients. It is suspected that the administration of large amounts of high molecular weight heparin chains via common routes of administration (i.e., intravenous or subcutaneous) increases the incidence of heparin-induced thrombocytopenia (HIT). HIT is a complication resulting from exposure to heparin that can lead to limb- and life-threatening thrombotic complications. In HIT, when heparin binds to platelet factor 4 (PF4), the immune system forms antibodies against heparin. The antibodies then form complexes with heparin / PF4 that bind to and activate platelets, resulting in the formation of clots and a decrease in platelet count. HIT can lead to venous thromboembolism and, in some cases, arterial thrombosis (referred to as HITT). Due to the risk of HIT suspected to be associated with high molecular weight heparin chains, low molecular weight heparins are preferred in medical clinical applications.

[0004] However, recent advances suggest that high molecular weight heparin may be useful in certain applications, for example because it is highly localized to eosinophils, a type of white blood cell that fights multicellular parasites and certain infections in vertebrates.

[0005] Normally, eosinophils are present in the bloodstream, lower gastrointestinal tract, and lymphatic system, but pathologically, they can infiltrate many more organs and sites, causing inflammation and various diseases and conditions. The distinguishing feature of eosinophils is their prominent cationic protein-containing granules. These granules consist of a central electron-dense nucleus and an electron radiolucent matrix. The nucleus primarily contains major basic protein 1 (MBP-1 or eMBP-1), while the matrix contains eosinophil peroxidase (EPO), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Granules also contain major basic protein 2 (MBP-2 or eMBP-2) in the nucleus and / or matrix. Upon degranulation, eosinophils release these proteins into the surrounding tissues, stimulating the release of histamine and causing inflammation. Studies have shown that MBP-1 is toxic to mammalian cells, bacteria, and certain parasites, and is deposited at sites of inflammation in many eosinophil-associated diseases, such as organ dysfunction (e.g., eosinophilic esophagitis). Heparin binds to MBP-1 and is effective in neutralizing the cytotoxic effects of MBP-1 in a dose-related manner. Furthermore, the binding strength of heparin to MBP-1 increases with molecular weight. Thus, high molecular weight heparin is useful for imaging, diagnosing, monitoring, and / or treating eosinophil-associated inflammation and / or eosinophil-associated pathologies.

[0006] Despite these advances, several challenges have hindered the production of high molecular weight heparin. The inherent lack of uniformity in chain length of heparin poses great difficulties in isolating chains of specific molecular weight. Due to the interest of low molecular weight heparins in the medical field, methods for producing low molecular weight heparin compounds have been successful. However, there has not been similar progress in developing methods to fractionate high molecular weight heparins.

[0007] Furthermore, even when heparin compounds having a target average molecular weight (e.g., low molecular weight heparins) were successfully produced, the molecular weights still varied widely and the compound may not have a high percentage of heparin chains within the target molecular weight range.

[0008] Therefore, it would be useful to have a method for producing a high molecular weight heparin compound that has a high average molecular weight and a high proportion of heparin chains having high molecular weight.

[0009] An embodiment of the present invention relates to a method for producing fractionated heparin, the method comprising: dissolving heparin in a solvent to form a heparin solution; and fractionating the heparin solution by tangential flow filtration using a fractionation membrane having a molecular weight cutoff of between about 8 kDa and about 12 kDa, thereby obtaining a fractionated heparin having a weight average molecular weight of about 20 kDa or more, wherein at least 50% of the heparin chains in the fractionated heparin have a molecular weight of 20 kDa or more.

[0010] An additional embodiment of the invention relates to a method for producing high molecular weight (HMW) heparin, the method comprising the steps of: dissolving a heparin salt in a salt solution to form a heparin solution; sterilizing the heparin solution by filtration through a sterile membrane having a pore size of about 0.2 μm, thereby obtaining a sterile heparin solution; fractionating the sterile heparin solution by tangential flow filtration using a fractionation membrane having a pore size of about 5 nm, thereby obtaining a fractionated heparin having a weight average molecular weight of about 20 kDa or greater, wherein at least 50% of the heparin chains in the fractionated heparin have a molecular weight of 20 kDa or greater; desalting the fractionated heparin by tangential flow filtration using a desalting membrane having a pore size of about 3 nm, thereby obtaining desalted heparin; and drying the desalted heparin by lyophilization to obtain the HMW heparin. [Brief description of the drawings]

[0011] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles, features and characteristics of the invention.

[0012] [Figure 1] FIG. 2 is a flow diagram of an exemplary method for producing a high molecular weight heparin compound, according to an embodiment. [Diagram 2] 2A and 2B are sensorgrams of signal responses of fractionated heparin samples (ie, analytes) binding to eMBP1 (ie, ligand), according to an embodiment. [Diagram 3] 1 is a plot graph of complex half-life versus molecular weight for seven different molecular weight heparin samples at a concentration of 100 ng / mL that bind to eMBP1 at a density of 1200 RU, according to an embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present disclosure is not limited to the specific systems, devices, and methods described. The terminology used herein is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope. Aspects of the present disclosure may be embodied in many different forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.

[0014] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. With respect to the use of virtually any plural and / or singular term herein, one of ordinary skill in the art can interpret the plural to the singular and / or the singular to the plural as appropriate to the context and / or application. Various singular / plural permutations may be expressly provided herein for clarity.

[0015] As will be understood by those of skill in the art, for all purposes, including in terms of providing a written description, all ranges disclosed herein are intended to encompass each intervening value between the upper and lower limits of the range, and any other stated or intervening value within that stated range. Also, all ranges disclosed herein encompass all possible subranges and combinations of subranges. Any stated range can be readily recognized as fully descriptive and allowing for the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, upper third, etc. Also, as will be understood by those of skill in the art, all phrases such as "up to," "at least," etc. refer to ranges that are inclusive of the number recited and can then be broken down into subranges as described above. Finally, as will be understood by those of skill in the art, ranges include individual members. Thus, for example, a group having 1 to 3 cells refers to groups having 1, 2, or 3 cells, and ranges of values ​​from 1 cell to 3 cells. Similarly, a group having 1 to 5 cells refers to groups having 1, 2, 3, 4, 5 cells, and groups having 1 to 5 cells, etc.

[0016] Further, even when a particular number is explicitly recited, one of ordinary skill in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the mere recitation of "two repeats" without other modifiers means at least two repeats, or more than two repeats). Furthermore, when an idiom similar to "at least one of A, B, and C, etc." is used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the idiom (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When phrases similar to "at least one of A, B, and C, etc." are used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the phrase (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). One of ordinary skill in the art will further understand that virtually any conjunctive word and / or phrase presenting two or more alternative terms, whether in the description, sample embodiments, or drawings, should be taken to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B," or "A and B."

[0017] Additionally, where features of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of individual members or subgroups of members of the Markush group.

[0018] All percentages, parts and ratios are based on the total weight of the compound and all measurements were made at about 25° C. unless otherwise noted.

[0019] The term "about" as used herein refers to variations in numerical quantities that occur, for example, due to real-world measurement or handling procedures, due to inadvertent errors in these procedures, due to differences in the manufacture, source, or purity of the composition or reagent, and the like. In general, the term "about" as used herein means greater or smaller than the stated value or range of values ​​by 1 / 10 of the stated value, e.g., ±10%. The term "about" also refers to variations that would be recognized as equivalent by one of ordinary skill in the art, unless they encompass known values ​​implemented by the prior art. Each value or range of values ​​preceded by the term "about" is also intended to encompass embodiments of the stated absolute value or range of values. Quantitative values ​​referred to in this disclosure, whether modified by the term "about", include equivalents to the stated value, e.g., variations in numerical quantities of such values ​​that may occur but would be recognized as equivalents by one of ordinary skill in the art. If the context of this disclosure indicates otherwise or is inconsistent with such an interpretation, the above interpretation can be modified as would be readily apparent to one of ordinary skill in the art. For example, in a list of numerical values ​​such as "about 49, about 50, about 55," "about 50" means less than half the interval between the preceding and succeeding values, e.g., a range from greater than 49.5 to less than 52.5. Furthermore, expressions of values ​​"less than about" or "greater than about" should be understood in light of the definition of the term "about" provided herein.

[0020] In general, it will be understood by those of skill in the art that the terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," and the term "includes" should be interpreted as "including, but not limited to"). Furthermore, the transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, non-reproduced elements or method steps. Although various compositions, methods, and devices are described with the term "comprising" various components or steps (which should be interpreted as meaning "including, but not limited to"), the compositions, methods, and devices can also "consist essentially of" or "consist of" various components and steps, and such terms should be interpreted as defining essentially closed-member groups. In contrast, the transitional term "consisting of" excludes elements, steps, or ingredients not specified in the claim. The transitional term "consist essentially of" limits the scope of the claim to specified materials or steps that "do not materially affect the basic and novel characteristics" of the claimed invention.

