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 of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 187, 624, “Methods of Manufacturing a High Molecular Weight Heparin Compound,” filed on 12 May 2021, which is incorporated herein by reference in its entirety. The following are prior art documents related to the invention of this application (including documents cited in the international phase after the international filing date and documents cited when the application entered the national phase in other countries): (Prior art document) (Patent Document) (Patent Document 1) European Patent Application Publication No. 3943513 (Non-patent literature) (Non-patent document 1) HUBER RENE ET AL, "Cellular and Molecular Effects of High-Molecular-Weight Heparin on Matrix Metalloproteinase 9 Expression", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES,Vol.{0}20,No.{0}7,30 March 2019 (2019-03-30),page 1595 [Overview of the Initiative] 【0002】 This disclosure generally relates to methods for producing high molecular weight heparin compounds. The disclosed subject matter applies to the production of compounds and / or compositions having them for imaging, diagnosis, monitoring, and / or treatment of various conditions. For example, 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 ocular diseases. 【0003】 Historically, high molecular weight heparin has been avoided in the medical field, while low molecular weight heparin has been preferred. Heparin is a polysaccharide, and the polymer chain lengths are inherently heterogeneous, containing heparin chains of various molecular weights. Generally, to lower the molecular weight, heparin is depolymerized and fractionated, and low molecular weight heparin is administered to patients. It is suspected that administering large amounts of high molecular weight heparin chains via common routes of administration (i.e., intravenously or subcutaneously) increases the incidence of heparin-induced thrombocytopenia (HIT). HIT is a complication resulting from heparin exposure that can cause limb and life-threatening thrombotic complications. In HIT, when heparin binds to platelet factor 4 (PF4), the immune system forms antibodies against heparin. These antibodies then form a complex with heparin / PF4, which binds to platelets and activates them, leading to thrombus formation and a decrease in platelet count. HIT can cause venous thromboembolism and, in some cases, arterial thrombosis (called HITT). Due to the suspected risk of HIT associated with high molecular weight heparin chains, low molecular weight heparin is preferred for clinical applications in the medical field. 【0004】 However, recent advances suggest that high molecular weight heparin may be useful for certain applications. For example, high molecular weight heparin is useful in this regard because it localizes with high probability to eosinophils, a type of white blood cell that fights multicellular parasites and certain infections in vertebrates. 【0005】 Normally, eosinophils are found in the bloodstream, lower gastrointestinal tract, and lymphatic system, but pathologically they infiltrate many more organs and sites, causing inflammation and various diseases and conditions. A distinctive feature of eosinophils is their granules, which contain prominent cationic proteins. These granules consist of an electron-dense central nucleus and an electron-transparent matrix. The nucleus mainly 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). The granules also contain major basic protein 2 (MBP-2 or eMBP-2) in the nucleus and / or matrix. During degranulation, eosinophils release these proteins into the surrounding tissue, stimulating histamine release and causing inflammation. Studies have shown that MBP-1 is toxic to mammalian cells, bacteria, and certain parasites, and that it deposits at inflammatory sites in many eosinophil-related diseases, such as organ dysfunction (e.g., eosinophilic esophagitis). Heparin binds to MBP-1 and is effective in dose-related neutralization of its cytotoxic effects. Furthermore, the binding affinity of heparin to MBP-1 increases with molecular weight. Therefore, high molecular weight heparin is useful for imaging, diagnosing, monitoring, and / or treating eosinophil-related inflammation and / or pathological conditions. 【0006】 Despite these advances, several challenges have hindered the production of high molecular weight heparin. The lack of uniformity in heparin chain length makes it difficult to isolate chains of specific molecular weights. While methods for producing low molecular weight heparin compounds have been successful due to the growing interest in low molecular weight heparin in the medical field, similar progress has not been made in developing methods for fractionating high molecular weight heparin. 【0007】 Furthermore, even when the production of heparin compounds with a target average molecular weight (e.g., low molecular weight heparin) was successful, the molecular weight still varied considerably, and the proportion of heparin chains within the target molecular weight range was sometimes not high. 【0008】 Therefore, a method for producing high molecular weight heparin compounds that have a high average molecular weight and a high proportion of heparin chains with high molecular weight would be useful. 【0009】 Embodiments of the present invention relate to a method for producing fractionated heparin, the method comprising the steps of: 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 fractionation molecular weight between approximately 8 kDa and approximately 12 kDa, thereby obtaining fractionated heparin having a weight-average molecular weight of approximately 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 present invention relates to a method for producing high molecular weight (HMW) heparin, the method comprising: a step of dissolving a heparin salt in a salt solution to form a heparin solution; a step of sterilizing the heparin solution by filtering it through a sterile membrane having a pore size of about 0.2 μm, thereby obtaining a sterile heparin solution; a step of fractionating the sterile heparin solution by tangential flow filtration using a fractionation membrane having a pore size of about 5 nm, thereby obtaining 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; a step of 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 a step of drying the desalted heparin by freeze-drying to obtain the HMW heparin. [Brief explanation of the drawing] 【0011】 The accompanying drawings are incorporated herein and constitute part of this specification, illustrating embodiments of the present invention and are useful together with this specification to illustrate the principles, features, and characteristics of the present invention. 【0012】 [Figure 1] This is a flowchart illustrating an exemplary method for producing a high molecular weight heparin compound according to the embodiment. [Figure 2] Figures 2A and 2B are sensorgrams of the signal response of a fractionated heparin sample (i.e., analyte) bound to eMBP1 (i.e., ligand) according to the embodiment. [Figure 3] This is a plot graph of the complex half-life versus molecular weight for seven different heparin samples with varying molecular weights, bound to eMBP1 at a density of 1200 RU and a concentration of 100 ng / mL, according to the embodiment. [Modes for carrying out the invention] 【0013】 This disclosure is not limited to the specific systems, apparatus, and methods described herein. The terms used herein are for illustrative purposes only and are not intended to limit the scope of any particular version or embodiment. Such embodiments as those described herein are embodied in many different forms. Rather, these embodiments are provided to ensure that the disclosure is thorough and complete and fully conveys its scope to those skilled in the art. 【0014】 As used herein, the singular forms “a,” “an,” and “the” include a plural reference unless the context clearly indicates otherwise. With regard to substantially any use of plural and / or singular terms herein, those skilled in the art can interpret them plural-to-singular and / or singular-to-plural depending on the context and / or use. Various singular / plural permutations may be explicitly defined herein for clarity. 【0015】 As will be understood by those skilled in the art, for all purposes, including in terms of providing written explanations, all ranges disclosed herein are intended to encompass each intermediate value between the upper and lower limits of that range, and any other described values ​​or intermediate values ​​within that described range. Furthermore, all ranges disclosed herein encompass any possible subranges and combinations thereof. Any described range can be readily recognized as fully explaining and enabling the decomposition of the same range into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can readily be decomposed into the lower third, middle third, upper third, etc. Also, as will be understood by those skilled in the art, all phrases such as “maximum,” “at least,” etc., include the number mentioned and refer to a range that can then be decomposed into subranges, as described above. Finally, as will be understood by those skilled in the art, ranges include individual members. Thus, for example, the group having 1 to 3 cells refers to the group having 1, 2, or 3 cells, and the range of values ​​between 1 and 3 cells. Similarly, the group having 1 to 5 cells refers to groups having 1, 2, 3, 4, or 5 cells, as well as groups having 1 to 5 cells. 【0016】 Furthermore, even when a specific number is explicitly recited, one of ordinary skill in the art will recognize that such a recitation should be construed to mean at least the recited number (e.g., a mere recitation of "two iterations" without any other modifiers means at least two iterations, or two or more iterations). Additionally, when a phrase similar to "at least one of A, B, and C, etc." is used, generally, such a construction is 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, a system 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). When a phrase similar to "at least one of A, B, and C, etc." is used, generally, such a construction is 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, a system 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). One of ordinary skill in the art will further understand that substantially any conjunctive word and / or phrase presenting two or more alternative terms in any of the description, sample embodiments, or drawings is intended to cover the possibility of including one of the terms, any of the terms, or both terms. For example, the phrase "A or B" is understood to cover the possibilities of "A" or "B", or "A and B". 