Method for preserving and administering pre-β high-density lipoprotein extracted from human plasma.

Abstract

Systems and methods for obtaining, storing, and administering delipidated plasma. Extracted delipidated plasma containing pre-β HDL is obtained and spot tested to establish a baseline amount or concentration of pre-β HDL. The batch is subjected to storage, stored, and then reprepared for use at a later date. A portion of the batch can be retested to determine whether the pre-β HDL in the delipidated plasma has deteriorated or become ineffective. [Selection diagram] None

Description

Cross - reference to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 611,098, filed on December 28, 2017, entitled "Method for Treating Cholesterol - Related Diseases", which is hereby incorporated by reference in its entirety.

Technical Field

[0002] The present invention generally relates to a system, apparatus, and method for removing lipids from HDL particles while substantially leaving LDL particles intact by extracorporeal treatment of plasma using either a single solvent or a plurality of solvents for treating chronic cardiovascular diseases and acute kidney diseases. More specifically, the present invention relates to a system and method for preserving and administering pre - βHDL derived from non - autologous defatted plasma.

Background Art

[0003] Familial hypercholesterolemia (FH) is an inherited autosomal dominant disorder characterized by a marked increase in low - density lipoprotein (LDL), tendon xanthomas, and premature coronary heart disease, caused by mutations in the "FH gene" including the low - density lipoprotein receptor (LDLR), apolipoprotein B - 100 (ApoB), or proprotein convertase subtilisin / kexin type 9 (PCSK9). FH presents a clinically recognizable phenotype consisting of the accumulation of LDL in plasma, severe hypercholesterolemia due to cholesterol deposition in tendons and skin, and almost invariably high - risk atherosclerotic arterial disease that appears as coronary artery disease (CAD). FH patients, due to this gene mutation, are unable to effectively metabolize (or remove) excessive plasma LDL, resulting in elevated LDL levels.

[0004] When an individual inherits the defective FH gene from one parent, the form of FH is called heterozygous FH. Heterozygous FH is inherited in an autochromatic dominant manner and is a common genetic disorder, occurring in approximately 1 in 500 people in most countries. When an individual inherits the defective FH gene from both parents, the form of FH is called homozygous FH. Homozygous FH is extremely rare, occurring in approximately 1 in 160,000 to 1,000,000 people worldwide, and is characterized by LDL levels exceeding 700 mg / dl, more than 10 times the ideal level of 70 mg / dl desired for CVD patients. Due to high LDL levels, patients with homozygous FH develop malignant atherosclerosis (narrowing and occlusion of blood vessels) and early heart attacks. This process begins before birth and progresses rapidly. It can affect the coronary arteries, carotid arteries, aorta, and aortic valve.

[0005] Heterozygous fecal hypertension (HeFH) is typically treated with statins, bile acid chelators, or other lipid-lowering drugs that reduce cholesterol levels, and / or by providing genetic counseling. Homozygous fecal hypertension (HoFH) often does not respond well to drug therapy and requires other treatments, including LDL apheresis (removing LDL in a manner similar to dialysis), jejunal bypass surgery that dramatically lowers LDL levels, and, in some cases, liver transplantation. In recent years, some medications have been approved for HoHF patients. However, these medications only lower LDL and, while they may contribute somewhat to slowing the further progression of atherosclerosis, they do not stop its progression. Furthermore, these medications are known to have serious side effects.

[0006] Cholesterol is synthesized in the liver or obtained from the diet. LDL is involved in the transport of cholesterol from the liver to tissues in various parts of the body. However, when LDL accumulates in the artery walls, it is oxidized by oxygen free radicals released from chemical reactions in the body, and interacts harmfully with blood vessels. Modified LDL causes white blood cells in the immune system to aggregate in the artery walls, forming fatty substances called plaque and damaging the cell layer that reinforces blood vessels. Oxidized LDL also reduces the level of nitric oxide, which allows blood to flow freely by relaxing blood vessels. If this process continues, the artery walls slowly constrict, leading to arteriosclerosis and thereby reducing blood flow. As plaque gradually accumulates, coronary arteries can become blocked, potentially leading to a heart attack. Plaque accumulation can also occur in peripheral blood vessels, such as those in the legs, a condition known as peripheral artery disease.

[0007] Obstructions can also occur in the blood vessels supplying blood to the brain, potentially leading to ischemic stroke. The underlying symptom of this type of obstruction is the development of fatty deposits covering the vessel walls. In the United States, it is known that at least 2.7% of men and women aged 18 and older have a history of stroke. The prevalence of stroke is also known to increase with age. With the increase in the elderly population, the prevalence of stroke survivors is projected to increase, particularly among older women. A significant proportion of all strokes (at least 87%) are ischemic in nature.

[0008] Furthermore, hypercholesterolemia and inflammation have been shown to be two major mechanisms involved in the development of atherosclerosis. Vascular risk factors for Alzheimer's disease and atherosclerosis overlap considerably. Inflammation is implicated in the pathogenesis of Alzheimer's disease, and abnormalities in cholesterol homeostasis may also be involved. Additionally, many contributing factors to atherogenesis also contribute to Alzheimer's disease. Specifically, in cell cultures, increased and decreased cholesterol levels promote and inhibit the formation of beta-amyloid (Aβ) from amyloid precursor protein (APP), respectively. Therefore, the use of therapies proven effective against the process of atherosclerosis may be one way to treat the progression of Alzheimer's disease.

[0009] Another common cardiovascular disease resulting from the development of atherosclerosis (hardening and narrowing of the arteries) in the elastic lining of the coronary arteries is coronary artery disease (CAD), also known as ischemic heart disease (IHD). According to statistical data collected from 2009 to 2012, an estimated 15.5 million Americans aged 20 and older have CAD. The overall prevalence of CAD in the United States is 6.2% of adults aged 20 and older.

[0010] A sudden decrease in blood flow to the coronary arteries can cause a portion of the heart muscle to lose its normal function. This condition is known as acute coronary syndrome (ACS). A conservative estimate of ACS discharges in 2010 was 625,000.

[0011] In contrast to LDL, high plasma HDL levels are desirable because they play a major role in "reverse cholesterol transport," where excess cholesterol moves from tissue sites to the liver where it is removed. Optimal total cholesterol levels are below 200 mg / dl, LDL cholesterol levels below 160 mg / dl, and HDL cholesterol levels of 45 mg / dl for men and 50 mg / dl for women. Lower LDL levels are recommended for individuals with elevated cholesterol levels, atherosclerosis, or a history of coronary artery disease. High levels of LDL increase the lipid content of coronary arteries, leading to the formation of rupture-prone, lipid-filled plaques. HDL, on the other hand, has been shown to reduce the lipid content of lipid-filled plaques, thus reducing the likelihood of rupture. In recent years, clinical trials of low-density lipoprotein (LDL) lowering drugs have clearly demonstrated that lowering LDL is associated with 30–45% of clinical cardiovascular disease (CVD) events. Cardiac vascular disease (CVD) events include those occurring in conditions such as HoFH, HeFH, and peripheral artery disease. However, many patients continue to experience cardiac events despite low LDL levels. Low HDL levels are frequently observed in subjects at high risk of CVD, and epidemiological studies have identified HDL as an independent risk factor modulating CVD risk. In addition to epidemiological studies, other evidence suggests that increasing HDL levels reduces the risk of CVD. There is growing interest in altering plasma HDL levels through diet, pharmacology, or genetic engineering as a potential strategy for treating CVD, including HoFH, HeFH, ischemic stroke, CAD, ACS, and peripheral artery disease, as well as for treating the progression of Alzheimer's disease.

[0012] The protein component of LDL known as apolipoprotein B (ApoB) and its products contain atherosclerotic elements. Elevated plasma LDL levels and decreased HDL levels are recognized as major causes of coronary artery disease. ApoB is most abundant in LDL particles and absent in HDL particles. Apolipoprotein AI (ApoA-I) and apolipoprotein A-II (ApoA-II) are found in HDL. Other apolipoproteins such as ApoC and its subtypes (CI, C-II, and C-III), ApoD, and ApoE are also found in HDL. ApoC and ApoE are also observed in LDL particles.

[0013] Numerous major classes of HDL particles have been reported, including HDL2b, HDL2a, HDL3a, HDL3b, and HDL3. Based on the electrophoretic mobility of agarose, the various forms of HDL particles have been described as two major groups: a major fraction with α-HDL mobility and a minority fraction exhibiting mobility similar to VLDL. This latter fraction is called pre-βHDL, and these particles are the most efficient HDL particle subclass for inducing cellular cholesterol efflux.

[0014] HDL lipoprotein particles are composed of ApoA-I, phospholipids, and cholesterol. Pre-βHDL particles are thought to be the first receptors for free cholesterol in cells and are essential for the eventual translocation of free cholesterol and esterified cholesterol to α-HDL. Pre-βHDL particles either translocate cholesterol to α-HDL or are converted to α-HDL. α-HDL then transports cholesterol to the liver, where excess cholesterol can be removed from the body.

[0015] HDL levels are inversely correlated with atherosclerosis and coronary artery disease. When cholesterol-carrying α-HDL reaches the liver, the α-HDL particles separate cholesterol and transport the free cholesterol to the liver. Subsequently, the α-HDL particles (separated from cholesterol) are converted into pre-β-HDL particles and excreted from the liver, where they are used to acquire further cholesterol in the body, then converted back to α-HDL, and the cycle repeats. Therefore, there is a need for a method to reduce or remove cholesterol from these various HDL particles, especially α-HDL particles, so that they can be used to remove further cholesterol from cells.