[0021] As used herein, the term "therapeutic" refers to an agent utilized to treat, combat, ameliorate, or ameliorate an undesirable condition or disease in a patient. In part, embodiments of the present invention are directed to the treatment of eosinophil-associated inflammation and / or other eosinophil-associated diseases and conditions.

[0022] The term "effective amount" as used herein refers to an amount of a compound that, when administered to a subject, is adequate to accomplish the compound's purpose, including imaging a subject's tissue, diagnosing a subject's disorder, and / or monitoring a subject's symptoms or disorder. The actual amount that constitutes an "effective amount" will vary depending on many conditions, including, but not limited to, the severity of the disorder, the size and health of the patient, the imaging modality, the method of diagnosis, the method of monitoring, and the route of administration. A skilled physician can easily determine the appropriate amount using methods known in the medical arts.

[0023] As used herein, the term "therapeutically effective amount" refers to an amount of a compound that, when administered to a subject, can alleviate symptoms of the subject's disorder or enhance the texture, appearance, color, feel, or hydration of the intended tissue treatment area. The actual amount that constitutes a "therapeutically effective amount" will vary depending on many conditions, including, but not limited to, the severity of the disorder, the size and health of the patient, and the route of administration. A skilled physician can easily determine the appropriate amount using methods known in the medical arts.

[0024] The phrase "pharmacologically acceptable" is used herein to refer to a subject agent / compound, salt, composition, dosage form, etc., that is suitable for use in contact with the tissues of humans and / or other mammals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. In some embodiments, pharma- ceutically acceptable means approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in mammals (e.g., animals), more particularly humans.

[0025] The terms "patient" and "subject" are intended to be interchangeable and mean any living organism to be treated with the compositions of the present invention. Thus, the terms "patient" and "subject" include, but are not limited to, any non-human mammal, primate, or human. In some embodiments, a "patient" or "subject" is a mammal, such as a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse, primate, or human. In some embodiments, the patient or subject is an adult, child, or infant. In some embodiments, the patient or subject is a human.

[0026] Where this disclosure refers to the term "doctor" and additional terms for various medical professionals with a particular job type or role, nothing in this disclosure is intended to limit the disclosure to a particular job type or function. A doctor or medical professional includes any physician, nurse, medical professional, or technician. Any of these terms or jobs may be used interchangeably with users of the systems disclosed herein unless expressly distinguished otherwise. For example, a reference to a doctor may, in some embodiments, also apply to a technician, nurse, or other medical provider.

[0027] "Tissue" refers to an association of similarly specialized cells that are united to perform a particular function.

[0028] In this disclosure, the term "disorder" means, and is used interchangeably with, the terms disease, condition, or illness, unless otherwise specified.

[0029] As used herein, the terms "administer," "administering," or "administration" refer to the direct administration of a compound (also called a substance of interest), a pharma- ceutically acceptable salt of a compound (substance of interest), or a composition to a subject, either by the subject or by a health care provider.

[0030] As used herein, the terms "treat", "treated" or "treating" refer to both therapeutic treatments, the purpose of which is to reduce the frequency or delay the onset of symptoms of a medical condition, or otherwise obtain beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, reversal, reduction or alleviation of symptoms of a pathology; reduction in the extent of a pathology, disorder or disease; stabilization (i.e., not worsening) of the condition, disorder or disease state; delay in onset or slowing of progression of the condition, disorder or disease; improvement in the condition of the condition, disorder or disease; and remission (whether partial or total), whether detectable or undetectable, or enhancement or amelioration of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without undue side effects. Treatment also includes prolonging survival compared to the expected survival if not receiving treatment.

[0031] As used herein, the terms "diagnose," "diagnosing," or "diagnosis" refer to a process of identifying the presence and / or nature of a disease, condition, or other physiological condition in a subject from its characteristics, signs, and symptoms. Diagnosis includes a statement or conclusion related to a disease, condition, or other physiological condition in a subject based on such a process.

[0032] The term "inhibiting" includes administering a composition of the invention to prevent the onset of the symptoms, alleviate the symptoms, relieve the symptoms, slow or reduce the progression of the disease and / or its symptoms, or eliminate the disease, condition, or disorder.

[0033] In some embodiments, the methods and compositions disclosed herein can be utilized with or on a subject in need of such testing, diagnosis, monitoring, and / or treatment, also referred to as "in need thereof." As used herein, the phrase "in need thereof" means that the subject has been identified as having a need for a particular method or treatment, or has been identified as having a condition, and that a method (e.g., imaging a tissue, diagnosing a condition, monitoring a condition) or treatment has been utilized with or on the subject for that particular purpose.

[0034] The compositions produced by the methods of the present invention can be administered in a conventional manner by any route where they are active. Administration can be systemic, topical, or oral. For example, administration can be, but is not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, buccal, or ocular, or intravaginal, inhalation, depot injection, or implant. Thus, the mode of administration (either alone or in combination with other medicaments) can be, but is not limited to, sublingual, injectable (including short-acting, depot, implant, and pellet forms injected subcutaneously or intramuscularly), topical (including nasal drops, ointments, or creams for application to the skin), and / or transdermal, such as vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, patches, creams, etc.

[0035] The specific administration method varies depending on the indication or purpose. The selection of the specific administration route and dosage is adjusted or titrated by the clinician according to known methods to obtain optimal clinical response. The amount of the compound administered is an effective amount. The dosage depends on the characteristics of the subject to be treated, such as the specific animal to be treated, age, weight, health condition, type of concurrent treatment if any, and frequency of treatment, and can be easily determined by those skilled in the art (e.g., clinician).

[0036] For oral administration, the composition can be easily formulated by combining the high purity high molecular weight heparin with pharma- ceutically acceptable carriers known in the art according to the methods herein. Such carriers allow the compounds of the present invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient to be imaged, diagnosed, and / or treated. Oral pharmaceutical preparations can be prepared by adding solid excipients, optionally grinding the resulting mixture, optionally adding suitable auxiliary agents, and then processing the mixture of granules to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, sugar fillers including lactose, sucrose, mannitol, sorbitol, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, cellulose preparations such as polyvinylpyrrolidone (PVP), etc. If desired, disintegrating agents can be added, such as, but not limited to, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.

[0037] Orally usable pharmaceutical compositions include, but are not limited to, push-fit capsules made of gelatin, and sealed capsules made of gelatin and plasticizers (such as glycerol or sorbitol). Push-fit capsules can contain the active ingredient mixed with a filler, such as lactose, a binder, such as starch, and / or a lubricant, such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active compound can be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers can be added. Capsules can also be coated with additional layers to protect the contents through one or more stages of digestion and / or to delay the release of the contents. For example, the capsule or other carrier can include an enteric coating (formed, for example, by a polymer) to prevent dissolution or disintegration in the gastric environment. All compositions for oral administration should be in a dosage suitable for such administration.

[0038] The term "carrier" as used herein includes carriers, excipients, and diluents, which means materials, compositions, or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, that are involved in carrying or transporting pharmaceutical, cosmetic, or other agents across tissue layers, such as the stratum corneum or stratum spinosum. Pharmaceutical compositions of compounds may also include suitable solid- or gel-phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers, such as polyethylene glycols.

[0039] As used herein, "mucosal tissue" refers to tissue lining various cavities in the body. Examples of mucosal tissue include, but are not limited to, mucosal tissue lining the nose, paranasal sinuses, bronchi, lungs, conjunctiva, oral cavity, tongue, esophagus, stomach, pylorus, duodenum, jejunum, ileum, ascending colon, cecum, appendix, transverse colon, descending colon, rectum, anus, urethra, and bladder. Mucosal tissue includes an epithelial surface, a glandular epithelium that secretes mucus, a basement membrane, and a submucosa that has connective tissue.

[0040] As used herein, "eosinophil granule protein" refers to a protein that constitutes the granules of eosinophils. When eosinophils are activated, granule proteins are released from the cells into the surrounding tissues. The released granule proteins cause pathological inflammatory responses in the surrounding tissues, such as the esophageal mucosal tissue. Examples of eosinophil granule proteins include, but are not limited to, major basic protein (MBP), major basic protein 1 (MBP-1), major basic protein 2 (MBP-2), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). Other examples of eosinophil granule proteins are described in Kita et al., Biology of Eosinophils, Chapter 19 of Immunology, which is incorporated herein by reference for the teaching of examples of eosinophil granule proteins.