【0017】 In addition, when features of the present disclosure are described from the perspective of a Markush group, one of ordinary skill in the art will recognize that the present disclosure thereby describes the features from the perspective of the individual members of the Markush group or subgroups of the members. 【0018】 Percentages, parts, and ratios are all based on the total weight of the compound, and unless otherwise specified, all measurements were made at about 25°C. 【0019】 As used herein, the term "about" refers to variations in numerical quantities that occur, for example, by the procedures of measurement or handling in the real world, by inadvertent errors in these procedures, or by differences in the manufacture, source, or purity of a composition or reagent. Generally, the term "about" as used herein means greater or less than the recited value by up to 1 / 10 of the recited value, e.g., ±10%. The term "about" also means variations that would be recognized as equivalent by one of ordinary skill in the art, unless the term "about" encompasses known values practiced by the prior art. Each value or range of values preceded by the term "about" is also intended to encompass embodiments of the recited absolute value or range of values. Whether or not modified by the term "about", quantitative values recited in the present disclosure include equivalents to the recited values, e.g., numerical quantity variations that may occur but would be recognized as equivalents by one of ordinary skill in the art. If the context of the present 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 a range that is less than half the interval between the preceding value and the following value, e.g., greater than 49.5 and less than 52.5. Further, expressions of values such as "less than about ~" or "greater than about ~" should be understood in view of the definition of the term "about" provided herein. 【0020】 In general, it will be understood by those skilled in the art that the terms used herein are generally intended to be “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “at least having,” 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 comprehensive or open-ended and does not exclude additional, non-reproducible elements or process steps. Various compositions, methods, and apparatuses are described using the term "comprising" various components or processes (to be interpreted as "including, but not limited to"), but compositions, methods, and apparatuses can also "consist essentially of" or "consist of" various components and processes, and such terms should be interpreted as defining a closed-member group. In contrast, the transitional term "consisting of" excludes elements, processes, or components not specified in the claims. The transitional term "consist essentially of" limits the scope of the claims to specified materials or processes that "do not substantially affect the basic and novel features" of the claimed invention. 【0021】 As used herein, the term “therapeutic” means an agent used to treat, combat, improve, or enhance an undesirable condition or disease in a patient. In part, embodiments of the present invention are directed toward the treatment of eosinophil-associated inflammation and / or other eosinophil-associated diseases and conditions. 【0022】 The term "effective amount" as used herein refers to the amount of the compound appropriate to perform the purpose of the compound, including imaging of the tissue of the subject, diagnosing the disorder of the subject, and / or monitoring the symptoms or disorder of the subject, when administered to the subject. The actual amount constituting the "effective amount" varies depending on many factors, including but not limited to the severity of the disorder, the patient's physique and health condition, imaging method, diagnostic method, monitoring method, and route of administration. A skilled physician can easily determine the appropriate amount using methods known in the medical technology field. 【0023】 In this specification, the term “therapeutically effective amount” is used to refer to the amount of a compound that, when administered to a subject, can alleviate the symptoms of the disorder in that subject or enhance the texture, appearance, color, sensation, or hydration of the intended tissue treatment area. The actual amount constituting the “therapeutic effective amount” varies depending on many factors, including but not limited to the severity of the disorder, the patient’s physique and health condition, and the route of administration. A skilled physician can readily determine the appropriate amount using methods known in the medical technology field. 【0024】 The term "pharmaceutically acceptable" is used herein to mean a drug / compound, salt, composition, dosage form, etc., of interest that, within the bounds of sound medical judgment, is suitable for use in contact with human and / or other mammalian tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio. In some embodiments, pharmaceutically acceptable means that it is approved by a federal or state regulatory agency or is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in mammals (e.g., animals), more specifically in humans. 【0025】 The terms “patient” and “subject” are interchangeable and are interpreted to mean any living organism treated with the composition 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, “patient” or “subject” is a mammal such as a mouse, rat, other rodent, rabbit, dog, cat, pig, cattle, 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 healthcare professionals by specific occupation or role, no part of this disclosure is intended to limit itself to any particular occupation or function. A physician or healthcare professional includes any physician, nurse, healthcare professional, or technician. Any of these terms or occupations can be used interchangeably with users of the systems disclosed herein, unless otherwise expressly distinguished. For example, a reference to a physician may, in some embodiments, also apply to a technician, nurse, or other healthcare provider. 【0027】 "Tissue" refers to a collection of similarly specialized cells that have come together to perform a specific function. 【0028】 In this disclosure, the term “disorder” means, unless otherwise specified, a disease, condition, or illness, and is used interchangeably with it. 【0029】 As used herein, the terms “administer,” “administering,” or “administration” refer to the direct administration of a compound (also called the substance of interest), a pharmaceutically acceptable salt of the compound (the substance of interest), or a composition to a subject by the subject or a healthcare provider. 【0030】 As used herein, the terms “treat,” “treated,” or “treating” refer to both therapeutic actions, the purpose of which is to reduce the frequency or delay the onset of symptoms of a medical condition, or otherwise to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, reversal, reduction, or mitigation of symptoms of a condition; reduction of the degree of a condition, disorder, or disease; stabilization (i.e., not worsening) of a condition, state, disorder, or disease; delay of the onset or progression of a condition, disorder, or disease; improvement of a condition, disorder, or disease; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of a condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive side effects. Treatment also includes extending survival compared to the survival expected if no treatment were received. 【0031】 As used herein, the terms “diagnose,” “diagnosing,” or “diagnosis” refer to the process of identifying the presence and / or nature of a disease, condition, or other physiological state in a subject based on its characteristics, signs, and symptoms. A diagnosis includes any statement or conclusion relating to a disease, condition, or other physiological state in a subject based on such a process. 【0032】 The term "inhibiting" includes, by administering the compositions of the present invention, preventing the onset of symptoms, alleviating symptoms, reducing symptoms, delaying or decreasing the progression of a disease and / or its symptoms, or eliminating a disease, condition or disorder. 【0033】 In some embodiments, the methods and compositions disclosed herein can be used with or on subjects requiring such examination, diagnosis, monitoring, and / or treatment, also referred to as “in need thereof.” As used herein, the phrase “in need thereof” means that a subject has been identified as having a particular need for method or treatment, or as having a condition, and that the method (e.g., imaging of tissue, diagnosis of a condition, monitoring of a condition) or treatment is used with or on the subject for that particular purpose. 【0034】 Compositions produced by the method of the present invention can be administered conventionally by any route through which they are active. Administration may be systemic, topical, or oral. For example, administration may be parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, transbuccal, or ocular, or vaginal, inhalation, depot injection, or implant. Thus, the mode of administration (either alone or in combination with other pharmaceuticals) may be sublingual, injectable (including short-acting subcutaneous or intramuscular injections, depot, implant, and pellet forms), topical (including nasal sprays, ointments, or creams for application to the skin), and / or transdermal, such as vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, patches, or creams. 【0035】 The specific method of administration varies depending on the efficacy or purpose. The choice of a specific route of administration and dosage is adjusted or escalated by the clinician according to known methods to obtain the optimal clinical response. The amount of compound administered is an effective amount. The dosage depends on the characteristics of the subject being treated, such as the specific animal being treated, age, weight, health condition, the type of concurrent treatment if any, and the frequency of treatment, and can be easily determined by those skilled in the art (e.g., clinicians). 