[0016] Renal artery stenosis refers to an obstruction of the arteries that supply blood to the kidneys and is characterized by two forms: a) smooth muscle plaque or b) cholesterol-filled plaque. Commonly known as renal artery stenosis, this condition reduces blood flow to the kidneys and can lead to high blood pressure. Renal artery plaque may be discovered during CT angiography. In some cases, renal artery stenosis is discovered during CT angiography for an aortic aneurysm. Traditionally, blood pressure gradually increases with age. However, a sudden onset of high blood pressure may also be associated with renal obstruction or renal artery stenosis. Reduced blood flow to the kidneys causes the kidneys to begin producing excessive cytokines, leading to vasoconstriction or high blood pressure.

[0017] Furthermore, "cholesterol embolism" can occur when cholesterol is released from the arteries (usually from atherosclerotic plaques), travels as an embolus in the bloodstream, and causes blockage (embolism) of blood vessels located further away. Once in circulation, cholesterol particles become immobile in small blood vessels or arterioles. They can reduce blood flow to tissues and cause inflammation and tissue damage that can damage the kidneys. Cholesterol embolism can lead to kidney failure and is a condition called atherotembolic kidney disease (AERD). AERD is one sign of a disease that can develop due to cholesterol-filled plaques. In patients with AERD, plaques can rupture in arteries, releasing cholesterol and other "junk" from within the plaque into the blood vessels. The released cholesterol and junk can travel through the arteries, block them, damage parts of the kidneys and their tissues, and thereby cause AERD. Atherosclerosis of the aorta is the most common cause of AERD.

[0018] Currently, treatment for renal artery stenosis, its symptoms such as AERD, and other cardiovascular diseases involves inserting a stent into the artery to open it up. This technique often normalizes blood pressure. However, stent placement is likely to treat only the symptoms, such as hypertension. In some patients, blood pressure may be normal while AERD is present. Therefore, it is necessary to address the underlying cause of the disease and treat renal artery stenosis in combination with or independently of the symptoms of hypertension.

[0019] Hyperlipidemia (or abnormally high blood lipid levels) can potentially be treated by changing a patient's diet. However, dietary therapy as the primary form of treatment requires significant effort from the patient, physician, dietitian, registered dietitian, and other healthcare professionals, and therefore unnecessarily burdens the qualifications of healthcare professionals. Another drawback of this treatment is that its success does not depend solely on diet. Rather, the success of dietary therapy depends on a combination of social, psychological, economic, and behavioral factors. Therefore, treatment based solely on correcting deficiencies within the patient's diet is not always successful.

[0020] When dietary modifications fail, drug therapy has been used as an adjunct. Such treatments include the use of commercially available lipid-lowering drugs, administered alone or in combination with other therapies, as a supplement to dietary management. These drugs, called statins, include lovastatin, pravastatin, simvastatin, fluvastatin, atorvastatin, and cerivastatin. Statins are particularly effective in lowering LDL levels and are also effective in lowering triglycerides, clearly inversely proportional to their LDL-lowering effect. Statins also raise HDL levels, but to a lesser extent than other anticholesterol drugs. Statins also increase nitric oxide, but this decreases in the presence of oxidized LDL, as mentioned above.

[0021] Another drug therapy, bile acid resins, works by binding to bile acids, which are substances produced by the liver using cholesterol as one of its main components. Because these drugs bind to bile acids in the gastrointestinal tract, they are not absorbed into the body but are excreted in the feces. As a result, the liver has to take in more cholesterol from the bloodstream to continue building bile acids, which leads to an overall decrease in LDL levels.

[0022] Nicotinic acid, also known as niacin or vitamin B3, is effective in lowering triglyceride levels and raising HDL levels higher than other cholesterol-lowering drugs. Nicotinic acid also reduces LDL cholesterol.

[0023] Fibrate derivatives or fibrates are used to lower triglyceride levels and increase HDL when other drugs commonly used for these purposes, such as niacin, are not effective.

[0024] Probucol lowers LDL cholesterol levels but also lowers HDL levels. This is generally used for specific genetic disorders that cause high cholesterol levels or when other cholesterol-lowering drugs are ineffective or unavailable.

[0025] PCSK9 lowers LDL cholesterol levels by increasing the cellular levels of LDL receptors present in the liver.

[0026] Lipid-lowering drugs have been successful to varying degrees in reducing blood lipids. However, there is no lipid-lowering drug that can successfully treat all types of hyperlipidemia. Some lipid-lowering drugs have been quite successful, but the medical community has found little conclusive evidence that lipid-lowering drugs cause regression of atherosclerosis. Furthermore, all lipid-lowering drugs have undesirable side effects. Atherosclerosis remains a major cause of death in many parts of the world as a result of unsuccessful diet management, drug therapy, and other treatments.

[0027] New treatments have been used to reduce the lipid mass in patients for whom drug therapy and diet therapy have not been sufficiently effective. For example, extracorporeal treatments such as plasma (plasma) pheresis and LDL apheresis have been employed and have been shown to be effective in reducing LDL.

[0028] Plasma pheresis therapy or plasma exchange therapy involves replacing a patient's plasma with donor plasma or more generally a plasma protein fraction. Plasma pheresis is a process of removing plasma from blood cells by a cell separator. The separator functions by rotating the blood at high speed to separate the cells from the fluid or by passing the blood through a membrane with pores small enough for only the fluid components of the blood to pass through. The cells are returned to the person being treated, while the plasma is discarded and replaced with other fluids.

[0029] This treatment has led to complications due to the introduction of foreign proteins and the transmission of infectious diseases. Also, plasma pheresis has the drawback that all serum lipoproteins such as VLDL, LDL, and HDL are non-selectively removed. Furthermore, plasma pheresis can cause several side effects, including allergic reactions in the form of fever, chills, rash, and in some cases even anaphylaxis.

[0030] As described above, it is not desirable to remove HDL, which is secreted from both the liver and the intestine as nascent discoidal particles containing cholesterol and phospholipids. HDL is thought to be involved in the reverse transport of cholesterol, which is the process of removing excess cholesterol from tissues and transporting it to the liver for reuse or disposal in bile.

[0031] In contrast to plasma apheresis, LDL apheresis selectively removes ApoB, which contains cholesterol such as LDL, while retaining HDL. Several methods have been developed for LDL apheresis. These techniques include the absorption of LDL into heparin agarose beads, the use of immobilized LDL antibodies, cascade filtration absorption with immobilized dextran sulfate, and LDL precipitation at low pH in the presence of heparin. Each of the above methods is effective in removing LDL. However, this treatment process has drawbacks, such as not having a positive effect on HDL or causing metabolic shifts that may promote atherosclerosis or other cardiovascular diseases. As its name suggests, LDL apheresis only treats LDL in patients with severe hyperlipidemia.

[0032] Another method to achieve a reduction in plasma cholesterol in patients with homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, and acquired hyperlipidemia is an extracorporeal lipid removal process called cholesterol apheresis. In cholesterol apheresis, blood is drawn from the patient, the plasma is separated from the blood, and the plasma is mixed with a solvent mixture. The solvent mixture extracts lipids from the plasma. The defatted plasma is then remixed with the patient's blood cells and returned to the patient. However, this procedure denatures LDL particles, and these denatured LDL particles may worsen the severity of heart disease. At the same time, this process also results in further delipidation of HDL particles.

[0033] However, conventional extracorporeal delipidation processes aim for the simultaneous removal of LDL and HDL. This process has many drawbacks, primarily because the delipidated LDL tends to aggregate and subsequently worsen rather than alleviate the condition of heart disease. Furthermore, extracorporeal systems are designed to subject body fluids to substantial treatment through as many multi-stage solvent exposure and extraction steps as possible.

[0034] Intense, multi-step solvent exposure and extraction have several drawbacks. Removing sufficient amounts of solvent from the defatted plasma to safely return it to the patient can be challenging.

[0035] Therefore, existing apheresis and extracorporeal systems for the treatment of plasma components have many drawbacks that limit their performance for clinical application. Improved systems, devices, and methods capable of removing lipids from blood components are needed to provide therapeutic and preventive measures for chronic cardiovascular disease. Methods have also been provided for selectively removing lipids from HDL particles, thereby creating modified HDL particles with enhanced cholesterol-receiving capacity.

[0036] In chronic diseases, methods have been provided to selectively remove lipids from HDL particles, thereby creating modified HDL particles with enhanced cholesterol-receiving ability without substantially affecting LDL particles. However, these methods assume immediate re-administration of modified HDL particles and do not provide means for storing, preserving, or using modified HDL particles over long periods.

[0037] Methods for preserving delipidated plasma and denatured HDL particles are also needed. Autologous and non-autologous plasma should be introduced into the patient within a few hours of derivation. However, there may be situations where it is not possible to introduce aspirated denatured HDL particles into the patient within the prescribed timeframe after derivation. This may be particularly true when a patient requires denatured HDL particles (autologous) that cannot be sufficiently aspirated from the patient, but a non-autologous plasma source is rather available. Access to life-saving treatment for more patients can be enhanced by preserving delipidated plasma so that it can be used as needed and when needed, and by making it readily available. [Overview of the project]

[0038] The following embodiments and aspects are described and illustrated in conjunction with systems, tools, and methods, but are intended to be illustrative and not to limit the scope of the invention.

[0039] A method for storing pre-β high-density lipoprotein for administration to patients, including the following steps: A step of obtaining a batch of defatted plasma containing the aforementioned pre-β high-density lipoprotein; A step of testing a portion of the batch of delipidated plasma in order to characterize the pre-β high-density lipoprotein; A step of storing the batch of degreased plasma; A step of preparing the stored delipidated plasma for administration to the patient; A step of testing the prepared delipidated plasma to characterize the pre-β high-density lipoprotein; and The step of administering the pre-β high-density lipoprotein to the patient.

[0040] Optionally, the method further includes changing the amount of pre-β high-density lipoprotein before the preservation step so as to ensure that the concentration of pre-β high-density lipoprotein is in the range of 1 mg / dl to 400 mg / dl.