[0041] As used herein, "high molecular weight heparin" refers to heparin and / or heparin salts (e.g., sodium heparin) having a molecular weight of about 20 kDa or greater. Heparin polymers typically consist of a mixture of polydisperse linear polymers, i.e., polymers with molecular chains of various lengths, where the molecular weights of the heparin chains vary and cannot be fully described by a single numerical value. Thus, high molecular weight heparins are more specifically described as having an average molecular weight of about 20 kDa or greater. The average molecular weight can be calculated as a number average (i.e., the total weight of the sample divided by the number of molecules in the sample). Additionally, high molecular weight heparins may have a different polydispersity than unfractionated heparin, as described further herein. Polydispersity can be quantified as the polydispersity index (PDI):

number

[0042] As used herein, "low molecular weight heparin" refers to heparin and / or heparin salts (e.g., heparin sodium) having a molecular weight of about 8 kDa or less. For example, Enoxaparin is a product of the low molecular weight heparin family and has a molecular weight of about 4.5 kDa. Heparin polymers usually consist of a mixture of polydisperse linear polymers, i.e., with molecular chains of various lengths, and the molecular weights of the heparin chains vary and cannot be fully described by a single numerical value. Thus, low molecular weight heparins are more specifically described as having an average molecular weight of less than about 8 kDa. The average molecular weight can be calculated as a number average (i.e., the total weight of the sample divided by the number of molecules in the sample). Furthermore, the polydispersity of low molecular weight heparins may vary based on the method of depolymerization. In certain embodiments, low molecular weight heparins have a lower polydispersity than unfractionated heparin, as further described herein. In other embodiments, the low molecular weight heparin has a polydispersity substantially equal to and / or greater than unfractionated heparin.

[0043] As used herein, "unfractionated heparin" or "heparin" refers to heparin polymers with molecular chains of various lengths and molecular weights between 3 and 30 kDa. "Unfractionated heparin" or "heparin" may have a greater polydispersity than high or low molecular weight heparins and has not been fractionated to isolate a fraction of molecules having a specific, limited range of molecular weights. In other instances, unfractionated heparin has a lower or substantially equal polydispersity than high or low molecular weight heparins.

[0044] As used herein, a "radiolabel" is an isotopic composition that can be attached to a substance, such as heparin, and can be tracked as the substance passes through a system or tissue. A non-limiting example of a radiolabeled substance is radiolabeled heparin, including but not limited to radiolabeled high molecular weight heparin, radiolabeled low molecular weight heparin, and radiolabeled unfractionated heparin. As provided herein, the methods described herein can be used with any of the radiolabeled heparins disclosed herein, including but not limited to radiolabeled high molecular weight heparin, radiolabeled low molecular weight heparin, and radiolabeled unfractionated heparin. In some embodiments, the radiolabeled heparin can be used with any of the radiolabeled heparins disclosed herein, including but not limited to radiolabeled high molecular weight heparin, radiolabeled low molecular weight heparin, and radiolabeled unfractionated heparin. 99m Examples of other radioactive labels include, but are not limited to, 111In, 14C, 3H, 13N, 18F, 51Cr, 125I, 133Xe, 81mKr, and 131I. Other radioactive labels that can be attached to a substance, such as heparin, are shown in Table 1. Radioactive labels, such as 99m Tc can be bound to a substance, such as heparin, using commercially available reagents well known to those of skill in the art. 99m Tc-heparin can be prepared as shown in Example 5 below. [Table 1] TIFF2024518174000004.tif193141

[0045] Less than the full scale of the disclosure may be claimed for any reason, by reserving the right to exclude or exclude by proviso any individual member of such group, including any subrange or combination of subranges within such group, which may be claimed according to ranges or similar methods hereby. Furthermore, less than the full scale of the disclosure may be claimed for any reason, by reserving the right to exclude any individual substituent, structure, or any member of the group or claimed group hereby. Throughout this disclosure, various patents, patent applications, and publications are referenced. The disclosures of these patents, patent applications, and publications are incorporated by reference in their entireties into this disclosure in order to more fully describe the state of the art as of the date of this disclosure to those skilled in the art. In the event of a conflict between the cited patents, patent applications, and publications and this disclosure, the present disclosure shall govern.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Nothing in this disclosure is to be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior invention.

[0047] As discussed herein, high molecular weight heparin is effective in localizing to eosinophil-associated inflammation sites. In addition, high molecular weight heparin is effective in neutralizing the toxic effects of MBP-1 and other eosinophil granule proteins, including MBP-2, EDN, ECP, and EPO. In some embodiments, high molecular weight heparin functions as a drug by application or delivery to one or more eosinophil-associated inflammation sites. Furthermore, since high molecular weight heparin is used to target eosinophil-associated inflammation, tracers and / or therapeutic agents are conjugated to high molecular weight heparin to provide targeted delivery to eosinophil-associated inflammation. High molecular weight heparin compounds are advantageous because high molecular weight heparin binds more strongly to eosinophil-associated inflammation sites than low molecular weight heparin. As a result, the amount of heparin (e.g., high molecular weight heparin) used to localize eosinophil-associated inflammation can be reduced in the hope that a greater proportion of heparin will localize to one or more inflammation sites.

[0048] Despite these advantages, several challenges have hindered the production of high molecular weight heparin. The lack of uniformity in chain length characteristic of heparin poses great difficulties in isolating chains of specific molecular weights. Given the interest of low molecular weight heparins in the medical field, methods for producing low molecular weight heparin compounds have been successful. However, similar progress has not been made in developing fractionation methods for high molecular weight heparins. Furthermore, even when heparin compounds (e.g., low molecular weight heparins) with a targeted average molecular weight are successfully produced, there is a large variation in molecular weight and the percentage of heparin chains within the targeted molecular weight range may not be high.

[0049] Method for producing high molecular weight heparin compounds Referring now to FIG. 1, a flow diagram of an exemplary method for producing a high molecular weight heparin (HMWH) compound according to an embodiment is depicted. As shown in FIG. 1, method 100 includes step 105 of dissolving heparin (i.e., starting material) to form a heparin solution, and step 115 of fractionating the heparin solution via tangential flow filtration (TFF) using a membrane having a molecular weight cut-off (MWCO) of between about 8 kDa and about 12 kDa, e.g., about 10 kDa. According to method 100, TFF results in a harvest comprising fractionated heparin, i.e., high molecular weight heparin compounds, having an average molecular weight of 20 kDa or more. In some embodiments, the fractionated heparin is of high purity, i.e., a substantial portion of the heparin chains in the fractionated heparin have a high molecular weight, as further described herein.

[0050] In some embodiments, the heparin starting material comprises unfractionated heparin (UFH). In some embodiments, the unfractionated heparin is a heparin salt. In some embodiments, the heparin salt comprises sodium heparin, calcium heparin, and / or additional heparin salts known to those of skill in the art. For example, the starting material may be USP sodium heparin, i.e., sodium heparin that meets the quality standards of the United States Pharmacopeia. Other types of commercially available heparin preparations are also contemplated herein.

[0051] 1, step 105 of dissolving the heparin starting material will now be described in further detail. In some embodiments, step 105 of dissolving the heparin starting material comprises dissolving heparin in a salt solution. In some embodiments, the salt solution comprises a sodium chloride (NaCl) solution. However, a variety of salt solutions may be utilized herein, as would be apparent to one of skill in the art.

[0052] In some embodiments, the salt solution is provided at a predetermined concentration, e.g., molar concentration. The concentration of the salt solution can affect the permeability of the membrane to heparin chains. Thus, the concentration of the salt solution can be refined based on the desired parameters of the heparin product. For example, a salt concentration that is too low will reduce the permeability, and heparin chains containing low molecular weight will be filtered out little. Thus, a salt concentration that is too low will reduce the purity of the heparin product. In another example, a salt concentration that is too high will increase the permeability, and all or substantially all of the heparin will be filtered through the membrane. Thus, a salt concentration that is too high will reduce the yield of the heparin product. Thus, the molar concentration of the salt solution is adjusted and regulated to improve the effective molecular weight cutoff of the membrane associated with heparin. In some embodiments, the salt solution is provided at a molar concentration of about 100 mM (i.e., about 0.1 mol / L). However, various concentrations of salt solutions are contemplated herein. For example, salt solutions include about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150 mM, about 200 mM, greater than about 200 mM molar, or any individual value or range therebetween.