【0036】 For oral administration, compositions can be readily formulated by combining high-purity, high-molecular-weight heparin with pharmaceutically acceptable carriers well known in the art, according to the methods herein. Such carriers enable the formulation of the compounds of the present invention as tablets, pills, coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral intake by patients being imaged, diagnosed, and / or treated. Oral pharmaceutical formulations can be obtained by adding solid excipients, optionally grinding the resulting mixture, adding suitable adjuvants as desired, and then processing the granular mixture to obtain tablet or coated tablet cores. Suitable excipients include, but are not limited to, sugar fillers including lactose, sucrose, mannitol, and sorbitol, and cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrants such as, but not limited to, cross-linked polyvinylpyrrolidone, agar, alginic acid or its salts (e.g., sodium alginate) may be added. 【0037】 Pharmaceutical compositions for oral use include, but are not limited to, press-fit capsules made of gelatin, and sealed capsules made of gelatin and a plasticizer (such as glycerol or sorbitol). Press-fit capsules may 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 may be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Further stabilizers may also be added. Capsules may also be coated with an additional layer to protect the contents through one or more stages of digestion and / or to delay the release of the contents. For example, capsules or other carriers may include an enteric coating (e.g., formed by a polymer) to prevent dissolution or disintegration in the gastric environment. All compositions for oral administration should be in a dose suitable for such administration. 【0038】 As used herein, the term “carrier” encompasses carriers, excipients, and diluents, and means materials, compositions, or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, that are involved in transporting or delivering pharmaceuticals, cosmetics, or other agents across tissue layers, such as the stratum corneum or stratum spinosum. A pharmaceutical composition of a compound may also contain a suitable solid or gel-phase carrier or excipient. Examples of such carriers or excipients, but not limited to, include calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycol. 【0039】 In this specification, “mucosal tissue” refers to the tissue that lines various cavities within the body. Examples of mucosal tissue include, but are not limited to, the mucosal tissue lining the nose, 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 the epithelial surface, mucus-secreting glandular epithelium, basement membrane, and submucosa containing connective tissue. 【0040】 As used herein, "eosinophil granule protein" refers to the proteins that make up the granules of eosinophils. When eosinophils are activated, granule proteins are released from the cell into the surrounding tissue. The released granule proteins trigger a pathological inflammatory response in the surrounding tissue, such as the esophageal mucosa. 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, and the teaching of examples of eosinophil granule proteins is incorporated herein by reference. 【0041】 As used herein, “high molecular weight heparin” refers to heparin and / or heparin salts (e.g., heparin sodium) having a molecular weight of approximately 20 kDa or greater. Heparin polymers are typically polydisperse linear polymers, i.e., mixtures of polymers with molecular chains of varying lengths, and the molecular weight of the heparin chains varies and cannot be fully described by a single number. Therefore, high molecular weight heparin is described in more detail as having an average molecular weight of approximately 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). Furthermore, high molecular weight heparin may have different polydispersity than unfractionated heparin, as further described herein. Polydispersity can be quantified as a polydispersity index (PDI): 【number】 Here, M w M is the weight-average molecular weight of the sample (i.e., the sum of the molecular weights of each molecule multiplied by the weight fraction of each molecule relative to the total weight of the sample). N is the number-average molecular weight of the compound. In some embodiments, high molecular weight heparin has lower polydispersity than unfractionated heparin. In some embodiments, high molecular weight heparin has higher polydispersity than unfractionated heparin. In other embodiments, high molecular weight heparin has substantially the same polydispersity as unfractionated heparin. 【0042】 As used herein, “low molecular weight heparin” refers to heparin and / or heparin salts (e.g., heparin sodium) having a molecular weight of approximately 8 kDa or less. For example, enoxaparin is a product of the low molecular weight heparin family with a molecular weight of approximately 4.5 kDa. Heparin polymers typically consist of a mixture of polydisperse linear polymers, i.e., those with molecular chains of varying lengths, and the molecular weight of the heparin chains varies and cannot be fully described by a single number. Therefore, low molecular weight heparin is described in more detail as having an average molecular weight of less than approximately 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 heparin may vary depending on the method of depolymerization. In some embodiments, low molecular weight heparin has lower polydispersity than unfractionated heparin, as further described herein. In other embodiments, low molecular weight heparin has substantially the same and / or higher polydispersity as unfractionated heparin. 【0043】 As used herein, "unfractionated heparin" or "heparin" refers to heparin polymers with molecular chains of varying lengths and molecular weights ranging from 3 to 30 kDa. "Unfractionated heparin" or "heparin" may have greater polydispersity than high-molecular-weight heparin or low-molecular-weight heparin, and is not fractionated to sequestrate fractions of molecules with a specific, limited range of molecular weights. In other examples, unfractionated heparin may have lower or substantially equal polydispersity to high-molecular-weight heparin or low-molecular-weight heparin. 【0044】 As used herein, “radiolabel” refers to an isotopic composition that can be bound to a substance, such as heparin, and can be tracked as the substance passes through a system or tissue. Non-exclusive examples of radiolabeled substances include, but are not limited to, radiolabeled high molecular weight heparin, radiolabeled low molecular weight heparin, and radiolabeled unfractionated heparin. As provided herein, the methods described herein are used with, but are not limited to, radiolabeled high molecular weight heparin, radiolabeled low molecular weight heparin, and radiolabeled unfractionated heparin. In some embodiments, radiolabeled heparin 99m This is Tc-heparin. Other examples of radioactive labels, but not limited to these, include 111In, 14C, 3H, 13N, 18F, 51Cr, 125I, 133Xe, 81mKr, and 131I. Table 1 shows other radioactive labels that can bind to substances, such as heparin. Radioactive labels, e.g. 99m Tc can be bound to a substance, such as heparin, using commercially available reagents well known to those skilled in the art. In some embodiments, 99m Tc-heparin can be prepared as shown in Example 5 below. [Table 1] TIFF0007873679000003.tif193141 【0045】 You may, for any reason, request less than the full scope of this disclosure, including any sub-scope or combination of sub-scopes within the group, which may be claimed in accordance with the scope or similar method herein, with the proviso reserved to exclude or omit any individual member of such group. Furthermore, you may, for any reason, request less than the full scope of this disclosure, with the proviso reserved to exclude any individual substituent, structure or group thereof, or any member of the claimed group. Throughout this disclosure, various patents, patent applications, and publications are referenced. The disclosures of these patents, patent applications, and publications are incorporated into this disclosure in their entirety by reference to more fully describe the state of the art known to those skilled in the art as of the date of this disclosure. In the event of any conflict between the cited patents, patent applications, and publications and this disclosure, this disclosure shall prevail. 【0046】 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Nothing in this disclosure shall be construed as acknowledging that the embodiments described herein do not have prior rights to such disclosure under prior art. 【0047】 As discussed herein, high molecular weight heparin is effective in localizing to eosinophil-associated inflammation sites. Furthermore, high molecular weight heparin is effective in neutralizing the toxic effects of MBP-1, as well as 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. Consequently, the amount of heparin (e.g., high molecular weight heparin) used for localization of eosinophil-associated inflammation can be reduced in the expectation that a larger 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 inherent to heparin makes it very difficult to isolate chains of a specific molecular weight. While methods for producing low molecular weight heparin compounds have been successful due to the growing interest in low molecular weight heparin in the medical field, similar progress has not been made in the development of fractionation methods for high molecular weight heparin. Furthermore, even when heparin compounds with a target average molecular weight (e.g., low molecular weight heparin) are successfully produced, there is often considerable variability in molecular weight, and the proportion of heparin chains within the target molecular weight range may not be high. 