[0041] Optionally, the preservation step may include freezing the batch at a temperature below -30°C.

[0042] Optionally, the preparation step may include thawing the stored defatted plasma in a temperature range of 2°C to 26°C.

[0043] Optionally, the preservation step may include exposing 1 milliliter to 2 liters of degreased plasma to a temperature below -30°C for less than 20 minutes.

[0044] Optionally, the step of testing a portion of the batch of degreased plasma to characterize the pre-β high-density lipoprotein includes determining a first concentration of the pre-β high-density lipoprotein. Optionally, the step of testing the prepared degreased plasma to characterize the pre-β high-density lipoprotein includes determining a second concentration of the pre-β high-density lipoprotein and comparing the second concentration of the pre-β high-density lipoprotein to the first concentration of the pre-β high-density lipoprotein to determine the degree of degradation. Optionally, the method further includes determining, based on the second concentration of the pre-β high-density lipoprotein, whether the prepared degreased plasma is suitable for administration.

[0045] Optionally, the method further includes adding a preservative to the delipidated plasma before storage.

[0046] Optionally, the preparation step may further include thawing the stored defatted plasma and storing the thawed defatted plasma at a temperature in the range of 1°C to 6°C for 5 days or less.

[0047] This specification also discloses a method for storing denatured high-density lipoprotein for administration to a patient, comprising the following steps: The process of obtaining a batch of degreased plasma containing denatured high-density lipoprotein by connecting at least one person to a device for collecting blood, collecting blood containing blood cells from the at least one person, separating the blood cells from the blood to produce a plasma fraction containing high-density lipoprotein and low-density lipoprotein, degreasing the high-density lipoprotein using a solvent, separating the low-density lipoprotein, and collecting the degreased plasma containing the denatured high-density lipoprotein; A step of testing a portion of the batch of delipidated plasma in order to characterize the denatured high-density lipoprotein; A step of storing the batch of degreased plasma; A step of preparing the stored delipidated plasma for administration to the patient; A step of testing the prepared delipidated plasma to characterize the denatured high-density lipoprotein; and A step of administering the denatured high-density lipoprotein to the patient.

[0048] Optionally, the method further includes changing the amount of denatured high-density lipoprotein before the preservation step so as to ensure that the concentration of the denatured high-density lipoprotein is in the range of 1 mg / dl to 400 mg / dl.

[0049] Optionally, the preservation step may include freezing the batch at a temperature below -30°C.

[0050] Optionally, the preparation step may include thawing the stored defatted plasma in a temperature range of 2°C to 26°C.

[0051] Optionally, the preservation step may include exposing 1 milliliter to 2 liters of degreased plasma to a temperature below -30°C for less than 20 minutes.

[0052] Optionally, the step of testing a portion of the batch of degreased plasma to characterize the denatured high-density lipoprotein includes determining a first concentration of the denatured high-density lipoprotein. Optionally, the step of testing the prepared degreased plasma to characterize the denatured high-density lipoprotein includes determining a second concentration of the denatured high-density lipoprotein and comparing the second concentration to the first concentration to determine the degree of degradation. Optionally, the method further includes determining, based on the second concentration of the denatured high-density lipoprotein, whether the prepared degreased plasma is suitable for administration.

[0053] Optionally, the method further includes adding a preservative to the delipidated plasma before storage.

[0054] Optionally, the preparation step may further include thawing the stored defatted plasma and storing the thawed defatted plasma at a temperature in the range of 1°C to 6°C for 5 days or less.

[0055] This specification also discloses a method for treating cardiovascular disease in a patient, comprising the steps of: obtaining a plasma fraction comprising high-density lipoprotein and low-density lipoprotein; mixing the plasma fraction with a lipid-removing agent to produce a mixture of lipids, the lipid-removing agent, denatured high-density lipoprotein, and low-density lipoprotein, wherein the denatured high-density lipoprotein is defatted high-density lipoprotein; separating the denatured high-density lipoprotein and low-density lipoprotein from the lipids and the lipid-removing agent; storing the denatured high-density lipoprotein for an extended period; preparing the stored denatured high-density lipoprotein for use; separating components of high-density lipoprotein particles from the denatured high-density lipoprotein prepared after storage; and delivering the components of the high-density lipoprotein particles to the patient.

[0056] Optionally, the method of preservation includes freezing. Optionally, the method of preparation for use includes thawing.

[0057] Optionally, the storage method may include storing DP in a volume of 1 milliliter to 2 liters.

[0058] Optionally, the mixing method includes mixing the plasma fraction with a lipid-removing agent that removes lipids associated with the high-density lipoprotein without substantially denaturing the low-density lipoprotein.

[0059] Optionally, a method for obtaining a plasma fraction containing the high-density lipoprotein and the low-density lipoprotein may include obtaining it from at least one of the patient and / or an individual other than the patient.

[0060] Optionally, the method of treating the cardiovascular disease includes treating at least one of the following: AERD, homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, ischemic stroke, coronary artery disease, acute coronary syndrome, and peripheral artery disease.

[0061] Optionally, treatment methods for cardiovascular disease include treatment methods for the progression of Alzheimer's disease.

[0062] Optionally, the step of mixing a lipid removal agent with the plasma fraction generates a denatured high-density lipoprotein in which the concentration of pre-β high-density lipoprotein is increased relative to the total protein.

[0063] Optionally, the process of obtaining a plasma fraction from the treatment of cardiovascular disease further includes: connecting at least one person to a device for collecting blood; collecting blood containing blood cells from the person; and separating the blood cells from the blood to produce a plasma fraction containing high-density lipoprotein and low-density lipoprotein.

[0064] Optionally, the step of separating components of high-density lipoprotein particles from the denatured high-density lipoprotein includes using affinity chromatography.

[0065] Optionally, the method using affinity chromatography includes: adding plasma containing defatted high-density lipoprotein to a column; dropping the plasma through a column containing an antibody for binding to the ApoA-I protein; washing the column to remove unwanted substances; and delivering a dissociation reagent through the column to break the binding between the antibody and the ApoA-I protein, thereby separating at least pre-βHDL.

[0066] Optionally, the step of separating components of high-density lipoprotein particles from the denatured high-density lipoprotein may include using ultracentrifugation.

[0067] Optionally, using ultracentrifugation includes centrifuging the denatured high-density lipoprotein to a density of 1.21, separating the bottom fraction containing pre-β high-density lipoprotein particles and plasma proteins, and centrifuging the bottom fraction to a density of 1.25 to separate the pre-β high-density lipoprotein particles from the plasma proteins.

[0068] Optionally, using ultracentrifugation includes centrifuging the denatured high-density lipoprotein to a density of 1.006, separating a bottom fraction containing low-density lipoprotein and plasma containing high-density lipoprotein, centrifuging the separated plasma containing low-density lipoprotein and high-density lipoprotein to a density of 1.063, separating a bottom fraction containing high-density lipoprotein particles, centrifuging the separated high-density lipoprotein particles to a density of 1.21, separating a bottom fraction containing pre-β high-density lipoprotein particles and plasma proteins, and centrifuging the bottom fraction to a density of 1.25 to separate pre-β high-density lipoprotein particles from plasma proteins.

[0069] Optionally, using ultracentrifugation involves centrifuging the denatured high-density lipoprotein to a density of 1.063, separating the bottom fraction containing plasma with α and pre-β high-density lipoprotein particles and plasma lipoprotein, and centrifuging the bottom fraction to a density of 1.25 to separate the α and pre-β high-density lipoprotein particles from the plasma protein.

[0070] This specification also discloses a method for treating cardiovascular disease in a patient, comprising the steps of: obtaining a plasma fraction comprising high-density lipoprotein and low-density lipoprotein; mixing the plasma fraction with a lipid-removing agent to produce a mixture of lipids, the lipid-removing agent, denatured high-density lipoprotein, and low-density lipoprotein, wherein the denatured high-density lipoprotein is defatted high-density lipoprotein; separating the denatured high-density lipoprotein and low-density lipoprotein from the lipids and the lipid-removing agent; separating the components of high-density lipoprotein particles from the denatured high-density lipoprotein; storing the components of the high-density lipoprotein particles for an extended period; preparing the stored components of high-density lipoprotein for use; and delivering the components of the high-density lipoprotein particles to the patient.

[0071] This specification also discloses a method for treating cardiovascular disease in a patient, comprising the steps of: obtaining a plasma fraction comprising high-density lipoprotein and low-density lipoprotein; mixing the plasma fraction with a lipid-removing agent to produce a mixture of lipids, the lipid-removing agent, denatured high-density lipoprotein, and low-density lipoprotein, wherein the denatured high-density lipoprotein is defatted high-density lipoprotein; separating the denatured high-density lipoprotein and low-density lipoprotein from the lipids and the lipid-removing agent; separating the components of high-density lipoprotein particles from the denatured high-density lipoprotein; and storing the components of the high-density lipoprotein particles for an extended period.

[0072] Optionally, the method further comprises the step of preparing the components of the stored high-density lipoprotein particles for use.

[0073] Optionally, the method further includes the step of delivering the components of the high-density lipoprotein particles to the patient.

[0074] Optionally, the preservation process may include freezing.

[0075] Optionally, the preparation step may include thawing.

[0076] Optionally, the components of the high-density lipoprotein particles include α-high-density lipoprotein and / or pre-β-high-density lipoprotein.

[0077] Optionally, the components of the high-density lipoprotein particles include pre-β high-density lipoprotein.