[0053] The heparin sodium and salt solution may be combined in various ratios during step 105 of dissolving the heparin starting material. In some embodiments, about 2 g of heparin sodium is combined with about 50 mL of salt solution, i.e., about 0.04 g / mL. However, it should be understood that this ratio is merely exemplary and may be varied as would be apparent to one of ordinary skill in the art. In some embodiments, the heparin sodium and salt solution are combined in a ratio of about 0.01 g / mL, about 0.02 g / mL, about 0.03 g / mL, about 0.04 g / mL, about 0.05 g / mL, about 0.1 g / mL, about 0.2 g / mL, greater than about 0.2 g / mL, and / or any individual value or range therebetween. Additionally, this process can be significantly scaled up to produce larger batches of heparin product. As will be further discussed, the methods described herein are advantageous because they can be significantly scaled up with only minor process modifications without compromising the average molecular weight and / or purity of the compound, thereby providing commercial advantages for the production of high molecular weight heparin over conventional methods.

[0054] 1, in some embodiments, the method further includes a step 110 of sterilizing the heparin solution by filtering through a submicron membrane to remove microorganisms and / or bacteria therefrom. For example, the heparin solution is filtered through a membrane having a pore size of about 0.2 μm or 0.22 μm. However, membranes having a variety of pore sizes configured to sterilize the fluid may be utilized.

[0055] Step 115 of fractionating a heparin solution via tangential flow filtration will now be described in more detail. To fully convey the improvements of the methods described herein, we first generally describe TFF in terms of its traditional usage. TFF (also called cross-flow filtration) is a rapid filtration method for the separation and purification of biomolecules. It has a wide range of biological applications, including fractionating large and small biomolecules. Typically, TFF is designed for the processing or separation of globular proteins with consistent structures.

[0056] In a typical TFF process using globular proteins, TFF involves concentrating target molecules in a feed solution by passing the feed solution tangentially over the surface of a membrane having pores with a defined molecular weight cut-off (MWCO). The feed side of the membrane can be subjected to a positive pressure (i.e., back pressure) to facilitate circulation and passage of molecules through the membrane. A portion of the molecules in the solution that are smaller than the MWCO permeate the membrane and are called the permeate or filtrate. Molecules in the solution that are larger than the MWCO are typically retained on the feed side of the membrane and are called the retentate. As smaller molecules are removed and the overall volume of the solution is reduced, the retained target molecules are concentrated in the retentate.

[0057] A typical TFF process further includes diafiltering the retentate by adding fresh solvent to the feed to replace the volume of permeate removed. In some embodiments, diafiltration is performed at intervals while concentration is performed continuously (i.e., discontinuous diafiltration), thereby cycling the solution through stages of concentration and dilution until the solution is sufficiently fractionated. In some embodiments, diafiltration is performed continuously. For example, solvent can be added at the same rate as the permeate flow rate, i.e., the concentration rate, so that the volume in the system remains substantially constant. In some embodiments, the total volume of solvent added to the system for filtration is approximately equal to the volume of the system (i.e., one diafiltration volume or DV). However, additional volumes may be utilized for diafiltration, for example, about 1 DV, about 2 DV, about 3 DV, about 4 DV, about 5 DV, about 10 DV, about 20 DV, greater than 20 DV, or individual values ​​or ranges therebetween. Each additional DV facilitates the removal of more molecules smaller than the MWCO, resulting in a more complete fractionation (i.e., a level of "purity" as defined and further described herein).

[0058] Turning again to the present embodiment, it should be understood that commercially available TFF systems (e.g., Minimate TFF Systems available from Pall Corporation, Port Washington, NY) are conventionally used to process globular proteins with consistent structure. Thus, the expected results, including the stated MWCO, are determined within this context. The stated MWCO of the membrane is defined as the expected MWCO for processing globular proteins based on pore size and other factors that would be known and understood by one of ordinary skill in the art. In contrast, heparin is a linear polysaccharide, and since the molecular weight of the heparin unit does not generally correspond to the diameter of the molecule, it may interact with the membrane pores in a different manner than globular proteins. As a result, the MWCO of the commercially available membrane is inaccurate when used with heparin. For example, heparin chains with molecular weights equal to or greater than the stated MWCO may pass through the membrane at a substantial rate such that the effective MWCO is greater than the stated MWCO (see, e.g., Examples 1-3 herein). This finding goes beyond the scope of conventional TFF processes and indicates that the pore size of the membrane is one of many factors that affect the MWCO of TFF of linear polysaccharides. Due to the inherent heterogeneity of heparin and the variable polymer chain length, many of the conditions under which TFF is performed can change the effective MWCO. For example, the effective MWCO can vary with a combination of factors such as membrane pore size, salt concentration, and applied pressure. These factors can therefore be modified to control the effective MWCO, i.e., to adjust the effective MWCO up or down relative to the listed effective MWCO. It should also be understood that molecules smaller than the MWCO may be retained to some extent and molecules larger than the effective MWCO may be removed to some extent, i.e., the effective MWCO under certain conditions is not absolute. Rather, under certain conditions, molecules smaller than the effective MWCO are generally more likely to be removed and molecules larger than the effective MWCO are more likely to be retained. For example, the MWCO and / or nominal molecular weight cut-off (NMWCO) may be generally defined for a membrane or other filtration component as the lowest molecular weight of a solute at which 90% or more of the solute is retained by the membrane.Thus, the MWCO can be an objective measure of membrane permeability as generally defined and understood by those skilled in the art. Furthermore, as discussed herein, since the MWCO is based on the processing of globular proteins, the effective MWCO may be different for non-globular proteins.

[0059] Referring again to FIG. 1, step 115 of fractionating the heparin solution via tangential flow filtration includes step 115A of concentrating the heparin solution and step 115B of diafiltering the heparin solution using a membrane having a predetermined MWCO. In some embodiments, TFF is performed using a membrane having a recited MWCO of about 10 kDa. In some embodiments, TFF is performed using a membrane having a recited MWCO ranging from about 8 kDa to about 12 kDa. However, other recited MWCOs are utilized in TFF under the appropriate set of conditions as described herein (i.e., with variations in salt concentration, applied pressure, total run time, total volume filtered, etc.) to generate the desired effective MWCO. For example, TFF is performed using membranes having a recited MWCO of about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 12 kDa, about 14 kDa, about 16 kDa, about 18 kDa, about 20 kDa, greater than about 20 kDa, or any individual value or range therebetween.

[0060] In some embodiments, MWCO is related to the size and / or diameter of the pores and / or openings that penetrate the membrane material. Thus, in some embodiments, the membranes used to perform TFF are described in terms of the nominal or average pore size of the membrane. In some embodiments, TFF is performed using membranes with an average pore size (i.e., diameter) of about 5 nm. In some embodiments, TFF is performed using membranes with an average pore size (i.e., diameter) of about 4 nm to about 6 nm. However, other pore sizes may be utilized in TFF under the appropriate set of conditions (i.e., with modifications to salt concentration, applied pressure, total run time, total filtration volume, etc.) as described herein to generate the desired effective MWCO. For example, TFF is carried out using membranes with average pore sizes of about 3 nm, about 3.5 nm, about 4 nm, about 4.5 nm, about 5 nm, about 5.5 nm, about 6 nm, about 6.5 nm, about 7 nm, about 7.5 nm, about 8 nm, about 8.5 nm, about 9 nm, about 9.5 nm, about 10 nm, greater than about 10 nm, or any individual value or range therebetween. It should also be understood that pore sizes are not constant within membrane materials and may vary significantly between membrane materials. Thus, pore sizes beyond the ranges expressly described herein may be carried out under appropriate conditions that produce the desired effective MWCO in some cases.

[0061] In some embodiments, the membrane is a polyethersulfone (PES) membrane. In one specific example, the membrane is a PES membrane with a polyolefin support in the form of a cassette or other standard membrane structure (e.g., T-Series TFF Cassettes available from Pall Corporation, Port Washington, NY). In additional embodiments, the membrane is a hollow fiber membrane (e.g., Microza Hollow Fiber Membrane Systems available from Pall Corporation, Port Washington, NY). For example, hollow fiber membranes are formed using polyvinylidene fluoride (PVDF) and / or polyacrylonitrile (PAN). In some embodiments, hollow fiber membranes provide a higher speed, efficiency, and / or overall yield of the filtration process compared to other conventional membrane materials. For example, hollow fiber membranes can improve the speed and efficiency of filtration by reducing clogging. However, it should be understood that a variety of membrane materials are available and can be selected to improve the scale, yield, speed, efficiency, capacity, cost, and / or other parameters of the filtration procedure, as known to those of ordinary skill in the art.