【0049】 Method for producing high molecular weight heparin compounds Referring here to Figure 1, a flow chart of an exemplary method for producing a high molecular weight heparin (HMWH) compound according to an embodiment is shown. As shown in Figure 1, Method 100 includes a step 105 of dissolving heparin (i.e., the starting material) to form a heparin solution, and a step 115 of fractionating the heparin solution via tangential flow filtration (TFF) using a membrane having a fractional molecular weight cutoff (MWCO) between about 8 kDa and about 12 kDa, for example, about 10 kDa. According to Method 100, the TFF yields a recovery product containing fractional heparin having an average molecular weight of 20 kDa or more, i.e., a high molecular weight heparin compound. In some embodiments, the fractional heparin is of high purity, i.e., a substantial portion of the heparin chains in the fractional heparin has 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 heparin sodium, heparin calcium, and / or additional heparin salts known to those skilled in the art. For example, the starting material may be USP heparin sodium, i.e., heparin sodium that meets the quality standards of the United States Pharmacopeia. Other types of commercially available heparin preparations are also intended herein. 【0051】 Referring again to Figure 1, step 105 for dissolving the heparin starting material is described in more detail here. In some embodiments, step 105 for dissolving the heparin starting material includes dissolving heparin in a salt solution. In some embodiments, the salt solution includes a sodium chloride (NaCl) solution. However, as will be apparent to those skilled in the art, various salt solutions can be used herein. 【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. Therefore, the concentration of the salt solution can be refined based on desired parameters of the heparin product. For example, if the salt concentration is too low, permeability decreases, and heparin chains containing low molecular weight are hardly filtered out. Therefore, if the salt concentration is too low, the purity of the heparin product decreases. In another example, if the salt concentration is too high, permeability increases, and all or substantially all of the heparin passes through the membrane and is filtered out. Therefore, if the salt concentration is too high, the yield of the heparin product decreases. Therefore, 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 solution are intended herein. For example, salt solutions include molar concentrations greater than approximately 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, and 200 mM, or individual values ​​or ranges in between. 【0053】 Heparin sodium and salt solution may be combined in various ratios during step 105, which involves dissolving the heparin starting material. In some embodiments, about 2 g of heparin sodium is combined with about 50 mL of salt solution, which is approximately 0.04 g / mL. However, it should be understood that this ratio is merely illustrative and may be modified as will be apparent to those skilled in the art. In some embodiments, the heparin sodium and salt solution may be combined in ratios of individual values ​​or ranges greater than or between approximately 0.01 g / mL, approximately 0.02 g / mL, approximately 0.03 g / mL, approximately 0.04 g / mL, approximately 0.05 g / mL, approximately 0.1 g / mL, approximately 0.2 g / mL, approximately 0.2 g / mL, and / or between. Furthermore, this step can be significantly scaled up to produce larger batches of heparin products. As will be further discussed, the method described herein is advantageous because it can be scaled up significantly with only minor modifications to the process, without compromising the average molecular weight and / or purity of the compound, thereby providing a commercial advantage over conventional methods for the production of high molecular weight heparin. 【0054】 As shown in Figure 1, in some embodiments, the method further includes a step 110 of sterilizing the heparin solution by filtering it 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 various pore sizes configured to sterilize the fluid may be utilized. 【0055】 Step 115, the fractionation of the heparin solution via tangential flow filtration, will now be described in more detail. To fully convey the improvements made by the methods described herein, TFF will first be described in general terms from the perspective of its conventional uses. TFF (also known as direct current filtration) is a rapid filtration method for the separation and purification of biomolecules. It can be applied to a wide range of biological fields, such as fractionating large and small biomolecules. Typically, TFF is designed for the processing or separation of globular proteins with a consistent structure. 【0056】 In a typical TFF process using globular proteins, the TFF involves concentrating target molecules in the feed solution by passing the feed solution tangentially over the surface of a membrane having pores with a predetermined molecular weight cutoff (MWCO). Positive pressure (i.e., back pressure) can be applied to the feed side of the membrane to facilitate molecular circulation and passage through the membrane. Some molecules in the solution smaller than the MWCO permeate the membrane and are called the permeate or filtrate. Molecules in the solution larger than the MWCO are generally retained on the feed side of the membrane and are called the retaining solution. As relatively small molecules are removed and the overall volume of the solution decreases, the retained target molecules become concentrated in the retaining solution. 【0057】 A typical TFF process further includes the step of dialysfiltration of the retained solution by adding fresh solvent to the feed solution to replace the amount of permeate removed. In some embodiments, dialysfiltration is performed intermittently (i.e., discontinuous dialysfiltration) while concentration is performed continuously, thereby circulating the solution through the concentration and dilution steps until the solution is sufficiently fractionated. In some embodiments, dialysfiltration is performed continuously. For example, the solvent can be added at the same rate as the permeate flow rate, i.e., the concentration rate, such 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., 1 dialysfiltration volume or DV). However, additional volumes may be utilized for dialysfiltration at individual values ​​or ranges greater than, for example, about 1 DV, about 2 DV, about 3 DV, about 4 DV, about 5 DV, about 10 DV, about 20 DV, or 20 DV. Each additional DV makes it easier to remove more molecules smaller than MWCO, resulting in a more complete fractionation (i.e., a level of "purity" as defined and further described herein). 【0058】 Returning to this embodiment, it should be understood that commercially available TFF systems (e.g., Minimate TFF Systems, available from Pall Corporation in Port Washington, New York) have conventionally been used to process globular proteins with a consistent structure. Therefore, the expected results, including the described MWCO, are determined within this context. The described MWCO of a membrane is defined as the expected MWCO for processing globular proteins, based on pore size and other factors that would be known and understood to those skilled in the art. In contrast, heparin is a linear polysaccharide, and the molecular weight of heparin units generally does not correspond to the molecular diameter, so it may interact with membrane pores in a different way than globular proteins. As a result, the MWCO of commercially available membranes becomes inaccurate when used with heparin. For example, heparin chains with a molecular weight greater than the described MWCO may pass through the membrane at a substantial rate such that the effective MWCO is greater than the described MWCO (see, e.g., Examples 1-3 of this specification). This finding goes beyond the scope of conventional TFF processes and demonstrates that membrane pore size is one of many factors affecting the MWCO of linear polysaccharide TFFs. Due to the inherent heterogeneity of heparin and variable polymer chain length, many of the conditions for performing TFF can alter the effective MWCO. For example, the effective MWCO can vary depending on a combination of factors such as membrane pore size, salt concentration, and applied pressure. Therefore, these factors can be modified to control the effective MWCO, i.e., to adjust it above or below the stated effective MWCO. It should also be understood that the effective MWCO under specific conditions is not absolute, meaning 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. Rather, under specific conditions, molecules smaller than the effective MWCO are generally more easily removed, and molecules larger than the effective MWCO are more easily retained. For example, MWCO and / or nominal molecular weight cutoff (NMWCO) are sometimes defined for membranes or other filtration components as the lowest molecular weight of the solute in which more than 90% of the solute is retained by the membrane.Therefore, MWCO can objectively measure membrane permeability in a manner generally defined and understood by those skilled in the art. Furthermore, as discussed herein, since MWCO is based on the treatment of globular proteins, effective MWCO may differ with respect to non-globular proteins. 【0059】 Referring again to Figure 1, step 115 for fractionating the heparin solution via tangential flow filtration includes step 115A for concentrating the heparin solution and step 115B for dialyseptic filtration of the heparin solution using a membrane having a predetermined MWCO. In some embodiments, the TFF is performed using a membrane having a described MWCO of about 10 kDa. In some embodiments, the TFF is performed using a membrane having a described MWCO in the range of about 8 kDa to about 12 kDa. However, other described MWCOs are used in the TFF under a suitable set of conditions as described herein (i.e., with modifications to salt concentration, applied pressure, total operating time, total filtration volume, etc.) to produce the desired effective MWCO. For example, TFF is performed using a membrane with MWCOs specified in individual values ​​or ranges greater than or between approximately 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 12kDa, 14kDa, 16kDa, 18kDa, 20kDa, and approximately 20kDa. 