[0078] This specification also discloses a method for treating cardiovascular disease in a patient, comprising the steps of: obtaining a plasma fraction comprising high-density lipoprotein and low-density lipoprotein; mixing the plasma fraction with a lipid-removing agent to produce a mixture of lipids, the lipid-removing agent, denatured high-density lipoprotein, and low-density lipoprotein, wherein the denatured high-density lipoprotein is defatted high-density lipoprotein; separating the denatured high-density lipoprotein and low-density lipoprotein from the lipids and the lipid-removing agent; storing the denatured high-density lipoprotein for a predetermined period or longer; preparing the stored denatured high-density lipoprotein for use; separating components of high-density lipoprotein particles from the denatured high-density lipoprotein prepared after storage; storing the components of the high-density lipoprotein particles for a longer period or a predetermined period; preparing the components of the stored high-density lipoprotein particles for use; and delivering the components of the high-density lipoprotein particles to the patient.

[0079] Optionally, the preservation process may include freezing.

[0080] Optionally, the preparation step may include thawing.

[0081] Optionally, the components of the high-density lipoprotein particles include α-high-density lipoprotein and / or pre-β-high-density lipoprotein.

[0082] Optionally, the components of the high-density lipoprotein particles include pre-β high-density lipoprotein.

[0083] Other embodiments described above in this specification are described in more detail in the drawings and detailed description provided below. [Brief explanation of the drawing]

[0084] These features and advantages of the present invention, as well as other features and advantages, will be better understood by referring to the following detailed description in conjunction with the accompanying drawings. [Figure 1] Figure 1A is a flowchart illustrating an exemplary process for separating pre-βHDL from degreased (denatured) HDL according to some embodiments of this specification. Figure 1B is a schematic diagram of a system comprising multiple components used according to some embodiments of this specification to achieve the process disclosed herein. Figure 1C is a picture of an exemplary embodiment of a system configuration of multiple components used according to some embodiments of this specification to achieve the process disclosed herein. Figure 1D is a flowchart illustrating an exemplary process used to increase the concentration of a desired substance in degreased plasma using affinity chromatography according to some embodiments of this specification. Figure 1E is a flowchart illustrating an exemplary process used to increase the concentration of a desired substance in degreased plasma using ultracentrifugation according to some embodiments of this specification. Figure 1F is a flowchart illustrating another exemplary process used to increase the concentration of a desired substance in degreased plasma using ultracentrifugation according to some embodiments of this specification. Figure 1G is a flowchart illustrating another exemplary series of steps used to increase the concentration of a desired substance in treated plasma using ultracentrifugation according to some embodiments of this specification. [Figure 2] This flowchart shows the process for testing, storing, and validating stored defatted plasma containing pre-βHDL. [Modes for carrying out the invention]

[0085] In some embodiments, this specification relates to systems, apparatus and methods for preserving modified HDL particles (also called defatted HDL) with reduced lipid content, particularly cholesterol content, primarily derived from non-autologous plasma for a patient, where the preserved product can be used later. Embodiments herein generate and preserve these modified HDL particles with reduced lipid content without substantially denaturing LDL particles. Embodiments herein denature the original α-HDL particles (present in defatted plasma) to produce modified HDL particles with increased concentrations of HDL components, including α-HDL and / or pre-β-HDL, compared to the original HDL. Furthermore, derivatives of the newly formed HDL particles (modified HDL) are processed to separate α-HDL and / or pre-β-HDL from the defatted plasma. In some embodiments, the defatted plasma is processed to produce a more concentrated solution of α-HDL and / or pre-β-HDL. Modified HDL, comprising a concentrated solution of α-HDL and / or pre-β-HDL, is, in one embodiment, stored by freezing and administered to a patient after thawing, in order to enhance cellular cholesterol and treat cardiovascular disease and / or other lipid-related disorders. In one embodiment, the modified HDL comprises a concentrated solution of about 20% α-HDL particles (present in delipidated plasma) and about 80% pre-β-HDL.

[0086] The therapeutic processes described herein make the methods and systems described herein more effective in the treatment of cardiovascular diseases, including homozygous familial hypercholesterolemia (HoFH), heterozygous familial hypercholesterolemia (HeFH), ischemic stroke, coronary artery disease (CAD), acute coronary syndrome (ACS), peripheral artery disease (PAD), AERD, renal artery stenosis (RAS) and its symptoms, as well as in the treatment of the progression of Alzheimer's disease.

[0087] This specification relates to multiple embodiments. The following disclosures enable those skilled in the art to practice the invention. The language used herein should not be construed as a general disclaimer of any particular embodiment, nor should it be used to limit the claims beyond the meaning of the terms used herein. The general principles defined herein can be applied to other embodiments and uses without departing from the spirit and scope of the invention. Furthermore, the terms and expressions used are for illustrative purposes only and should not be considered restrictive. Accordingly, the invention should be given the broadest scope, encompassing many alternatives, modifications, and equivalents that conform to the disclosed principles and features. For clarity, details relating to technical materials known in the art relating to the invention are not described in detail so as not to unnecessarily obscure the invention. In the description and claims of this application, the words “comprise,” “include,” and “have,” and their forms, are not necessarily limited to the enumerated components to which the words may relate.

[0088] It should be noted that, unless otherwise specified, any features or components described herein in relation to a particular embodiment may be used and implemented in other embodiments.

[0089] The term "fluid" is defined as a fluid from an animal or human containing lipids or lipid-containing particles, a fluid from a cultured tissue and cell containing lipids, and a fluid mixed with lipid-containing cells. For the purposes of the present invention, a reduction in the amount of lipids in a fluid includes a reduction in lipids in plasma and particles contained in plasma (but not limited to HDL particles). Examples of fluids include, but are not limited to, biological fluids such as blood, plasma, serum, lymph, cerebrospinal fluid, peritoneal fluid, pleural fluid, pericardial fluid, and various fluids of the reproductive system (e.g., but not limited to semen, ejaculate, follicular fluid, and amniotic fluid); cell culture reagents such as normal serum, fetal bovine serum, or serum from any animal or human; and immunological reagents such as various preparations of antibodies and cytokines from cultured tissues and cells, fluids mixed with lipid-containing cells, and fluids containing lipid-containing organisms such as physiological saline containing lipid-containing organisms. A preferred fluid to be processed by the method of the present invention is plasma.

[0090] The term "lipid" is defined as any one or more of the group of fats or fatty substances that occur in humans or animals. Fats or fatty substances are characterized by insolubility in water and solubility in organic solvents. The term "lipid" is known to those skilled in the art and includes, but is not limited to, complex lipids, simple lipids, triglycerides, fatty acids, glycerophospholipids (phospholipids), true fats (e.g., esters of fatty acids), glycerol, cerebrosides, waxes, and sterols (e.g., cholesterol and ergosterol).

[0091] The term "extraction solvent" is defined as one or more solvents used to extract lipids from a fluid or from particles within a fluid. The solvent enters the fluid and remains in it until removed by other subsystems. Suitable extraction solvents include, but are not limited to, solvents that extract or dissolve lipids, such as phenols, hydrocarbons, amines, ethers, esters, alcohols, halogenated hydrocarbons, halocarbons, and combinations thereof. Suitable extraction solvents include, but are not limited to, ethers, esters, alcohols, halogenated hydrocarbons, or halocarbons, such as diisopropyl ether (DIPE) (also known as isopropyl ether), diethyl ether (DEE) (also known as ethyl ether), lower alcohol pairs (e.g., butanol, especially n-butanol), ethyl acetate, dichloromethane, chloroform, isoflurane, sevoflurane (1,1,1,3,3,3-hexafluoro-2-(fluoromethoxy)propane-d3), perfluorocyclohexanes, trifluoroethane, cyclofluorohexanol, and combinations thereof.

[0092] The term "patient" refers to animals and humans, which may be sources of fluids processed by the method of the present invention, or recipients of derivatives of HDL particles with reduced lipid content and / or plasma.

[0093] The term "HDL particles" includes several types of particles defined based on various methods, such as methods for measuring charge, density, size, and immunoaffinity, for example, but not limited to, electrophoretic mobility, ultracentrifugation, immunoreactivity, and other methods known to those skilled in the art. Such HDL particles include, but are not limited to, α-HDL, pre-βHDL (including pre-β1HDL, pre-β2HDL, and pre-β3HDL), HDL2 (including HDL2a and HDL2b), HDL3, VHDL, LpA-I, LpA-II, and LpA-I / LpA-II (see Barrans et al., Biochemica Biophysica Acta 1300;73-85, 1996). Accordingly, denatured HDL particles are produced by carrying out the method of the present invention. These denatured derivatives of HDL particles can be denatured in many ways, including, but are not limited to, changes in metabolic and / or physicochemical properties (see Barrans et al., Biochemica Biophysica Acta 1300; 73-85, 1996); molecular weight (kDa); charge; diameter; shape; density; hydration density; buoyancy properties; cholesterol content; free cholesterol content; esterified cholesterol content; molar ratio of free cholesterol to phospholipids; immunoaffinity; content, activity, or helical structure of one or more enzymes or proteins (ApoA-I, ApoA-II, ApoD, ApoE, ApoJ, ApoA-IV, cholesterol ester transporter protein (CETP), lecithin; cholesterol acyltransferase (LCAT)); and changes in one or more of their cholesterol binding ability and / or cholesterol transport ability and / or rate.

[0094] The terms "denatured high-density lipoprotein" and "defatted high-density lipoprotein" are used interchangeably and refer to high-density lipoproteins with reduced lipid blood products, particularly those with reduced lipid content. This can be present in the resulting plasma when a defatting process is performed. Similarly, the term "processed plasma" refers to the plasma obtained after a defatting process has been performed.

[0095] Figure 1A is a flowchart illustrating an exemplary process for separating pre-βHDL from denatured HDL according to several embodiments herein. In 102, the plasma delipidation process is initiated for a subject or patient suffering from cardiovascular disease or related conditions. This process is typically initiated after one or more observations by the physician treating the patient. Observations may be based on a combination of symptoms and, but not limited to, test results such as blood tests and imaging analyses. Observations may lead the physician to conclude that the reduction of harmful lipids from the patient's physiological systems is necessary for the patient's treatment.