[0062] In some embodiments, the membrane utilizes a porous support and / or a non-woven support. For example, the support is formed from a polyolefin. In another example, the support is formed from acrylonitrile butadiene styrene (ABS). In another example, the support is formed from polyvinyl chloride (PVC). However, it should be understood that a variety of supports may be utilized as would be known to one of ordinary skill in the art.

[0063] In some embodiments, the applied pressure during TFF is about 29 psi (see Examples 1-2). In some embodiments, the applied pressure during TFF is about 30 psi (see Example 3). However, the applied pressure may be varied with appropriate corresponding conditions to produce a desired effective MWCO. For example, the applied pressure may be about 1 psi, about 5 psi, about 10 psi, about 15 psi, about 20 psi, about 25 psi, about 30 psi, or any individual value or range therebetween.

[0064] In some embodiments, the total filtration volume used in diafiltration step 115B is about 1800 mL, i.e., 36 DV. In some embodiments, the total filtration volume used in diafiltration is about 2050 mL, i.e., 41 DV. It should be understood that the total filtration volume selected also affects the "purity" of the fractionated heparin as defined and described herein, since the total filtration volume affects the amount of low molecular weight particles removed through TFF. In some embodiments, the purity of the fractionated heparin is directly related to the total filtration volume. Thus, the total filtration volume can be varied with appropriate corresponding conditions to generate a desired effective MWCO. For example, the total filtration volume can be about 5 DV, about 10 DV, about 20 DV, about 30 DV, about 40 DV, about 50 DV, about 100 DV, or any individual value or range therebetween.

[0065] In some embodiments, fractionating the heparin solution via TFF, step 115, results in a retentate comprised of fractionated heparin having an average molecular weight greater than the average molecular weight of the heparin starting material. In some embodiments, the fractionated heparin has an average molecular weight of at least about 20 kDa, i.e., an HMWH compound (see Examples 1-3, where the average molecular weight is 20 kDa or greater). However, in some embodiments, the fractionated heparin comprises an average molecular weight greater than about 20 kDa. For example, the fractionated heparin may comprise an average molecular weight of about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 35 kDa, about 40 kDa, greater than about 40 kDa, or any individual value or range therebetween.

[0066] In some embodiments, the fractionated heparin has a high purity. The purity of the fractionated heparin is defined as the amount of heparin chains having a molecular weight equal to or greater than a predetermined threshold. For example, the predetermined threshold may be about 20 kDa, and the purity of the fractionated heparin is determined accordingly based on the fraction, percentage, or ratio of heparin chains having a molecular weight equal to or greater than 20 kDa (i.e., the percentage that is high molecular weight heparin) compared to heparin chains having a molecular weight less than about 20 kDa. In some embodiments, the fractionated heparin has a purity of heparin chains equal to or greater than 20 kDa of at least about 50%, i.e., "high purity" (see Examples 1-3, where the average molecular weight is equal to or greater than 20 kDa). In further embodiments, the fractionated heparin has a purity of heparin chains equal to or greater than 20 kDa of about 60%, about 70%, about 80%, about 90%, about 95%, greater than about 95%, or any individual value or range therebetween.

[0067] In some embodiments, the fractionated heparin is additionally characterized by a maximum amount of molecular chains having a molecular weight below a certain threshold. For example, the fractionated heparin comprises a percentage of heparin chains having a molecular weight below 20 kDa of about 50% or less, about 40% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, less than about 5%, or any individual value or range therebetween. In additional embodiments, the fractionated heparin is additionally characterized by a maximum amount of low molecular weight heparin chains therein, i.e., the amount of heparin chains having a molecular weight below the cutoff value (e.g., about 8 kDa) that defines the low molecular weight heparin. For example, fractionated heparin includes a percentage of heparin chains having a molecular weight of less than about 8 kDa that is about 50% or less, about 40% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, less than about 5%, or any individual value or range therebetween.

[0068] Further, in some embodiments, the predetermined threshold is a value other than about 20 kDa. For example, the predetermined threshold is set based on a minimum desired average molecular weight of the fractionated heparin. In some embodiments, the predetermined threshold for assessing the purity of the fractionated heparin is about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 35 kDa, about 40 kDa, greater than about 40 kDa, or any individual value or range therebetween. Similarly, the cutoff value for low molecular weight chains may be a value other than about 8 kDa. For example, the fraction may be about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, greater than about 12 kDa, or any individual value or range therebetween.

[0069] As described herein, the properties of the resulting fractionated heparin are controlled by the conditions of the TFF process in fractionating step 115. For example, the average molecular weight and / or purity of the fractionated heparin will vary based on a combination of factors including membrane pore size (i.e., MWCO as described), salt concentration, applied pressure, total run time, and total filtered volume. Thus, these factors can be adjusted to produce fractionated heparin with desired properties.

[0070] In a particular example, the average molecular weight of the fractionated heparin is selected as 20 kDa or more, and the purity of the fractionated heparin is selected as 50% or more. Thus, the dissolving step 105 can be performed using a NaCl solution of about 100 mM. Furthermore, the concentrating heparin solution step 115A is performed using a membrane with a stated MWCO of about 10 kDa and an applied pressure of about 29-30 psi. Furthermore, the diafiltration step 115B of the heparin solution can be performed using a NaCl solution of about 100 mM and a total filtration volume of about 36-41 DV, for example, about 1800-2050 mL. As shown in Examples 1-3, these conditions result in fractionated heparin with an average molecular weight of 20 kDa or more and a purity of 50% or more.

[0071] Once the fractionation step 115 is complete, the retentate can be collected from the feed side of the membrane. Additionally, the membrane can be washed with deionized water to obtain a wash solution. Since the wash solution may contain high molecular weight heparin that was collected on the membrane during the fractionation step 115, the wash solution may be combined with the backflow solution to improve the yield of fractionated heparin.

[0072] 1, the method 100 further comprises desalting the fractionated heparin 120. In some embodiments, the desalting step 120 is performed via TFF at suitable selected conditions.

[0073] In some embodiments, the desalting step 120 generally includes a step 120A of concentrating the fractionated heparin using a membrane having a MWCO configured to prevent the passage of the fractionated heparin and allow the passage of salts and / or their ions in the salt solution. In some embodiments, a membrane having a MWCO of about 3 kDa is used. However, membranes having MWCOs of about 1 kDa, about 3 kDa, about 5 kDa, greater than about 5 kDa, or any individual value or range therebetween are utilized herein. In some embodiments, the fractionated heparin is concentrated 120A under an applied pressure of about 29-30 psi. However, the applied pressure may be varied as would be apparent to one of ordinary skill in the art.

[0074] In some embodiments, the desalting step 120 further comprises a step 120B of diafiltering the fractionated heparin with deionized water. In some embodiments, the diafiltering step 120B is performed with a total filtration volume of about 10 DV, e.g., about 500 mL. However, the total filtration volume may vary as would be apparent to one of ordinary skill in the art.

[0075] Although exemplary steps of step 120 of desalting fractionated heparin are described herein, it should be understood that a variety of conventional methods of desalting may be utilized herein, as would be apparent to one of ordinary skill in the art.

[0076] 1, method 100 further includes a step 125 of drying the fractionated heparin, thereby producing an isolated HMWH compound. In some embodiments, drying step 125 is performed by lyophilization. However, it should be understood that drying step 125 may be performed by any conventional means as would be apparent to one of ordinary skill in the art.

[0077] As shown in Examples 1-3, the disclosed method produces HMWH compounds with a yield of about 15-18%. In some embodiments, the method is modified in various ways to improve the yield of HMWH compounds. In some embodiments, the membrane pore size, salt concentration, applied pressure, and / or total filtration volume used for fractionation are adjusted in a manner that improves the yield. For example, lowering the applied pressure improves the yield and slows the fractionation (i.e., longer total run time for a given volume of filtration). In another example, decreasing the total volume of filtration results in faster fractionation (i.e., shorter total run time for a given volume of filtration) and improves the yield. In another example, decreasing the membrane pore size results in fractionation with improved yield. Adjusting the salt concentration may also lead to improved yields of the methods disclosed herein.

[0078] In some embodiments, using a heparin starting material having a higher average molecular weight will similarly result in a higher yield. For example, the use of a heparin starting material that has been pre-filtered by molecular weight may result in a higher yield. In another example, the use of a heparin starting material that has been pre-filtered by a property that approximately correlates with molecular weight may also result in a higher yield.