【0060】 In some embodiments, MWCO is related to the size and / or diameter of pores and / or openings penetrating the membrane material. Therefore, in some embodiments, the membrane used to carry out TFF is described with respect to the nominal or average pore diameter of the membrane. In some embodiments, TFF is carried out using a membrane having an average pore size (i.e., diameter) of about 5 nm. In some embodiments, TFF is carried out using a membrane having an average pore diameter (i.e., diameter) of about 4 nm to about 6 nm. However, other pore diameters may be utilized for TFF under a suitable set of conditions (i.e., with modifications to salt concentration, applied pressure, total operating time, total filtration rate, etc.) as described herein, in order to produce the desired effective MWCO. For example, TFF is carried out using films with average pore sizes having individual values ​​or ranges greater than or between approximately 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm, 7 nm, 7.5 nm, 8 nm, 8.5 nm, 9 nm, 9.5 nm, 10 nm, and 10 nm. It should also be understood that pore sizes are not constant within a film material and can vary significantly between film materials. Therefore, pore sizes exceeding the ranges explicitly described herein can, in some cases, be carried out under appropriate conditions to produce the desired effective MWCO. 【0061】 In some embodiments, the membrane is a polyethersulfone (PES) membrane. In one specific example, the membrane is a PES membrane having a polyolefin support in the form of a cassette or other standard membrane structure (e.g., T-Series TFF Cassettes available from Pall Corporation in Port Washington, New York). In additional embodiments, the membrane is a hollow fiber membrane (e.g., Microza Hollow Fiber Membrane Systems available from Pall Corporation in Port Washington, New York). For example, hollow fiber membranes are formed using polyvinylidene fluoride (PVDF) and / or polyacrylonitrile (PAN). In some embodiments, hollow fiber membranes offer higher filtration speed, efficiency, and / or overall yield compared to other conventional membrane materials. For example, hollow fiber membranes can improve filtration speed and efficiency by reducing clogging. However, as is known to those with a normal level of skill in the art, 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. 【0062】 In some embodiments, the membrane utilizes a porous support and / or a nonwoven support. For example, the support is formed from a polyolefin. In another example, the support is formed from acrylonitrile butadiene styrene (ABS). In yet another example, the support is formed from polyvinyl chloride (PVC). However, it should be understood that a variety of supports are available, as will be known to those skilled in the art. 【0063】 In some embodiments, the applied pressure in the TFF is approximately 29 psi (see Examples 1-2). In some embodiments, the applied pressure in the TFF is approximately 30 psi (see Example 3). However, the applied pressure may be varied under appropriate corresponding conditions to generate the desired effective MWCO. For example, the applied pressure may be approximately 1 psi, approximately 5 psi, approximately 10 psi, approximately 15 psi, approximately 20 psi, approximately 25 psi, approximately 30 psi, or any individual value or range in between. 【0064】 In some embodiments, the total filtration volume used in step 115B for dialysfiltration is approximately 1800 mL, or 36 DV. In some embodiments, the total filtration volume used for dialysfiltration is approximately 2050 mL, or 41 DV. Since the total filtration volume affects the amount of low molecular weight particles removed through the TFF, it should be understood that the selected total filtration volume also affects the "purity" of the fractionated heparin as defined and described herein. In some embodiments, the purity of the fractionated heparin is directly related to the total filtration volume. Therefore, the total filtration volume can be varied under appropriate corresponding conditions to produce the desired effective MWCO. For example, the total filtration volume may be approximately 5 DV, approximately 10 DV, approximately 20 DV, approximately 30 DV, approximately 40 DV, approximately 50 DV, approximately 100 DV, or any individual value or range in between. 【0065】 In some embodiments, step 115, which fractionates the heparin solution via TFF, yields a retention solution consisting of fractionated heparin having an average molecular weight greater than the average molecular weight of the heparin starting material. In some embodiments, the average molecular weight of the fractionated heparin is 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 consists of an average molecular weight greater than about 20 kDa. For example, the fractionated heparin may include average molecular weights of individual values ​​or ranges greater than or between 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, about 40 kDa, or greater than or between these values. 【0066】 In some embodiments, fractionated heparin has high purity. The purity of fractionated heparin is defined as the amount of heparin chains having a molecular weight above a predetermined threshold. For example, the predetermined threshold may be about 20 kDa, and accordingly, the purity of fractionated heparin is determined based on the fraction, proportion, or ratio (i.e., the proportion of high molecular weight heparin) of heparin chains having a molecular weight of 20 kDa or more compared to heparin chains having a molecular weight of less than about 20 kDa. In some embodiments, fractionated heparin has a purity of at least about 50% heparin chains of 20 kDa or more, i.e., "high purity" (see Examples 1-3, where the average molecular weight is 20 kDa or more). In further embodiments, fractionated heparin has a purity of heparin chains of 20 kDa or more in individual values ​​or ranges greater than or between about 60%, about 70%, about 80%, about 90%, about 95%, about 95%, or about 95%. 【0067】 In some embodiments, fractionated heparin is further characterized by the maximum amount of molecular chains having a molecular weight below a predetermined threshold. For example, fractionated heparin includes a proportion of heparin chains with a molecular weight of less than 20 kDa in individual values ​​or ranges between 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 of the individual values ​​or ranges in between. In additional embodiments, fractionated heparin is further characterized by the maximum amount of low molecular weight heparin chains within it, i.e., the amount of heparin chains having a molecular weight below a fractionation value that defines low molecular weight heparin (e.g., about 8 kDa). For example, fractionated heparin includes the proportion of heparin chains having a molecular weight of less than approximately 8 kDa, in individual values ​​or ranges within the following categories: approximately 50% or less, approximately 40% or less, approximately 30% or less, approximately 25% or less, approximately 20% or less, approximately 15% or less, approximately 10% or less, approximately 5% or less, less than approximately 5%, or any of the individual values ​​or ranges in between. 【0068】 Furthermore, in some embodiments, the predetermined threshold is a value other than approximately 20 kDa. For example, the predetermined threshold is set based on the minimum desired average molecular weight of the fractionated heparin. In some embodiments, the predetermined threshold for evaluating the purity of the fractionated heparin is an individual value or range greater than or between approximately 20 kDa, approximately 21 kDa, approximately 22 kDa, approximately 23 kDa, approximately 24 kDa, approximately 25 kDa, approximately 26 kDa, approximately 27 kDa, approximately 28 kDa, approximately 29 kDa, approximately 30 kDa, approximately 35 kDa, approximately 40 kDa, approximately 40 kDa, or approximately 40 kDa. Similarly, the fractionation value of the low molecular weight chain may be a value other than approximately 8 kDa. For example, the fractions may be values ​​greater than approximately 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 11kDa, 12kDa, or 12kDa, or any individual value or range in between. 【0069】 As described herein, the properties of the resulting fractionated heparin are controlled by the conditions of the TFF step in the fractionation step 115. For example, the average molecular weight and / or purity of the fractionated heparin varies based on a combination of factors including membrane pore size (i.e., MWCO as described), salt concentration, applied pressure, total operating time, and total filtration amount. These factors can therefore be adjusted to produce fractionated heparin with desired properties. 【0070】 In certain cases, the average molecular weight of the fractionated heparin is selected to be 20 kDa or higher, and the purity of the fractionated heparin is selected to be 50% or higher. Therefore, the dissolution step 105 can be carried out using a NaCl solution of about 100 mM. Furthermore, the concentration step 115A of the heparin solution is carried out using a MWCO of about 10 kDa and a membrane with an applied pressure of about 29-30 psi. Furthermore, the dialysieving step 115B of the heparin solution can be carried out 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, under these conditions, fractionated heparin with an average molecular weight of 20 kDa or higher and a purity of 50% or higher can be obtained. 【0071】 Once the fractionation step 115 is complete, the retaining solution can be recovered from the supply side of the membrane. Furthermore, the membrane can be washed with deionized water to obtain a washing solution. Since the washing solution may contain high molecular weight heparin recovered on the membrane during the fractionation step 115, the washing solution may be combined with the backflow solution to improve the yield of fractionated heparin. 【0072】 Referring again to Figure 1, method 100 further includes a step 120 of desalting the fractionated heparin. In some embodiments, the desalting step 120 is carried out via TFF under appropriately selected conditions. 【0073】 In some embodiments, the desalting step 120 typically includes a step 120A of concentrating the fractionated heparin using a membrane having a MWCO configured to obstruct the passage of the fractionated heparin and allow the passage of salts and / or their ions in a salt solution. In some embodiments, a membrane having an MWCO of about 3 kDa is used. However, membranes having MWCOs of individual values ​​or ranges in between, such as about 1 kDa, about 3 kDa, about 5 kDa, greater than about 5 kDa, or in between, 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 is apparent to those with a normal level of skill in the art. 