[0096] In step 104, a blood fraction is obtained, which in one embodiment is plasma. According to embodiments herein, the blood fraction is obtained from either a patient (autologous) or a non-autologous source. Blood fractions from non-autologous sources are collected from healthy, voluntary donors. The process of blood fractionation is typically carried out by filtration, centrifugation of the blood, aspiration, or any other method known to those skilled in the art. Blood fractionation separates plasma from blood. In one embodiment, blood fractionation is carried out remotely from the method described in relation to Figure 1A. In one embodiment, blood is taken from the patient or donor in an amount sufficient to produce about 12 ml / kg of plasma based on body weight. During the fractionation process, optionally, the blood can be combined with an anticoagulant such as sodium citrate and centrifuged with a force equal to about 2,000 times gravity. The blood is separated into plasma and red blood cells using a method commonly known to those skilled in the art, such as plasmapheresis. In one embodiment, the red blood cells are then aspirated from the plasma. In one embodiment, the blood fractionation process is carried out by collecting blood from a patient who has cardiovascular disease and / or related conditions and / or is being treated by a physician. In an alternative embodiment, the blood fractionation process is carried out by collecting blood from a person other than a patient who has cardiovascular disease and / or related conditions and is being treated by a physician. Thus, the plasma obtained as a result of the blood fractionation process may be either autologous or non-autologous.

[0097] Following fractionation, the red blood cells are stored in a suitable storage solution or, preferably, returned to the patient during plasmapheresis. Optionally, saline may be administered to the patient to replenish volume. If the blood is obtained from an individual other than the patient, the cells are returned to that individual, sometimes referred to as the donor.

[0098] Plasma, obtained from blood, is typically a pale yellow fluid containing the extracellular matrix of blood cells. Plasma is usually 95% water and contains soluble proteins, which make up about 6-8% of its composition. Plasma also contains glucose, clotting factors, electrolytes, hormones, carbon dioxide, and oxygen. The density of plasma is approximately 1006 kg / m³. 3That is, 1.006 g / ml.

[0099] In some other embodiments, low-density lipoprotein (LDL) is also separated from the plasma. The separated LDL is usually discarded. In other embodiments, the LDL is left in the plasma. According to embodiments herein, the blood fraction or plasma obtained in 104 also includes plasma containing high-density lipoprotein (HDL) and may or may not contain other protein particles. In embodiments, autologous or non-autologous plasma collected from a patient or donor is then isolated by an approved plasmapheresis device. The plasma may be transported using a continuous or batch process.

[0100] In step 106, the blood fraction or plasma obtained in step 104 is mixed with one or more solvents, for example, lipid removers. In one embodiment, the solvents used may be either or both of the organic solvents sevoflurane and n-butanol. In an embodiment, the plasma and solvent are introduced into at least one of a mixer, agitator, or other device that brings the plasma and solvent into contact. In an embodiment, the solvent system is optimally designed so that only HDL particles are treated and their lipid levels are reduced, while LDL levels are not affected. The solvent system involves analyzing variability factors such as the solvent used, the mixing method, time, and temperature. In this step, the type, proportion, and concentration of the solvent may vary. The acceptable proportion of solvent to plasma includes any combination of solvent and plasma. In some embodiments, the proportions used are 1 part solvent to 2 parts plasma, 1 part solvent to 1 part plasma, or 2 parts solvent to 1 part plasma. In one embodiment, when using a solvent containing 5 parts n-butanol to 95 parts sevoflurane, a proportion of 2 parts solvent per 1 part plasma is used. Furthermore, in one embodiment, when a solvent containing n-butanol is used, this specification uses a ratio of solvent to plasma such that the final solvent / plasma mixture contains at least 3% n-butanol. In one embodiment, the final concentration of n-butanol in the final solvent / plasma mixture is 3.33%. The plasma and solvent are introduced into at least one of a mixer, agitator, or other device that brings the plasma and solvent into contact. The plasma may be transported using a continuous or batch process. In addition, various detection means may be included to monitor pressure, temperature, flow rate, and solvent level, etc. The solvent dissolves lipids from the plasma. In embodiments of this specification, the solvent dissolves lipids to produce treated plasma containing denatured HDL particles with reduced lipid content. The process is designed to treat HDL particles to reduce their lipid levels and produce denatured HDL particles without destroying plasma proteins or substantially affecting LDL particles. It should be noted that there is no clinically significant reduction in blood components after plasmapheresis.

[0101] Energy is introduced into the system in the form of various mixing methods, times, and rates. In 108, the bulk solvent is removed from the modified HDL particles by centrifugation. In embodiments, any remaining soluble solvent is removed by carbon adsorption, evaporation, or hollow fiber contractor (HFC) pervaporation. The mixture may optionally be tested for the remaining solvent using gas chromatography (GC) or similar means. Testing for the remaining solvent may be optionally excluded based on statistical validation.

[0102] In the extracted denatured HDL solution, the concentration of pre-βHDL is increased. It is estimated that denatured HDL in delipidated plasma contains approximately 80–85% pre-β particles and approximately 15–20% αHDL particles. The concentration of pre-βHDL is higher in denatured HDL compared to the original HDL present in the plasma before treatment with the solvent. The concentration of pre-βHDL particles is substantially increased compared to the plasma solution initially separated from the blood fraction, which typically contains approximately 5% pre-βHDL particles.

[0103] Figure 1B shows exemplary embodiments of the system and its components used to achieve the method described herein. This figure represents a flow chart of exemplary basic elements defining the elements of the HDL denaturation system 200. Embodiments of the components of system 200 are used after obtaining a blood fraction from a patient or another individual (donor). Plasma separated from the blood is brought to system 200 in a sterile bag for further processing. A fluid inlet 205 is provided and connected to a mixing device 220 via a tube. A solvent inlet 210 is provided and similarly connected to the mixing device 220 via a tube. In embodiments, valves 215 and 216 are used to control the flow of fluid from the fluid inlet 205 and solvent from the solvent inlet 210, respectively. It should be understood that the fluid inlet 205 contains any fluid containing HDL particles, or not containing LDL particles, as described above. Furthermore, it should be understood that the solvent inlet 210 may contain a single solvent, a mixture of solvents, or multiple different solvents mixed in the solvent inlet 210. Although the solvent input section 210 is depicted as a single solvent container, it may include multiple separate solvent containers. Embodiments of the types of solvents that can be used are described above.

[0104] The mixer 220 mixes the fluid from the fluid input section 205 and the solvent from the solvent input section 210 to produce a fluid-solvent mixture. In embodiments, the mixer 220 can use a shaker bag mixing method with the input fluid and the input solvent in multiple batches, e.g., one, two, three, or more batches. An exemplary mixer is the Barnstead Labline orbital shaker table. Once produced, the fluid-solvent mixture is delivered through a tube to the separator 225, controlled by at least one valve 215a. In one embodiment, the separator 225 can separate the bulk solvent by gravity separation in a funnel-shaped bag.

[0105] In the separator 225, the fluid-solvent mixture is separated into a first layer and a second layer. The first layer contains a mixture of the solvent and lipids removed from the HDL particles. The first layer is transported to the first waste container 235 via valve 215b. The second layer contains a mixture of the remaining solvent, modified HDL particles, and other elements of the introduced fluid. Those skilled in the art will understand that the compositions of the first and second layers differ based on the properties of the introduced fluid. Once the first and second layers are separated in the separator 225, the second layer is transported via a tube to the solvent extraction device 240. In one embodiment, a pressure sensor 229 and a valve 230 are placed in the flowstream to control the flow of the second layer to the solvent extraction device 240.

[0106] The opening and closing of valves 215, 216 to allow fluid flow from input containers 205, 210 can be time-controlled using mass equilibrium calculations obtained from the weight determination of the fluid input sections 205, 210 and the separator 225. For example, valve 215b between the separator 225 and the first waste container 235, and valve 230 between the separator 225 and the solvent extraction device 240 are opened after sufficient time has elapsed for the input mass (fluid and solvent) to substantially equilibrium with the mass in the separator 225 and to allow separation of the first and second layers. Depending on the solvent used, and therefore which layer settles at the bottom of the separator 225, either valve 215b between the separator 225 and the first waste container 235 or valve 230 between the separator 225 and the solvent extraction device 240 is opened. Those skilled in the art will understand that the timing of opening depends on the amount of fluid present in the first and second layers, and further understand that it is preferable to keep the valve 215b between the separator 225 and the first waste container 235 open for just long enough time to remove all of the first layer and some of the second layer, thereby ensuring that as much solvent as possible is removed from the fluid sent to the solvent extraction device 240.

[0107] In this embodiment, the glucose input section 255 and one or more saline input sections 260 are fluidly connected to a flow path 221 that connects the separator 225 to the solvent extraction device 240. Multiple valves 215c and 215d are also incorporated into the flow stream from the glucose input section 255 and the saline input section 260, respectively, to the tube forming the flow path 221 from the separator 225 to the solvent extraction device 240. The glucose and saline are incorporated in this embodiment to prime the solvent extraction device 240 before system operation. If such priming is not required, the addition of glucose and saline is not necessary. Furthermore, those skilled in the art will understand that the addition of glucose and saline can be replaced with other priming agents if required by the solvent extraction device 240.

[0108] In some embodiments, the solvent extraction device 240 is a charcoal column designed to remove a specific solvent used in the solvent input section 210. An exemplary solvent extraction device 240 is an Asahi Hemosorber charcoal column. A pump 250 is used to move the second layer from the separator 225 through the solvent extraction device 240 to the product container 245. In embodiments, the pump 250 is a rotary peristaltic pump, for example, a Masterflex model 77201-62.