[0079] In some embodiments, the methods disclosed herein produce additional useful by-products. For example, the filtrate or permeate (i.e., material removed from the retentate via TFF) contains heparin with a substantially reduced average molecular weight. In some embodiments, the permeate contains a LMWH. In some embodiments, the permeate is processed to produce a LMWH via an additional fractionation step. Such fractionation steps are known to those skilled in the art. Thus, the methods disclosed herein can be used, with or without additional steps, to produce a LMWH compound as a by-product along with a HMWH compound.

[0080] While the exemplary methods described herein utilize tangential flow filtration to produce fractionated heparin from unfractionated heparin starting material, it should be understood that additional types of filtration may be utilized to accomplish this step. In additional embodiments, alternative types of mechanical filtration, as known to those of skill in the art, are utilized to fractionate the heparin. Thus, some or all of the remaining steps as described herein may be used in combination with such alternative filtration methods to produce the final high molecular weight heparin compound.

[0081] It should be appreciated that the presently disclosed method is advantageous because attempts to produce isolated HMWH compounds using conventional methods of filtering by molecular weight encounter various difficulties. In general, the inherent heterogeneity of heparin's linear structure and polymer chain length (and therefore molecular weight) precludes the ability to fractionate higher molecular weight heparins with substantial purity using conventional methods. However, the methods disclosed herein demonstrate the unexpected discovery that fractionation of heparin by TFF using a membrane for globular proteins produces a heparin fraction with an average molecular weight of 20 kDa or more, with more than 50% of the heparin chains having a molecular weight of 20 kDa or more. Furthermore, these properties of the resulting heparin fraction can be carefully tuned by adjusting the conditions of TFF, such as membrane pore size, salt concentration, applied pressure, and filtration volume.

[0082] The disclosed method is further advantageous due to its scalability. For example, it may be possible to produce HMWH compounds by gel filtration chromatography, but such processes would be difficult to scale up due to their relatively high cost and additional difficulties when using large volumes. Other types of membrane filtration can involve high levels of fouling (accumulation on the membrane), causing clogging and additional difficulties when using large filtration volumes and / or run times. In contrast, TFF is relatively low cost and by its nature, has a low degree of fouling. Thus, TFF is highly scalable and can be used to fractionate thousands of liters of solution at little additional cost. Thus, the methods disclosed herein are highly scalable for manufacturing purposes compared to other processes.

[0083] The methods described herein are not intended to be limited with respect to the specific embodiments described, which are intended only as illustrations of various features. As will be apparent to those skilled in the art, many modifications, variations, and additions to the methods are possible without departing from the spirit and scope thereof.

[0084] The HMWH compounds are useful for forming HMWH compositions for various medical applications. In some embodiments, the method further comprises combining the HMWH compounds with a pharma- ceutically acceptable excipient to produce a HMWH composition. In some embodiments, the HMWH compositions are useful for imaging, diagnosing, and / or treating a medical condition.

[0085] In some embodiments, the HMWH composition is configured for binding and / or localization to manifestations of eosinophil-associated inflammation or eosinophil-associated conditions. In some embodiments, the average molecular weight of the HMWH compound and / or the purity of the HMWH compound are selected to optimize binding to sites that manifest eosinophil-associated inflammation. Since HMWH exhibits a higher affinity for MBP-1 than low molecular weight heparin (LMWH) or unfractionated heparin (UFH), HMWH binds more strongly to sites of eosinophil-associated inflammation than LMWH or unfractionated heparin UFH.

[0086] In some embodiments, the binding affinity of the HMWH composition is directly related to molecular weight and increases with the average molecular weight of the HMWH compound. Thus, as the average molecular weight of the HMWH increases, the amount of heparin required to localize eosinophil-associated inflammation can be reduced, with the expectation that a greater proportion of the administered heparin will localize to the site of inflammation.

[0087] In some embodiments, the localization rate of HMWH compositions increases with increasing purity of the HMWH compound, since it is directly related to molecular weight. Thus, as the purity of the HMWH increases, the amount of heparin required for proper localization of eosinophil-associated inflammation can be reduced, with the expectation that a greater proportion of the administered heparin will be localized at the site of inflammation. Similarly, if a higher molecular weight threshold is used to define purity as described herein, the localization rate can similarly increase.

[0088] The HMWH compositions produced by the methods herein can be configured for administration in a conventional manner by any route in which they are active. Administration may be systemic, topical, or oral. For example, administration may be, but is not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, buccal, or ocular, or intravaginal, inhalation, depot injection, or implant. Thus, the mode of administration (either alone or in combination with other medicaments) may be, but is not limited to, sublingual, injectable (including short-acting, depot, implant, and pellet forms injected subcutaneously or intramuscularly), topical (including nasal drops, ointments, or creams for application to the skin), and / or transdermal, such as vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, patches, and creams. The specific method of administration will vary depending on the indication and purpose. It should also be noted that the risk of HIT and / or HITT suspected to be associated with high molecular weight heparin is limited to systemic administration. Thus, topical and / or oral administration is advantageous in that it does not pose a significant risk of HIT and / or HITT.

[0089] In some embodiments, the HMWH composition is configured to image an eosinophil-associated condition and / or other target condition in which the HMWH is localized. Thus, the method further includes attaching a tracer, such as a radiolabeled imaging agent, to the HMWH compound. For example, the radiolabeled imaging agent is 99mThe HMWH composition may be administered and used to visualize the target condition using conventional imaging modalities, including but not limited to single photon emission computed tomography (SPECT), positron emission tomography (PET) scans, conventional or computed tomography (CT), magnetic resonance imaging (MRI), or a combination thereof. The HMWH composition with the tracer may also be utilized to diagnose and / or monitor the target condition based on the images taken as described. In some embodiments, the HMWH composition allows for a reduction in the amount of tracer that must be administered to the patient for adequate imaging of the site of the target condition (e.g., eosinophil-associated inflammation). For example, due to the avidity and localization rate of the HMWH to the site, a greater percentage of the HMWH composition is localized to the site, as compared to unfractionated or low molecular weight heparins, and thus the amount (or dose) of tracer administered is reduced. Thus, if the tracer is radioactive, the amount of radioactive material required for adequate imaging is reduced, improving the safety of the composition and limiting any effects associated with administration of a radiolabeled imaging agent.

[0090] In some embodiments, the HMWH composition is configured for the treatment of eosinophil-associated conditions and / or other target conditions expressing toxins that the HMWH localizes. Thus, the method further comprises binding a therapeutic agent to the HMWH composition. In some embodiments, the HMWH composition further comprises a therapeutically effective amount of a therapeutic agent for administration to the patient. In some embodiments, the therapeutic agent is configured to have a therapeutic effect on the target condition. Due to the binding activity and localization rate of the HMWH compound to the site of the target condition (e.g., eosinophil-associated inflammation), the amount (or dose) of the therapeutic agent required for proper care can be reduced, thereby limiting any side effects associated with administration of the therapeutic agent. Thus, the therapeutically effective amount of the therapeutic agent is less than the therapeutically effective amount typically associated with administration of the therapeutic agent in the absence of the HMWH compound or another targeting mechanism. In some embodiments, the therapeutic agent is a glucocorticoid. In some embodiments, the glucocorticoid is one or more of mometasone, fluticasone, budesonide, and solumedrol. Additional therapeutic agents are contemplated herein, as will be apparent to those of skill in the art.

[0091] In some embodiments, the HMWH comprises various additional ingredients or additives as would be known to one of ordinary skill in the art. In some embodiments, the method further comprises adding a stabilizer to the HMWH composition. In some embodiments, the method further comprises adding a flavoring agent to the HMWH composition.

[0092] Although the present invention has been described in some detail with reference to certain preferred embodiments thereof, other versions are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description and preferred versions contained within. Various aspects of the present invention will now be described with reference to the following non-limiting examples. EXAMPLES

[0093] Example 1 - Tangential Flow Filtration for the Production of HMWH Composition | Batch 1 Method: 2.0132 g of USP heparin was dissolved in 50 mL of 100 mM NaCl solution and filtered through a 0.22 micron membrane. The heparin solution was filtered through a tangential flow filtration (TFF) system. A 10 kDa molecular weight cut-off (MWCO) membrane was installed in the TFF system and washed with deionized water before processing. The heparin solution was then pumped through the TFF system and filtration was performed at a back pressure of 29 psi. A 100 mM NaCl replacement solution was added during the filtration to keep the retentate volume constant (i.e., diafiltration step). The process was stopped when the total volume of the permeate reached 1800 mL. The retentate was then desalted and dried to obtain the HMWH composition as described in Example 4.