【0074】 In some embodiments, the desalting step 120 further includes the step 120B of dialysfiltration of the fractionated heparin using deionized water. In some embodiments, the dialysfiltration step 120B is carried out with a total filtration volume of about 10 DV, for example, about 500 mL. However, the total filtration volume may be varied as is apparent to those of the usual level of skill in the art. 【0075】 While an exemplary step 120 for desalting fractionated heparin is described herein, it should be understood that various conventional methods of desalting are also used herein, as will be apparent to those of the ordinary level of art. 【0076】 Referring again to Figure 1, Method 100 further includes a step 125 of drying the fractionated heparin and a step of producing the HMWH compound isolated thereby. In some embodiments, the drying step 125 is carried out by freeze-drying. However, it should be understood that the drying step 125 may be carried out by any conventional means as would be obvious to a person of the ordinary level of art in the art. 【0077】 As shown in Examples 1-3, the disclosed method produces HMWH compounds in a yield of approximately 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., the total operating time is longer for a given filtration volume). In another example, reducing the total filtration volume results in faster fractionation (i.e., a shorter total operating time for a given filtration volume) and improves the yield. In yet another example, reducing the membrane pore size results in a fraction with improved yield. Adjusting the salt concentration may also lead to an improvement in the yield of the method disclosed herein. 【0078】 In some embodiments, using heparin starting materials with a higher average molecular weight also results in higher yields. For example, using heparin starting materials pre-filtered by molecular weight may result in higher yields. In another example, using heparin starting materials pre-filtered by properties that correlate approximately with molecular weight may also result in higher yields. 【0079】 In some embodiments, the method disclosed herein produces further useful by-products. For example, the filtrate or permeate (i.e., the substance removed from the holding solution via TFF) contains heparin with a substantially reduced average molecular weight. In some embodiments, the permeate contains LMWH. In some embodiments, the permeate is treated to produce LMWH through an additional fractionation step. Such fractionation steps are known to those skilled in the art. Thus, the method disclosed herein can be used, with or without additional steps, to produce LMWH compounds as a by-product along with HMWH compounds. 【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 used to achieve this step. In additional embodiments, alternative types of mechanical filtration, as known to those skilled in the art, are used to fractionate heparin. Thus, some or all of the remaining steps described herein may be used in combination with such alternative filtration methods to produce the final high molecular weight heparin compound. 【0081】 When attempting to produce HMWH compounds isolated by conventional methods of molecular weight filtration, various difficulties are encountered, and it should be understood that the currently disclosed method is advantageous. In general, the inherent heterogeneity of heparin's linear structure and polymer chain length (and therefore molecular weight) hinders the ability of conventional methods to fractionate heparin of higher molecular weight with substantial purity. However, the method disclosed herein shows 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, where more than 50% of the heparin chains have a molecular weight of 20 kDa or more. Furthermore, these properties of the resulting heparin fraction can be carefully controlled by adjusting the TFF conditions, such as membrane pore size, salt concentration, applied pressure, and filtration rate. 【0082】 The disclosed method is further advantageous due to its scalability. For example, while it may be possible to produce HMWH compounds by gel filtration chromatography, such a process is relatively high-cost and presents further difficulties when utilizing large volumes, making scale-up difficult. Other types of membrane filtration can involve a high degree of fouling (accumulation on the membrane), causing clogging, which is even more difficult when dealing with large volumes and / or operating times. In contrast, TFF is relatively low-cost and, by its nature, has a low degree of fouling. Therefore, TFF is highly scalable and can be used to fractionate thousands of liters of solution with only a small additional cost. Thus, the method disclosed herein is highly scalable for production purposes compared to other processes. 【0083】 The methods described herein are not intended to be limited to the specific embodiments described, but are intended only as examples 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 their spirit and scope. 【0084】 HMWH compounds are useful for forming HMWH compositions for various medical applications. In some embodiments, the method further includes the step of preparing an HMWH composition by combining an HMWH compound with a pharmaceutically acceptable excipient. In some embodiments, the HMWH composition is useful for imaging, diagnosing, and / or treating medical conditions. 【0085】 In some embodiments, the HMWH composition is configured for binding to and / or localization to the expression of eosinophil-associated inflammation or eosinophil-associated conditions. In some embodiments, the average molecular weight and / or purity of the HMWH compound are selected to optimize binding to sites expressing 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 eosinophil-associated inflammation sites than LMWH or unfractionated heparin UFH. 【0086】 In some embodiments, the binding affinity of the HMWH composition is directly related to its molecular weight and increases with the average molecular weight of the HMWH compound. Therefore, as the average molecular weight of the HMWH increases, the amount of heparin required for the localization of eosinophil-associated inflammation can be reduced, with the expectation that a larger proportion of the administered heparin will be localized to the site of inflammation. 【0087】 In some embodiments, the localization rate of an HMWH composition is directly related to its molecular weight and therefore increases with increasing purity of the HMWH compound. 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 larger proportion of the administered heparin will localize to the site of inflammation. Similarly, if a higher molecular weight threshold is used to define purity as described herein, the localization rate may increase accordingly. 【0088】 HMWH compositions manufactured by the methods described herein can be configured to be administered conventionally by any route through which they are active. Administration may be systemic, topical, or oral. For example, administration may be parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, transbuccal, or ocular, or vaginal, inhalation, depot injection, or implant. Thus, the mode of administration (either alone or in combination with other pharmaceuticals) may be sublingual, injectable (including short-acting subcutaneous or intramuscular injections, depot, implant, and pellet forms), topical (including nasal sprays, ointments, or creams for application to the skin), and / or transdermal, such as vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, patches, or creams. The specific method of administration will vary depending on the indication and purpose. It should also be noted that the risks of HIT and / or HITT suspected to be associated with high molecular weight heparin are limited to systemic administration. Therefore, 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 eosinophil-associated states and / or other target states to which HMWH is localized. Therefore, the method further includes conjugating a tracer, such as a radiolabeled contrast agent, to the HMWH compound. For example, a radiolabeled contrast agent 99mThis is Tc. Additional tracers, such as tracers used in positron emission tomography, may be employed to detect the binding of HMWH to eosinophil-associated inflammation sites. In some embodiments, the tracer is any tracer or label listed in Table 1. Thus, HMWH compositions are administered and used to visualize target conditions using conventional imaging modalities, including, but not limited to, single-photon emission computed tomography (SPECT), positron emission (PET) scans, conventional or computed tomography (CT), magnetic resonance imaging (MRI), or combinations thereof. HMWH compositions with tracers can also be used for the diagnosis and / or monitoring of target conditions based on images acquired as described. In some embodiments, HMWH compositions allow for a reduction in the amount of tracer that must be administered to the patient for proper imaging of the site of the target condition (e.g., eosinophil-associated inflammation). For example, due to the binding activity and localization rate of HMWH to the site, a larger proportion of the HMWH composition is localized to the site compared to unfractionated heparin or low molecular weight heparin, thus reducing the amount (or dosage) of tracer administered. Therefore, if the tracer is radioactive, the amount of radioactive material required for sufficient imaging is reduced, improving the safety of the composition and limiting any effects associated with the administration of radiolabeled contrast agents. 【0090】 In some embodiments, the HMWH composition is configured for the treatment of eosinophil-associated conditions and / or other target conditions that express toxins to which HMWH localizes. Therefore, the method further comprises conjugating a therapeutic agent to the HMWH composition. In some embodiments, the HMWH composition further comprises a therapeutically effective amount of the therapeutic agent for administration to a patient. In some embodiments, the therapeutic agent is configured to have a therapeutic effect against 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 adequate care can be reduced, thereby limiting any side effects associated with the administration of the therapeutic agent. Therefore, the therapeutically effective amount of the therapeutic agent is less than the therapeutically effective amount typically associated with the 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 solu-medrol. Further therapeutic agents are intended herein, as will be apparent to those skilled in the art. 