[0109] The first layer is delivered to a waste container 235, which is fluidly connected to a separator 225 by a tube and at least one valve 215b. Furthermore, any other waste liquid, if generated, can be delivered to a second waste container 255 through a flow path connecting the solvent extraction device 240 and the product container 245. Optionally, in one embodiment, a valve 215f is included in the flow path from the solvent extraction device 240 to the product container 245. Optionally, in one embodiment, a valve 215g is included in the flow path from the solvent extraction device 240 to the second waste container 255.

[0110] In one embodiment of this specification, gravity is used, where practical, to move the fluid through each of the multiple components. For example, the introduced plasma 205 and introduced solvent 210 are discharged into a mixer using gravity. If the mixer 220 includes a shaker bag and the separator 225 includes a funnel-shaped bag, the fluid is moved from the shaker bag to the funnel-shaped bag and then, where appropriate, to the first waste container 235 using gravity.

[0111] In further embodiments not shown in Figure 1B, the generated fluid in the product container 245 is passed through a solvent detection system or a lipid removal agent detection system to determine whether any solvent or other undesirable components are present in the generated fluid. In one embodiment, the generated fluid is subjected to a sensor capable of determining the concentration of a solvent introduced in the solvent input section, such as n-butanol or diisopropyl ether. In embodiments, the sensor can provide such concentration information in real time without the need to physically transport a sample of the generated fluid or air in headspace to a remote device.

[0112] In one embodiment, the generated fluid is further processed in a second step to separate or isolate at least pre-βHDL particles, and optionally both α and pre-βHDL particles. In one embodiment, the second step (described below) takes place in a separate area distinct from the degreasing process, where the final product, the generated fluid, is transported to a processing laboratory or room. In an alternative embodiment, the second step processing takes place inline with the degreasing system, thereby connecting the system to an affinity column subsystem or an ultracentrifugation subsystem. The resulting separated α and / or pre-βHDL particles are then introduced into the patient's bloodstream.

[0113] In one embodiment, a surface acoustic sensor is activated using molecular imprint polymer technology. The surface acoustic sensor receives input through interaction between its surface and the surrounding environment, and produces an electrical response generated by the piezoelectricity of the sensor substrate. Molecular imprint polymer technology is used to enable this interaction. Molecular imprint polymers are plastics programmed to recognize target molecules in complex biological samples, such as drugs, toxins, or environmental pollutants. Molecular imprint technology is made possible by polymerizing an excess amount of crosslinked monomers with one or more functional monomers in the presence of the target molecule to be recognized, i.e., a target template molecule exhibiting a structure similar to that of the target solvent.

[0114] The use of molecular imprint polymer technology to enable surface acoustic sensors allows for greater specificity to the concentration of the target solvent and enables the differentiation of such target solvents from other possible interfering substances. As a result, the presence of acceptable interfering substances, which may have similar structures and / or properties to the target solvent, does not prevent the sensor from accurately reporting the concentration of each solvent present.

[0115] Alternatively, if the introduced solvent contains certain solvents, such as n-butanol, the solvent concentration can be measured using electrochemical oxidation. Electrochemical measurements have several advantages. They are simple, precise, rapid, and have a wide dynamic range. Measurements are straightforward and unaffected by humidity. In one embodiment, the target solvent, for example, n-butanol, is oxidized on a platinum electrode using cyclic voltammetry. This technique is based on changing the applied voltage at a predetermined scanning rate with bidirectional working electrodes while monitoring the current. A single full cycle, a partial cycle, or a series of cycles can be performed. Platinum is a preferred electrode material, but other electrodes, such as gold, silver, iridium, or graphite, can be used. While cyclic voltammetry is used, other pulsed techniques, such as differential pulsed voltammetry or square wave voltammetry, may increase the speed and sensitivity of the measurement.

[0116] The embodiments herein explicitly cover any and all forms of automatically sampling, measuring, detecting, and analyzing the generated fluid or the headspace above the generated fluid. For example, such automated detection can be achieved by incorporating a mini gas chromatography (GC) analyzer that automatically samples the air in the product container, sends it to a GC instrument optimized for the specific solvent used in the degreasing process, and analyzes the sample for the presence of the solvent using known GC techniques.

[0117] Referring again to Figure 1B, suitable materials for use in any of the device components described herein include biocompatible materials approved for medical applications, including contact with bodily fluids, and conforming to USPV1 or ISO10993 standards. Furthermore, the material is not substantially decomposed by exposure to the solvents used herein, for example, during at least one use. The material is sterilizable by either radiation or ethylene oxide (EtO) sterilization. Such suitable materials can be molded into the object by conventional processes, for example, but not limited to, extrusion molding, injection molding, etc. Materials that satisfy these requirements include, but are not limited to, nylon, polypropylene, polycarbonate, acrylic, polysulfone, polyvinylidene fluoride (PVDF), fluoroelastomers, e.g., VITON (available from DuPont Dow Elastomers LLC), thermoplastic elastomers, e.g., SANTOPRENE (available from Monsanto), polyurethane, polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), polyphenylene ether (PFE), perfluoroalkoxy copolymer (PFA) (available as TEFLON PFA from E. DuPont de Nemours and Company), and combinations thereof.

[0118] The valves 215, 215a, 215b, 215c, 215d, 215e, 215f, 215g, 216, and other valves used in each embodiment may consist of pinch, globe, ball, gate, or other conventional valves, but are not limited to these. In some embodiments, the valve is a shut-off valve, for example, Acro Associates' Model 955 valve. However, this specification is not limited to valves having a particular style. Furthermore, the components of each system described according to the embodiments herein may be physically connected or connected using conduits, tubes, or other such devices known to those skilled in the art, which may consist of flexible or rigid pipes.

[0119] Figure 1C shows an exemplary configuration of a system used according to some embodiments herein to accomplish the procedures disclosed herein. Referring to Figure 1C, the arrangement of the basic components of the HDL modification system 300 is shown. A fluid inlet 305 is provided and connected to a mixer 320 via a tube. A solvent inlet 310 is provided and also connected to the mixer 320 via a tube. Preferably, a valve 316 is used to control the flow of fluid from the fluid inlet 305 and solvent from the solvent inlet 310. It should be understood that the fluid inlet 305 preferably contains any fluid, including plasma containing HDL particles and having or lacking LDL particles, as described above. It should be further understood that the solvent inlet 310 may contain a single solvent, a mixture of solvents, or multiple different solvents mixed in the solvent inlet 310. Although depicted as a single solvent vessel, the solvent inlet 310 may contain multiple separate solvent vessels. Preferred types of solvents used are described above.

[0120] The mixer 320 mixes the fluid from the fluid input section 305 and the solvent from the solvent input section 310 to produce a fluid-solvent mixture. Preferably, the mixer 320 can use a shaking vessel mixing method for the input fluid and the input solvent in multiple batches, for example, one, two, three, or more batches. Once the fluid-solvent mixture is produced, it is delivered to the separator 325 through a tube, controlled by at least one valve 321. In a preferred embodiment, the separator 325 can perform large-scale solvent separation by gravity separation in a funnel-shaped container.

[0121] In separator 325, the fluid-solvent mixture is separated into a first and second layer. The first layer contains a mixture of the solvent and lipids removed from the HDL particles. The second layer contains a mixture of the remaining solvent, modified HDL particles, and other elements of the introduced fluid. Those skilled in the art will understand that the compositions of the first and second layers differ based on the properties of the introduced fluid. Once the first and second layers are separated in separator 325, the second layer is transported through a tube to solvent extractor 340. Preferably, a pressure sensor 326 and a valve 327 are positioned in the fluid flow to control the flow of the second layer to the solvent extractor 340.

[0122] Preferably, the glucose input section 330 and the saline input section 350 are fluidly connected to a flow path from the separator 325 to the solvent extraction device 340. A number of valves 331 are also preferably incorporated into the fluid flow from the glucose input section 330 and the saline input section 350 to the tube forming the flow path from the separator 325 to the solvent extraction device 340. Glucose and saline are incorporated into the invention to prime the solvent extraction device 340 before system operation. If such priming is not required, the addition of glucose and saline is not necessary. Furthermore, those skilled in the art will understand that the addition of glucose and saline can be replaced with other priming agents if required by the solvent extraction device 340.

[0123] The solvent extraction apparatus 340 is preferably a charcoal column intended to remove a specific solvent used in the solvent input section 310. An exemplary solvent extraction apparatus 340 is an Asahi Hemosorber charcoal column. A pump 335 is used to move the second layer from the separator 325 through the solvent extraction apparatus 340 to the product container 315. The pump is preferably a peristaltic pump, such as a Masterflex model 77201-62.

[0124] The first layer is delivered to a waste container 355, which is fluidly connected to the separator 325 by a tube and at least one valve 356. Furthermore, any other waste liquid, if generated, can be delivered to the waste container 355 through a flow path connecting the solvent extractor 340 and the product container 315.

[0125] Preferably, embodiments of the present invention use gravity to move the fluid through each of the multiple components, where practically possible. For example, preferably, gravity is used to drain the introduced plasma 305 and introduced solvent 310 into the mixer 320. If the mixer 320 includes a shaking vessel and the separator 325 includes a funnel vessel, the fluid moves from the shaking vessel to the funnel vessel, and then, where appropriate, into the waste container 355 using gravity.

[0126] Generally, the present invention preferably includes a configuration in which all input sections (such as plasma input sections and solvent input sections), disposable elements (such as mixing bags, separation bags, waste bags, solvent extraction devices, and solvent detection devices), and product containers are located in an easily accessible position and can be easily removed and replaced by a technician.