[0094] Results: The process yielded 297 mg of heparin with a yield of 15%. The average molecular weight of this composition was 23.9 kDa (i.e., high molecular weight overall). Additionally, 56% of the heparin chains in the HMWH composition had a molecular weight greater than 20 kDa (i.e., high purity). Anti-factor Xa and anti-factor IIa assays were also performed on the composition to determine the ratio of anti-Xa:anti-IIa activity that is related to the therapeutic benefit of heparin as an anticoagulant. The results of Example 1 are summarized in Table 2. [Table 2] EXAMPLES

[0095] Example 2 - Tangential Flow Filtration for the Production of HMWH Composition | Batch 2 Method: 2.0801 g of USP heparin was dissolved in 52 mL of 100 mM NaCl solution and filtered through a 0.22 micron membrane. The heparin solution was filtered through a TFF system. A 10 kDa MWCO membrane was installed in the TFF system and washed with deionized water before processing. The heparin solution was then pumped through the TFF system and filtration was performed at a back pressure of 29 psi. A 100 mM NaCl replacement solution was added during the filtration to keep the retentate volume constant (i.e., diafiltration step). The process was stopped when the total volume of permeate reached 2050 mL. The retentate was then desalted and dried to obtain the HMWH composition as described in Example 4.

[0096] Results: The process yielded 327 mg of heparin with a yield of 16%. The average molecular weight of this composition was 23.5 kDa (i.e., high molecular weight overall). Additionally, 54% of the heparin chains in the HMWH composition had a molecular weight greater than 20 kDa (i.e., high purity). Anti-factor Xa and anti-factor IIa assays were also performed on the composition to determine the ratio of anti-Xa:anti-IIa activity associated with therapeutic benefit. The results of Example 2 are summarized in Table 2. EXAMPLES

[0097] Example 3 - Tangential Flow Filtration for the Production of HMWH Composition | Batch 3 Method: 2.1476 g of USP heparin was dissolved in 54 mL of 100 mM NaCl solution and filtered through a 0.22 micron membrane. The heparin solution was filtered through a TFF system. A 10 kDa MWCO membrane was installed in the TFF system and washed with deionized water before processing. The heparin solution was then pumped through the TFF system and filtration was performed at a back pressure of 30 psi. A 100 mM NaCl replacement solution was added during the filtration to keep the retentate volume constant (i.e., diafiltration step). The process was stopped when the total volume of permeate reached 2050 mL. The retentate was then desalted and dried to obtain the HMWH composition as described in Example 4.

[0098] Results: The process yielded 358 mg of heparin with a yield of 18%. The average molecular weight of this composition was 23.3 kDa (i.e., high molecular weight overall). Additionally, 53% of the heparin chains in the HMWH composition had a molecular weight greater than 20 kDa (i.e., high purity). Anti-factor Xa and anti-factor IIa assays were also performed on the composition to determine the ratio of anti-Xa:anti-IIa activity associated with therapeutic benefit. The results of Example 3 are summarized in Table 2. EXAMPLES

[0099] Example 4 - Desalting and drying of the retentate Methods: After TFF using a 10 kDa MWCO membrane, the retentate was collected from the system. The membrane was washed separately with deionized water before being removed from the TFF system to obtain a wash solution. This wash solution was combined with the retentate for desalting. A 3 kDa MWCO membrane was installed in the TFF system and washed with pure water. The retentate / wash mixture was then pumped through and diafiltered against deionized water to remove salts. The desalting process was stopped when the total volume of permeate reached 10 times the volume of the retentate. EXAMPLES

[0100] Example 5 - 99m Preparation of Tc-heparin Stannous chloride solution (40 mg / mL, Sigma 243523) was prepared in deionized water under a stream of nitrogen. A 0.5 mL aliquot was filtered and mixed with 1.00 mL of NaCl (1.00 M) and 150 mg of preservative-free heparin (10,000 IU / mL). Approximately 100 mCi of freshly eluted 99m Tc was added and mixed at room temperature for 30 minutes. 99m An aliquot containing Tc and 20 mg of heparin was removed for tissue studies.

[0101] Results: Label affinity was measured by paper chromatography Whatman 31 with acetone and heparin. 99m The binding with Tc was confirmed to be greater than 97%.

[0102] Heparin was also analyzed by Sephadex G25 column chromatography (HiTrap 5mL desalting column, GE healthcare, 17140801), using 0.15M NaCl as the elution buffer and collecting fractions of approximately 1mL. 99m It was shown that all of the Tc elutes in the void volume, and there is no unbound Tc in the radiolabeled heparin. 99m The absence of Tc was confirmed.

[0103] In acidic environments 99mThe stability of Tc-heparin was tested by dilution in artificial gastric fluid (Carolina, 864603) and confirmed to be unchanged in its properties using both paper chromatography and Sephadex G25. EXAMPLES

[0104] Example 6 - Heparin binding to eMBP-1 by SPR The purpose of this study was to determine the apparent dissociation rate constants (k) of seven heparin samples bound to recombinant human (rhu) eMBP using surface plasmon resonance (SPR) Biacore technology. d The goal is to determine whether there is a correlation between the half-life of the complex and the molecular weight of heparin.

[0105] Methods: Unfractionated and fractionated heparin samples (i.e., analytes) were evaluated for binding to the surface of recombinant human (rhu) eMBP1 (i.e., ligand).

[0106] Assay conditions: Biosensor analysis was performed using a Biacore3000 optical biosensor equipped with a CM4 sensor chip (GE, Marlborough, MA; BR100539) in an HBS buffer system (10 mM HEPES, pH 7.4, and 150 mM NaCl) at 25° C. The autosampler was used at room temperature.

[0107] Surface treatment: eMBP1 was immobilized on the chip surface using thiol coupling chemistry. Following the protocol of the thiol coupling kit (Cytiva Life Sciences, Marlborough, MA), the surface was first activated with 0.2 M EDC and 0.05 M NHS for 2 min, followed by injection of 80 mM PDEA in 50 mM sodium borate buffer (pH 8.5) for 4 min. eMBP1 was diluted to 0.6 μM or 0.06 μM in 10 mM sodium acetate (pH 5.25) and injected until the targeted immobilization level was reached. Finally, 50 mM L-cysteine ​​in 0.1 M sodium acetate, 1.0 M sodium chloride (pH 4.0) was injected at 10 μL / min for 4 min to block remaining free cysteines. A reference flow cell was prepared using the same immobilization procedure, but without the addition of eMBP1. Rhu eMBP1 was captured in flow cells 2-4 (i.e., FC2, FC3, and FC4) of the sensor chip at several different densities in relative units (low density (1000 RU), medium density (3000 RU), and high density (4000 RU), respectively). The second chip was prepared in a similar manner, with low density (500 RU), medium density (800 RU), and high density (1200 RU), respectively.

[0108] Analyte preparation: A series of analyte concentrations from 10 μg / mL to 10 ng / mL was prepared by 10-fold dilution with running buffer.

[0109] Interaction parameters: Analytes were injected in duplicate or triplicate in the order of sample number. Multiple blank (buffer) injections were performed and used to evaluate and subtract system artifacts. For all analyte concentrations, the association phase was monitored for 600 s at a flow rate of 25 μL / min, and the dissociation phase was collected for 1800 s at a flow rate of 25 μL / min.

[0110] Surface regeneration: At the end of each binding cycle, the surface was regenerated with a 2-3 (s) pulse of 6 M guanidine at a flow rate of 100 μL / min.

[0111] Data analysis: Data were aligned, double-referenced, and fitted using Scrubber v2.0 software (BioLogic Software Pty Ltd, Campbell, Australia), an SPR data processing and nonlinear least-squares regression fitting program. Dissociation phase data were fitted globally to a simple exponential decay model for each sample and assay condition. This simple decay model oversimplified the complex dissociation that occurs between multiple dissociation events resulting from polydispersed analytes complexed to the eMBP1 surface.

[0112] Results: Overall, a clear linear response was observed between complex half-life and heparin molecular weight under certain conditions that depended on eMBP1 surface density and heparin concentration. Summary data of the correlation between complex half-life and heparin molecular weight are shown in Table 3. [Table 3]

[0113] 2A-2B, there are depicted sensorgrams of signal responses of fractionated heparin samples (i.e., analytes) binding to rhu eMBP1 (i.e., ligand) according to embodiments. FIG. 2A depicts signal response curves over time for seven heparin samples of different molecular weights at a concentration of 100 ng / mL that bind to eMBP1 at a density of 1200 RU according to embodiments. FIG. 2B depicts normalized signal response curves over time for seven heparin samples of different molecular weights at a concentration of 100 ng / mL that bind to eMBP1 at a density of 1200 RU. Referring now to FIG. 3, there is depicted a plot graph of complex half-life versus molecular weight for seven heparin samples of different molecular weights at a concentration of 100 ng / mL that bind to eMBP1 at a density of 1200 RU according to embodiments. FIGs. 2A-2B and 3 show the correlation between complex half-life and molecular weight of heparin as discussed herein.