【0091】 In some embodiments, the HMWH includes various additional components or additives that are known to those with an ordinary level of skill in the art. In some embodiments, the method further includes adding a stabilizer to the HMWH composition. In some embodiments, the method further includes adding a flavoring or deodorizing agent to the HMWH composition. 【0092】 Although the present invention has been described in considerable detail with reference to certain preferred embodiments, other versions are also possible. Therefore, the spirit and scope of the appended claims should not be limited to the descriptions and preferred versions contained herein. Various aspects of the present invention will be described with reference to the following non-limiting embodiments. [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. This heparin solution was filtered using a tangential flow filtration (TFF) system. A membrane with a molecular weight cutoff (MWCO) of 10 kDa was attached to the TFF system and washed with deionized water before processing. The heparin solution was then flowed through the TFF system and filtered under a back pressure of 29 psi. A 100 mM NaCl substitution solution was added during filtration to maintain a constant volume of retained liquid (i.e., a dialysis filtration step). The process was stopped when the total volume of permeate reached 1800 mL. The retained liquid was then desalted and dried as described in Example 4 to obtain the HMWH composition. 【0094】 Results: This process yielded 297 mg of heparin in a yield of 15%. The average molecular weight of this composition was 23.9 kDa (i.e., high molecular weight overall). Furthermore, 56% of the heparin chains in the HMWH composition had a molecular weight greater than 20 kDa (i.e., high purity). Anti-Xa and anti-IIa factor assays were also performed on the composition to determine the anti-Xa:anti-IIa activity ratio 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. This heparin solution was filtered using a TFF system. A 10 kDa MWCO membrane was attached to the TFF system and washed with deionized water before processing. The heparin solution was then flowed through the TFF system and filtered under a back pressure of 29 psi. A 100 mM NaCl substitution solution was added during filtration to maintain a constant volume of retained liquid (i.e., a dialysis filtration step). The process was stopped when the total volume of permeate reached 2050 mL. The retained liquid was then desalted and dried as described in Example 4 to obtain the HMWH composition. 【0096】 Results: This process yielded 327 mg of heparin in a yield of 16%. The average molecular weight of this composition was 23.5 kDa (i.e., high molecular weight overall). Furthermore, 54% of the heparin chains in the HMWH composition had a molecular weight greater than 20 kDa (i.e., high purity). Anti-Xa and anti-IIa factor assays were also performed on the composition to determine the anti-Xa:anti-IIa activity ratio relevant to 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. This heparin solution was filtered using a TFF system. A 10 kDa MWCO membrane was attached to the TFF system and washed with deionized water before processing. The heparin solution was then flowed through the TFF system and filtered under a back pressure of 30 psi. A 100 mM NaCl substitution solution was added during filtration to maintain a constant volume of retained liquid (i.e., a dialysis filtration step). The process was stopped when the total volume of permeate reached 2050 mL. The retained liquid was then desalted and dried as described in Example 4 to obtain the HMWH composition. 【0098】 Results: This process yielded 358 mg of heparin in 18% yield. The average molecular weight of this composition was 23.3 kDa (i.e., high molecular weight overall). Furthermore, 53% of the heparin chains in the HMWH composition had a molecular weight greater than 20 kDa (i.e., high purity). Anti-Xa and anti-IIa factor assays were also performed on the composition to determine the anti-Xa:anti-IIa activity ratio relevant to therapeutic benefit. The results of Example 3 are summarized in Table 2. [Examples] 【0099】 Example 4 - Desalting and drying of the retaining liquid Method: After TFF using a 10 kDa MWCO membrane, the retention solution was recovered from the system. The membrane was separately washed with deionized water before removal from the TFF system to obtain a washing solution. This washing solution was combined with the retention solution for desalting. A 3 kDa MWCO membrane was attached to the TFF system and washed with pure water. Then, the mixture of the retention solution / washing solution was fed and diafiltration was performed against deionized water to remove salts. The desalting process was stopped when the total amount of the permeate reached 10 times the amount of the retention solution. 【Example】 【0100】 Example 5- 99m Preparation of Tc heparin A tin chloride solution (40 mg / mL, Sigma 243523) was prepared in deionized water under a nitrogen stream. A 0.5 mL aliquot was filtered and mixed with 1.00 mL of NaCl (1.00 M) and heparin (10,000 IU / mL) without preservatives at 150 mg. Approximately 100 mCi of fresh eluted 99m Tc was added and mixed at room temperature for 30 minutes. An aliquot containing approximately 10 mCi of 99m Tc and 20 mg of heparin was removed for tissue experiments. 【0101】 Results: The labeling affinity was measured by paper chromatography Whatman No. 31 using acetone, and it was confirmed that the binding of heparin and 99m Tc was greater than 97%. 【0102】 Heparin was also analyzed by Sephadex G25 column chromatography (HiTrap 5 mL desalting column, GE healthcare, 17140801), using 0.15 M NaCl as the elution buffer, and fractions of approximately 1 mL were collected. This test showed that 99m Tc eluted at all void volumes, confirming that there was no unbound 99m Tc in the radiolabeled heparin. 【0103】 In an acidic environment 99mThe stability of Tc heparin was tested by diluting it with artificial gastric juice (Carolina, 864603), and its properties remained unchanged using both paper chromatography and Sephadex G25. [Examples] 【0104】 Example 6 - Heparin binding to eMBP-1 by SPR The objective of this study is to determine the apparent dissociation rate constant (k) of seven heparin samples that bind to recombinant human (rhu)eMBP using surface plasmon resonance (SPR) Biacore technology. d The objective 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 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 minutes, followed by injection of 80 mM PDEA in 50 mM sodium borate buffer (pH 8.5) for 4 minutes. eMBP1 was diluted to 0.6 μM or 0.06 μM with 10 mM sodium acetate (pH 5.25) and injected until the target immobilization level was reached. Finally, 50 mM L-cysteine ​​in 0.1 M sodium acetate and 1.0 M sodium chloride (pH 4.0) was injected at 10 μL / min for 4 minutes to block the remaining free cysteine. A reference flow cell was prepared using the same immobilization procedure, but without the addition of eMBP1. The rhu eMBP1 was captured in flow cells 2-4 of the sensor chip (i.e., FC2, FC3, FC4) to several different densities in relative units (low density (1000RU), medium density (3000RU), and high density (4000RU), respectively). The second chip was prepared in a similar manner to low density (500RU), medium density (800RU), and high density (1200RU), respectively. 【0108】 Analyte preparation: Analytes in concentrations ranging from 10 μg / mL to 10 ng / mL were prepared by diluting them 10-fold with running buffer. 【0109】 Interaction parameters: Analytes were injected in double or triple in 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 seconds at a flow rate of 25 μL / min, and the dissociation phase was collected for 1800 seconds at a flow rate of 25 μL / min. 【0110】 Surface regeneration: At the end of each binding cycle, the surface was regenerated by pulses of 6M guanidine at a flow rate of 100 μL / min for 2-3 seconds. 【0111】 Data Analysis: Data alignment, double referencing, and fitting were performed 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 globally fitted to a simple exponential decay model for each sample and assay condition. This simple decay model oversimplifies the complex dissociation that occurs between multiple dissociation events resulting from the polydisperse analyte compounded with the eMBP1 surface. 【0112】 Results: Overall, a clear linear relationship was observed between the complex half-life and heparin molecular weight under specific conditions dependent on eMBP1 surface density and heparin concentration. Summary data of the correlation between the complex half-life and heparin molecular weight are shown in Table 3. [Table 3] 【0113】 Referring now to Figures 2A-2B, sensorograms of the signal response of fractionated heparin samples (i.e., analytes) bound to rhu eMBP1 (i.e., ligand) are depicted according to the embodiment. Figure 2A shows the time-course signal response curves of seven heparin samples with different molecular weights at a concentration of 100 ng / mL bound to eMBP1 at a density of 1200 RU, according to the embodiment. Figure 2B shows the time-course normalized signal response curves of seven heparin samples with different molecular weights at a concentration of 100 ng / mL bound to eMBP1 at a density of 1200 RU. Next, referring to Figure 3, a plot graph of complex half-life versus molecular weight is depicted for seven heparin samples with different molecular weights at a concentration of 100 ng / mL bound to eMBP1 at a density of 1200 RU, according to the embodiment. Figures 2A-2B and 3 show the correlation between complex half-life and heparin molecular weight, as discussed herein. 【0114】 Furthermore, several assay conditions deviated from this correlation, highlighting the complexity of multimeric and polydisperse heparin samples binding to the eMBP1 surface. While a general correlation was observed where higher heparin molecular weight correlated with higher binding reactivity, this reaction was not seen under all conditions, including those with the highest binding activity. Under the conditions with the highest binding activity (i.e., high eMBP1 density and low heparin concentration), no clear correlation was observed. This phenomenon can be explained by the fact that the greater the number of subunits binding to each heparin molecule, the more similar the half-lives of the complexes across different molecular weight species become, thereby reducing the dynamic range of the assay. 