[0127] To enable the operation of the above embodiments of the present invention, it is preferable to supply users of such embodiments with a packaged set of components in the form of a kit, containing each component necessary to carry out the embodiments herein. The kit may include an input fluid container (i.e., a supply container for high-density lipoprotein), a supply container for lipid remover (i.e., a solvent container), disposable components for a mixer (such as bags or other containers), disposable components for a separator (such as bags or other containers), disposable components for a solvent extraction device (i.e., a charcoal column), a product container, a disposable component for a waste container (such as bags or other containers), a solvent detection device, and a plurality of tubes and a plurality of valves. The plurality of tubes and a plurality of valves may include those for controlling the flow of input fluid (high-density lipoprotein) from the input container and lipid remover (solvent) from the solvent container to the mixer, those for controlling the flow of a mixture of lipid remover, lipids, and particulate derivatives to the separator, those for controlling the flow of lipids and lipid remover to the waste container, those for controlling the flow of residual lipid remover, residual lipids, and particulate derivatives to the extraction device, and those for controlling the flow of particulate derivatives to the product container.

[0128] In one embodiment, the kit may include disposable components for a mixer (such as bags or other containers), disposable components for a separator (such as bags or other containers), disposable components for a waste container (such as bags or other containers), as well as a plurality of tubes and a plurality of valves. The plurality of tubes and a plurality of valves may include those for controlling the flow of input liquid (high-density lipoprotein) from the input container and the lipid remover (solvent) from the solvent container to the mixer, those for controlling the flow of a mixture of lipid remover, lipids, and particulate derivatives to the separator, those for controlling the flow of lipids and lipid remover to the waste container, those for controlling the flow of residual lipid remover, residual lipids, and particulate derivatives to the extraction device, and those for controlling the flow of particulate derivatives to the product container. The disposable components for the solvent extraction device (i.e., charcoal column), input liquid, input solvent, and solvent extraction device may be provided separately.

[0129] Extracting denatured HDL by delipidating plasma may be referred to as the first step of the methods described in the embodiments herein. According to the embodiments herein, in 110, the delipidated plasma is stored for subsequent use. In one embodiment, the delipidated plasma may be used after a long period of time, for example, at least one year, more preferably at least two years. In one embodiment, the delipidated plasma may be stored for a predetermined period, the period of which may depend on the storage process used.

[0130] Skimmed plasma (DP) can be stored in any aliquot or volume by utilizing appropriate storage methods, including but not limited to freezing. In various embodiments, any storage method can be used as long as the method retains a predetermined amount of potency of the final product compared to freshly skimmed plasma. Therefore, it is essential to evaluate skimmed plasma containing pre-βHDL using a 2D gel spot test or quality test to demonstrate that the pre-βHDL has not been degraded into free Apo A1. More specifically, the present invention provides a 2D gel test on a predetermined portion of stored skimmed plasma containing pre-βHDL, demonstrating that if 80% or less of the pre-βHDL has been degraded into Apo A1, the batch associated with the tested skimmed plasma is acceptable for administration to a patient.

[0131] Referring to Figure 2, process 400 for obtaining, storing, and thawing the extracted defatted plasma is shown. The extracted defatted plasma containing pre-βHDL is obtained in 405 using the process described herein. To establish a baseline amount or concentration of pre-βHDL, a spot test 410 is performed on a portion of a given batch of the extracted defatted plasma. The spot test 410 can be performed using 2D gel electrophoresis, in which a sample of the batch of extracted defatted plasma is solubilized, loaded onto a gel, and exposed to an electric field that causes the migration of proteins through the gel according to their isoelectric points. The separated proteins are then solubilized again and separated on a second axis orthogonal to their molecular weight. Thus, the spot test 410 quantifies the concentration or amount of proteins, such as pre-βHDL, in the sample along two axes, namely isoelectric point and molecular weight. Further clinical evaluations are described below.

[0132] After spot testing 410, the batch is subjected to storage 420, preferably by rapid freezing as further described below. In one embodiment, prior to storage 420, the batch is optionally modified 415 so that the protein concentration of pre-βHDL is within a predetermined range, as further described below. Stored pre-βHDL has been found to have low stability if the concentration of the preparation is too low, and reduced potency if the concentration of the preparation is too high. Therefore, the batch is optionally modified by dilution or concentration so that the concentration of pre-βHDL in defatted plasma is in the range of 0.5 mg / dl to 500 mg / dl, preferably in the range of 1 mg / dl to 400 mg / dl, or any increment therein. Preferably, the concentration of pre-βHDL in defatted plasma is 500 mg / dl or less.

[0133] After storage 420, the delipidated plasma is stored, which can range from one week to a maximum of three years or any increment therein. At some point, the delipidated plasma is thawed 430. In one embodiment, thawing is achieved by removing the frozen delipidated plasma and placing it in an environment with a temperature range of 2°C to 26°C. In one embodiment, the delipidated plasma during thawing is maintained in an environment with a temperature range of 3°C to 5°C, more preferably 4°C, for a period of 48 hours or less. Preferably, the thawed delipidated plasma is used within 48 hours, more preferably within 24 hours, of thawing and is not refreezed. After thawing 430, a portion of the batch can be retested 440 to determine whether the pre-βHDL in the delipidated plasma has deteriorated or is no longer effective, as will be further described below.

[0134] Optionally, additives may be included as part of the preservation process. Additives may be added to either the precursor or the final product to enhance the preservation process. In embodiments, DP is preserved using methods and standards similar to those applicable to the preservation of plasma. In one embodiment, preservation is achieved by freezing. Some of these standard methods and practices are defined in the CFR and AABB, ABC, ARC Circulars on the Use of Human Blood and Blood Components, as well as in the European Pharmacopoeia Guidelines on the Preparation of Plasma for Production. Volumes or aliquots of defatted source plasma are placed in the freezer within a few hours after the completion of the defatting process. In some embodiments, DP is placed in the freezer within eight hours of the defatting process, or within the time frame specified in the instructions for the use of the blood collection, processing, and storage system. In embodiments, volumes of DP are frozen at a temperature of approximately -18°C to -80°C by standard means for fresh frozen plasma.

[0135] In this specification, the volumes or aliquots described above are merely illustrative, and it should be noted that any amount (volume or aliquot) of delipidated plasma sample can be stored using the systems and methods described herein. It should also be noted that multiple volumes or aliquots can be stored in series (each volume or aliquot stored sequentially) or in parallel (multiple volumes or aliquots stored simultaneously).

[0136] Returning to the storage process, in one embodiment, as an example, 50 ml of degreased plasma (DP) is frozen by flash freezing using liquid nitrogen and then stored at -80°C. In another embodiment, as an example, 50 ml of degreased plasma (DP) is frozen at -80°C using a slower freezing method. In yet another embodiment, as an example, 50 ml of degreased plasma (DP) is frozen at -20°C in a frostless freezer using a slower freezing method. In one embodiment, as an example, 100 ml of DP is frozen using flash freezing. In another embodiment, as an example, up to 400 ml of DP is frozen using flash freezing. In other embodiments, DP volumes (units) in the range of at least 1 ml to 400 ml or more are frozen together using the above storage methods; any freezing method may be used to store any volume of DP. The time required to freeze the sample depends on its size; therefore, the length of the freezing time varies depending on the size of the aliquot. In embodiments, the time required to freeze the aliquot size is also a function of the method used for freezing. In the embodiments, the time required to freeze the sample is predetermined and depends on the sample size and / or the freezing method. In one embodiment, the time required to flash freeze concentrated pre-βHDL to -80°C is less than 30 minutes, less than 20 minutes, preferably less than 10 minutes. In another embodiment, the flash freezing temperature is less than -30°C.

[0137] In various embodiments of this specification, other freezing methods may be used. The chosen freezing method ensures that important components of DP are preserved. The shelf life of frozen defatted plasma may vary depending on the freezing temperature. Generally, at lower freezing temperatures, the product can be stored for a longer period and therefore has a longer "storage period" (later expiration date).

[0138] In this embodiment, each unit of source plasma is evaluated individually before and after the degreasing process. The plasma is evaluated for parameters including, but not limited to, the following: 1. Concentration and size of pre-β and αHDL particles. In some embodiments, 2D gel electrophoresis techniques are used in conjunction with both heavy and light gels, as well as immunoblotting for ApoA-I. 2. Clinical chemistry of plasma. The various properties to be determined may include total cholesterol, HDL, LDL, ApoA-I, ApoB, triglycerides, CBC, sodium, potassium, chloride, calcium, phosphorus, creatinine, BUN, fibrinogen, aPTT, PT, ALT, AST, ALP, bilirubin, uric acid, glucose, and LDH. 3. Furthermore, the DP fractions before freezing, and after freezing and thawing in the next step, are evaluated for cholesterol content by UV absorbance using known methods such as high-performance protein liquid chromatography (FPLC). 4. Selected samples / fractions, including but not limited to those depicted in Figures 1A and 1G, are assayed for cholesterol efflux capacity.

[0139] In some embodiments, the above parameters are also evaluated before storing or freezing the degreased plasma and after thawing the degreased plasma. In some embodiments, the potency of the thawed degreased plasma is in the range of 1% to 100% of the potency of the degreased plasma before storage. In some embodiments, the potency of the thawed degreased plasma is in the range of 1% to 150% of the potency of the degreased plasma before storage. In some embodiments, the potency of the thawed degreased plasma is in the range of 1% to 200% of the potency of the degreased plasma before storage. In some embodiments, the potency of the thawed degreased plasma is lower than the potency of the degreased plasma before storage. In some embodiments, the potency of the thawed degreased plasma is higher than the potency of the degreased plasma before storage.