[0114] Moreover, some assay conditions deviated from this correlation, highlighting the complexity of multimeric and polydisperse heparin samples binding to the eMBP1 surface. Although a general correlation was observed in which the higher the molecular weight of heparin, the higher the binding response, this response was not seen under all conditions, including the most avid conditions. In the most avid conditions (i.e., high density of eMBP1 and low concentration of heparin), no clear correlation was observed. This phenomenon can be explained by the fact that the more subunits bound to each heparin molecule, the more similar the half-lives of the complexes between the various molecular weight species, thereby reducing the dynamic range of the assay.

[0115] Moreover, a clear decay of the signal response was observed after each binding cycle, therefore the complex half-life, as opposed to the response signal, is a better evaluation tool for this assay.

[0116] In the above detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, similar reference numerals generally identify similar components unless otherwise indicated by the context. The exemplary embodiments described in this disclosure are not intended to be limiting. Other embodiments may be used and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the various features of the present disclosure, as generally described herein and illustrated in the figures, may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0117] The present disclosure is not limited in terms of the specific embodiments described in this application, which are intended as illustrations of various features. Instead, this application is intended to cover any variations, uses, or adaptations of the present teachings and to use their general principles. Moreover, this application is intended to cover departures from the present disclosure as are within known or customary practice in the art to which these teachings pertain. Many modifications and variations can be made to the specific embodiments described without departing from the spirit and scope of the present disclosure, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. It is to be understood that the present disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0118] The various features and functions disclosed above, or alternatives thereof, may be combined into many other different systems or applications. Various alternatives, modifications, variations, or improvements not presently foreseen or anticipated, may subsequently be made by those skilled in the art, each of which is also intended to be encompassed by the disclosed embodiments.

Claims

1. 1. A method for producing fractionated heparin, the method comprising: dissolving heparin in a solvent to form a heparin solution; and fractionating the heparin solution by tangential flow filtration using a membrane having a molecular weight cut-off of between about 3 kDa and about 12 kDa, thereby obtaining a fractionated heparin having a weight average molecular weight of about 20 kDa or more; said fractionating step wherein at least 50% of the heparin chains in said fractionated heparin have a molecular weight of 20 kDa or greater; A method comprising:

2. 10. The method of claim 1, wherein the heparin is USP heparin.

3. The method according to any one of claims 1 to 2, wherein the solvent is a sodium chloride (NaCl) solution.

4. 4. The method of claim 3, wherein the NaCl solution has a concentration of about 100 mM.

5. The method of claim 1, wherein the molecular weight cutoff of the fractionation membrane is about 5 kDa.

6. 2. The method of claim 1, wherein fractionating the heparin solution by tangential flow filtration comprises permeating at least a portion of the heparin solution through the fractionation membrane under an applied pressure to obtain a retentate comprising the fractionated heparin.

7. The method of claim 6, wherein the applied pressure is from about 10 psi to about 15 psi.

8. 8. The method of claim 6, wherein the step of fractionating the heparin solution by tangential flow filtration further comprises adding one or more diafiltration volumes (DV) of the solvent during the tangential flow filtration to maintain the volume of the retentate, thereby defining a total filtration volume.

9. The method of any one of claims 1-2 and 5-7, further comprising filtering the heparin solution through a submicron membrane to sterilize the heparin solution.

10. The method of any one of claims 1-2 and 5-7, further comprising the step of desalting the fractionated heparin.

11. 11. The method of claim 10, wherein desalting the fractionated heparin comprises performing tangential flow filtration using a desalting membrane having a molecular weight cut-off of between about 1 kDa and about 5 kDa.

12. 12. The method of claim 11, wherein the molecular weight cut-off of the desalting membrane is about 5 kDa.

13. The method of any one of claims 1-2 and 5-7, further comprising the step of drying the fractionated heparin.

14. 14. The method of claim 13, wherein drying the fractionated heparin comprises lyophilizing the fractionated heparin.

15. The method according to any one of claims 1-2 and 5-7, wherein the weight average molecular weight of the fractionated heparin is about 30 kDa or more.

16. 16. The method of claim 15, wherein the fractionated heparin has a weight average molecular weight of about 40 kDa or greater.

17. The method of any one of claims 1-2 and 5-7, wherein at least 60% of the heparin chains in the fractionated heparin have a molecular weight of 20 kDa or greater.

18. 18. The method of claim 17, wherein at least 70% of the heparin chains in the fractionated heparin have a molecular weight of 20 kDa or greater.

19. 1. A method for producing high molecular weight (HMW) heparin, the method comprising: dissolving a heparin salt in a salt solution to form a heparin solution having a concentration of about 0.02 g / mL to about 0.03 g / mL; fractionating the heparin solution by tangential flow filtration using a fractionation membrane having a molecular weight cutoff of about 5 kDa, thereby obtaining a fractionated heparin having a weight average molecular weight of about 20 kDa or greater, wherein at least 50% of the heparin chains in the fractionated heparin have a molecular weight of 20 kDa or greater; thereby obtaining said HMW heparin.

20. 20. The method of claim 19, wherein the heparin salt is selected from the group consisting of sodium heparin and calcium heparin.

21. The method of any one of claims 19 to 20, wherein the heparin salt is a USP heparin salt.

22. 20. The method of claim 19, wherein the salt solution is a sodium chloride (NaCl) solution.

23. 23. The method of claim 22, wherein the NaCl solution has a concentration of about 100 mM.

24. 20. The method of claim 19, wherein the molecular weight cut-off of the fractionation membrane is about 10 kDa.

25. 20. The method of claim 19, wherein fractionating the heparin solution by tangential flow filtration comprises permeating at least a portion of the heparin solution through the fractionation membrane under an applied pressure to obtain a retentate comprising the fractionated heparin.

26. 26. The method of claim 25, wherein the applied pressure is from about 10 psi to about 15 psi.

27. 27. The method of any one of claims 25-26, wherein fractionating the sterile heparin solution by tangential flow filtration further comprises adding one or more diafiltration volumes (DV) of the salt solution during the tangential flow filtration to maintain the volume of the retentate, thereby defining a total filtrate volume, the total filtrate volume comprising about 10 DV to about 20 DV.

28. 27. The method of any one of claims 19 to 20 and 22 to 26, further comprising the step of desalting the fractionated heparin by tangential flow filtration using a desalting membrane, the molecular weight cutoff of the desalting membrane being about 5 kDa.

29. The method according to any one of claims 19 to 20 and 22 to 26, wherein the weight average molecular weight of the fractionated heparin is about 30 kDa or more.

30. 30. The method of claim 29, wherein the fractionated heparin has a weight average molecular weight of about 40 kDa or greater.

31. 27. The method of any one of claims 19-20 and 22-26, wherein at least 60% of the heparin chains in the fractionated heparin have a molecular weight of 20 kDa or greater.

32. 32. The method of claim 31, wherein at least 70% of the heparin chains in the fractionated heparin have a molecular weight of 20 kDa or greater.

33. 27. The method according to any one of claims 19 to 20 and 22 to 26, wherein the step of fractionating the heparin solution by tangential flow filtration comprises permeating at least a portion of the heparin solution through the fractionation membrane under a transmembrane pressure of about 10 psi to about 15 psi, wherein (1) heparin chains in the heparin having a molecular weight of less than 20 kDa permeate the filtration membrane as a filtrate, and (2) heparin chains in the heparin having a molecular weight of 20 kDa or more do not permeate the filtration membrane, thereby obtaining a retentate comprising the fractionated heparin; The method, wherein the heparin salt comprises sodium heparin and the salt solution comprises a sodium chloride (NaCl) solution at a concentration of about 100 mM.

34. The method of claim 8, wherein the total filtration volume comprises about 10 DV to about 20 DV.

35. A method according to any one of claims 1 to 2 and 5 to 7, wherein the step of dissolving heparin in a solvent to form a heparin solution comprises dissolving the heparin in the solvent at a concentration of about 0.01 g / mL to about 0.05 g / mL.

36. 36. The method of claim 35, wherein the concentration comprises about 0.02 g / mL to about 0.03 g / mL.

37. The method according to any one of claims 1 to 2, 5 to 7, 19 to 20, and 22 to 26, wherein the fractionation membrane comprises any one of a hollow fiber membrane and a polyethersulfone (PES) membrane.

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