【0115】 Furthermore, a clear decay of the signal response was observed after each binding cycle. Therefore, the complex half-life, in contrast to the response signal, is a better evaluation tool for this assay. 【0116】 In the detailed description above, reference is made to the accompanying drawings, which constitute part of this specification. In the drawings, unless otherwise indicated in the context, similar reference numerals generally identify similar components. The exemplary embodiments described herein are not intended to be limiting. Other embodiments may be used and other modifications made without departing from the spirit or scope of the subject matter presented herein. It will be readily apparent that various features of this 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 intended herein. 【0117】 This disclosure is not limited in terms of the specific embodiments described in this application, which are intended to be illustrative of various features. Instead, this application is intended to cover any variations, uses, or adaptations of the teachings and to utilize their general principles. Furthermore, this application is intended to cover deviations from this disclosure such that these teachings are within the realm of prior art or common practice. As will be apparent to those skilled in the art, many modifications and variations can be made to the specific embodiments described without departing from the spirit and scope of this disclosure. In addition to those enumerated herein, functionally equivalent methods and apparatus within the scope of this disclosure will be apparent to those skilled in the art from the foregoing description. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems. It should also be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit them. 【0118】 The various features and functions disclosed above, or their substitutes, may be combined into many other different systems or applications. Various substitutes, modifications, variations, or improvements not currently foreseen or anticipated by those skilled in the art may subsequently be made, each of which is also intended to be encompassed by the disclosed embodiments.

Claims

[Claim 1] A method for producing fractionated heparin, wherein the method is: To form a heparin solution, the process involves dissolving heparin in a solvent, and A step of fractionating the heparin solution by tangential flow filtration using a fractionation membrane having a fractionation molecular weight between 3 kDa and 12 kDa, thereby obtaining fractionated heparin having a weight-average molecular weight of 20 kDa or more. The fractionation step, wherein at least 50% of the heparin chains in the fractionated heparin have a molecular weight of 20 kDa or more, Methods that include... [Claim 2] The method according to claim 1, wherein the heparin is USP heparin. [Claim 3] A method according to any one of claims 1 to 2, wherein the solvent is a sodium chloride (NaCl) solution. [Claim 4] The method according to claim 3, wherein the NaCl solution has a concentration of 100 mM. [Claim 5] The method according to claim 1, wherein the fractionated molecular weight of the fractionated film is 5 kDa. [Claim 6] A method according to claim 1, wherein the step of fractionating the heparin solution by tangential flow filtration includes the step of permeating at least a portion of the heparin solution through the fractionation membrane under applied pressure in order to obtain a holding solution containing the fractionated heparin. [Claim 7] The method according to claim 6, wherein the applied pressure is 29 psi to 30 psi. [Claim 8] The method according to claim 6, wherein the applied pressure is 10 psi. [Claim 9] The method according to claim 6, wherein the applied pressure is 15 psi. [Claim 10] A method according to any one of claims 6 to 9, wherein the step of fractionating the heparin solution by tangential flow filtration further comprises adding one or more dialysate filtration volumes (DV) of the solvent during the tangential flow filtration in order to maintain the volume of the retaining solution, thereby defining the total filtration volume. [Claim 11] A method according to any one of claims 1 to 2 and 5 to 9, further comprising the step of filtering the heparin solution through a submicron membrane to sterilize the heparin solution. [Claim 12] A method according to any one of claims 1 to 2 and 5 to 9, further comprising the step of desalting the fractionated heparin. [Claim 13] The method according to claim 12, wherein the step of desalting the fractionated heparin includes the step of performing tangential flow filtration using a desalting membrane having a fractionated molecular weight between 1 kDa and 5 kDa. [Claim 14] The method according to claim 13, wherein the fractional molecular weight of the desalting membrane is 5 kDa. [Claim 15] A method according to any one of claims 1 to 2 and 5 to 9, further comprising the step of drying the fractionated heparin. [Claim 16] A method according to claim 15, wherein the step of drying the fractionated heparin includes the step of freeze-drying the fractionated heparin. [Claim 17] A method according to any one of claims 1 to 2 and 5 to 9, wherein the weight-average molecular weight of the fractionated heparin is 30 kDa or more. [Claim 18] The method according to claim 17, wherein the weight-average molecular weight of the fractionated heparin is 40 kDa or more. [Claim 19] A method according to any one of claims 1 to 2 and 5 to 9, wherein at least 60% of the heparin chains in the fractionated heparin have a molecular weight of 20 kDa or more. [Claim 20] A method according to claim 19, wherein at least 70% of the heparin chains in the fractionated heparin have a molecular weight of 20 kDa or more. [Claim 21] A method for producing high molecular weight (HMW) heparin, wherein the method is: To form a heparin solution with a concentration of 0.01 g / mL to 0.2 g / mL, the process involves dissolving heparin salt in a salt solution, A step of fractionating the heparin solution by tangential flow filtration using a fractionation membrane having a molecular weight cutoff of 5 kDa, thereby obtaining fractionated heparin having a weight-average molecular weight of 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, and the fractionation step, A method comprising, thereby obtaining the HMW heparin. [Claim 22] The method according to claim 21, wherein the heparin salt is selected from the group consisting of heparin sodium and heparin calcium. [Claim 23] A method according to any one of claims 21 to 22, wherein the heparin salt is a USP heparin salt. [Claim 24] The method according to claim 21, wherein the salt solution is a sodium chloride (NaCl) solution. [Claim 25] The method according to claim 24, wherein the NaCl solution has a concentration of 100 mM. [Claim 26] The method according to claim 21, wherein the fractionated molecular weight of the fractionated film is 10 kDa. [Claim 27] A method according to claim 21, wherein the step of fractionating the heparin solution by tangential flow filtration includes the step of permeating at least a portion of the heparin solution through the fractionation membrane under applied pressure in order to obtain a holding solution containing the fractionated heparin. [Claim 28] The method according to claim 27, wherein the applied pressure is 29 psi to 30 psi. [Claim 29] The method according to claim 27, wherein the applied pressure is 10 psi. [Claim 30] A method according to claim 27, wherein the applied pressure is 15 psi. [Claim 31] A method according to any one of claims 27 to 30, wherein the step of fractionating the sterile heparin solution by tangential flow filtration further comprises adding one or more dialysate filtration volumes (DV) of the salt solution during the tangential flow filtration in order to maintain the volume of the retaining solution, thereby defining the total filtration volume, the total filtration volume being 10 DV to 20 DV. [Claim 32] A method according to any one of claims 21 to 22 and 24 to 30, further comprising the step of desalting the fractionated heparin by performing tangential flow filtration using a desalting membrane, wherein the fractionation molecular weight of the desalting membrane is 5 kDa. [Claim 33] A method according to any one of claims 21 to 22 and 24 to 30, wherein the weight-average molecular weight of the fractionated heparin is 30 kDa or more. [Claim 34] The method according to claim 33, wherein the weight-average molecular weight of the fractionated heparin is 40 kDa or more. [Claim 35] A method according to any one of claims 21 to 22 and 24 to 30, wherein at least 60% of the heparin chains in the fractionated heparin have a molecular weight of 20 kDa or more. [Claim 36] The method according to claim 35, wherein at least 70% of the heparin chains in the fractionated heparin have a molecular weight of 20 kDa or more. [Claim 37] In the method according to any one of claims 21 to 22 and 24 to 30, the step of fractionating the heparin solution by tangential flow filtration includes the step of permeating at least a portion of the heparin solution through the fractionation membrane under a transmembrane pressure of 10 psi to 15 psi, wherein (1) heparin chains in the heparin having a molecular weight of less than 20 kDa permeate the filtration membrane as 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 retaining solution containing the fractionated heparin. The method wherein the heparin salt contains heparin sodium, and the salt solution contains a 100 mM sodium chloride (NaCl) solution. [Claim 38] The method according to claim 10, wherein the total filtration amount includes 10 DV to 20 DV. [Claim 39] A method according to any one of claims 1 to 2 and 5 to 9, wherein the step of dissolving heparin in a solvent to form a heparin solution includes the step of dissolving the heparin in the solvent at a concentration of 0.01 g / mL to 0.05 g / mL. [Claim 40] The method according to claim 39, wherein the concentration comprises 0.02 g / mL to 0.03 g / mL. [Claim 41] A method according to any one of claims 1 to 2, 5 to 9, 21 to 22, and 24 to 30, wherein the fractionated film comprises one of a hollow fiber film and a polyethersulfone (PES) film.

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