[0140] In 112, the stored DP is prepared for further processing or normal use for the treatment of a patient. The patient treated with the DP may be or may not be an individual from whom plasma was obtained for the delipidation process (it may be autologous or non-autologous). If the DP was stored by freezing in 110, the DP is prepared by thawing in 112. In one embodiment, the frozen DP is thawed in a water bath at a temperature range of 30°C to 37°C for about 30 minutes. In some embodiments, the thawed plasma is maintained at 1°C to 6°C for 1 to 5 days. In another embodiment, the frozen DP is slowly thawed at room temperature. In yet another embodiment, the frozen DP is rapidly thawed in a refrigerator at about 5°C. Other methods for thawing frozen DP may be available for various amounts and compositions of DP.

[0141] In various embodiments of this specification, the second step involves further processing of the degreased or denatured HDL to separate or isolate components of the HDL particles, such as pre-βHDL particles or a combination of α and pre-βHDL particles. In 114, the processed and thawed plasma containing the denatured HDL particles with reduced lipid content, separated from the solvent in 108, is further processed with the solvent to produce a solution containing higher concentrations of α and pre-βHDL particles. Exemplary methods for separating α and pre-β particles from degreased plasma are described below.

[0142] According to some arbitrary embodiments of this specification, in 116, the separated α and pre-β HDL particles can be preserved by freezing and used long after thawing in 118. In one embodiment, thawing is achieved by removing the frozen degreased plasma and placing it in an environment with a temperature range of 2°C to 26°C. In one embodiment, the degreased plasma during thawing is maintained in an environment with a temperature range of 3°C to 5°C, more preferably 4°C, for a period of 48 hours or less. Preferably, the thawed degreased plasma is used within 48 hours, more preferably within 24 hours, of thawing and is not refreezable. In embodiments, the process for preserving and preparing the aspirated (derived) α and pre-β HDL particles is similar to the method for preserving and preparing DP. In one embodiment, a flash freezing method is preferably used to freeze the aspirated (derived) α and pre-β HDL particles.

[0143] (Method for separating pre-β particles from delipidated plasma) In one embodiment, affinity chromatography may be used to reduce the amount of unwanted substances (e.g., plasma proteins and certain lipoproteins such as LDL and VLDL) in delipidated plasma, and thus increase the concentration of a desired substance (e.g., pre-βHDL particles).

[0144] Figure 1D is a flowchart illustrating an exemplary sequence of steps used to increase the concentration of a desired substance in treated plasma using affinity chromatography, according to several embodiments herein. In one embodiment, an affinity column is used to capture ApoA-I protein particles (and therefore pre-βHDL particles) while removing undesirable particles from treated plasma. In 120, treated plasma is added to a column for affinity chromatography utilizing an antibody against ApoA-I, so that ApoA-I binds to the antibody as the treated plasma passes through the column. A solid medium such as a resin can be used to bind the desired substance in the form of HDL particles. The desired HDL particles may include a mixture of α-HDL and pre-βHDL particles. The composition of these particles is approximately 15% α-HDL and approximately 85% pre-β particles. In 122, unbound unwanted substances, including LDL and VLDL, pass through the column (as they do not bind to the antibody against ApoA-I) and are removed from the treated plasma solution. In step 124, the washing buffer can be passed through the column to remove unwanted proteins. Next, the dissociation reagent or solution is passed through the column so that pre-βHDL particles (or ApoA-I containing pre-βHDL particles) no longer bind to the antibody against ApoA-I and are effectively separated.

[0145] Ultracentrifugation is another method that can be used to reduce the amount of unwanted substances (e.g., plasma proteins and certain lipoproteins such as LDL and VLDL, as well as αHDL particles in this case) and thus increase the concentration of desired substances (e.g., pre-βHDL particles). This method utilizes the principle of centrifugation to separate the components of a solution by rotating the solution at a very high speed.

[0146] The density of the degreased plasma solution used as the starting material is 1006 kg / m³. 3That is, 1.006 g / ml. In order to separate various fractions during ultracentrifugation, it may be necessary to adjust the density of the starting material at each step, as described below. In one embodiment, the density can be adjusted by adding a denser substance or solvent (such as potassium bromide (KBr)) to the treated plasma. In one embodiment, any solution of any density that can be used to adjust the density of the starting material may be used.

[0147] In one embodiment, a concentrated stock solution of potassium bromide (KBr) and sodium chloride (NaCl) with a total density of 1.346 is added to a defatted / treated plasma solution to adjust the density of the treated plasma solution to a desired density. The density of the stock solution is higher than 1.25 g / mL (for example, the density of an aqueous solution of 4.62 M KBr is 1.37 g / mL), and the density of the treated plasma solution is approximately 1.006 g / mL. The solution resulting from the addition of a high-density substance to the treated plasma has a specific density that allows for the separation of various fractions during ultracentrifugation. Methods for adjusting density are well known to those skilled in the art and are also described in Havel et al., J. Clin. Invest. 1955, 34; pp. 1345-1353, which describes a method for separating and purifying lipoprotein fractions on a preparative scale for analysis and use in metabolic studies and is incorporated herein by reference.

[0148] In one embodiment, the combined plasma and solvent are introduced into an ultracentrifuge tube (or may be combined within the tube itself). The tube may be held by a rotor. The tube is centrifuged at high speed for a sufficient time to produce separation, then the rotor is smoothly stopped, and the gradient is gently pumped out of each tube to isolate the separated components. In one embodiment, ultracentrifugation is performed at a speed of about 105,000 × g for about 16 to 20 hours. It should be noted that the centrifugation time is adjusted based on the density of the starting material and the relative density of the desired fraction to be separated. For example, if the density of the material to be extracted in the first step is 1.21 g / mL and the subsequent density required in the second step is 1.25 g / mL, it will take longer to separate the two materials of similar density than to separate a material of density 1.006 g / mL from a starting material of density 1.21 g / mL.

[0149] In the embodiment, the desired fraction obtained contains at least pre-βHDL particles, which are separated from the treated plasma.

[0150] Figure 1E is a flowchart illustrating an exemplary sequence of steps used to increase the concentration of a desired substance in treated plasma using ultracentrifugation, according to several embodiments herein. In one embodiment, the method described in this flowchart may be used to more rapidly separate pre-βHDL using ultracentrifugation. In 130, the starting material, treated plasma, can be centrifuged at a adjusted density of 1.21, corresponding to the density of HDL. The upper fraction will contain VLDL, LDL, and αHDL, and pre-βHDL will be contained in the bottom fraction. In one embodiment, centrifugation is performed over 18–24 hours. In 132, the bottom fraction containing pre-βHDL and plasma proteins is isolated. In 134, the isolated bottom fraction containing pre-βHDL is centrifuged at a density of 1.25 to produce an upper fraction containing pre-βHDL and a bottom fraction containing plasma proteins. In one embodiment, centrifugation is performed over 24–48 hours. In step 136, the obtained upper and lower fractions containing pre-βHDL are isolated from the lower fractions containing plasma proteins to produce a concentrated pre-βHDL product.

[0151] At the end of the ultracentrifugation process, the pre-βHDL particles float to the top and are separated, yielding a solution containing a high concentration of pre-βHDL particles. The tube containing the concentrated pre-βHDL particles can then be removed.

[0152] Figure 1F is a flowchart showing another exemplary series of steps used to increase the concentration of a desired substance in treated plasma using ultracentrifugation, according to some embodiments herein.

[0153] In some embodiments, ultracentrifugation is a stepwise process performed at different densities. Because lipoproteins are lighter than proteins, ultracentrifugation at different densities can be used to separate them from defatted (treated) plasma to achieve sequential fractionation. At 140°F, the defatted (treated) plasma is separated from the plasma at a density of (1006 kg / m³). 3Centrifuge for 18-24 hours at a density of 1.006 (corresponding to 1.006 g / ml). At this stage, proteins are at the bottom of the centrifuge tube, and VLDL is at the top. Furthermore, plasma containing LDL and HDL is at the very bottom of the tube.

[0154] In step 142, the plasma fraction containing LDL / HDL is separated from the other components in the tube.

[0155] In step 144, plasma containing LDL / HDL (and a solution to adjust density) is centrifuged to a density of 1.063, corresponding to the density of LDL. The result is a fraction containing LDL at the top of the tube and a fraction containing HDL at the bottom of the tube.

[0156] In step 146, the bottom fraction containing HDL is separated from the other components in the tube.

[0157] In step 148, the HDL fraction is centrifuged at a density of 1.21, corresponding to the density of HDL. The final result is a fraction containing HDL at the top of the tube and pre-βHDL at the bottom of the tube.

[0158] In step 150, the bottom fraction containing pre-βHDL is centrifuged at a density of 1.25. The final result is a fraction containing pre-βHDL at the top of the tube and the remaining plasma components at the bottom of the tube. At the end of the ultracentrifugation process, the pre-βHDL particles float to the top and are separated to obtain a solution containing a high concentration of pre-βHDL particles. The tube containing the concentrated pre-βHDL particles can then be removed. The isolated lipoprotein fraction is separated from KBr using dialysis, gel filtration, or other suitable method.

[0159] Those skilled in the art will understand that the fractions obtained by centrifugation of the processed plasma can be separated by different methods and are not limited to the methods described in relation to Figures 1E and 1F.

[0160] Figure 1G is a flowchart showing another exemplary set of steps used to increase the concentration of a desired substance in treated plasma using ultracentrifugation, according to some embodiments herein. In one embodiment, the method described in this flowchart may be used to more rapidly separate both α(α-1) and pre-βHDL from delipidated plasma using ultracentrifugation. At 170, the starting material, treated plasma, may be centrifuged at a adjusted density of 1.063, corresponding to the density of LDL. The result is a fraction containing LDL at the top of the tube and a fraction containing HDL at the bottom of the tube. In one embodiment, centrifugation is performed over 18–24 hours. At 172, the bottom fraction containing α-1 and pre-βHDL and plasma proteins is isolated. At 174, the isolated bottom fraction containing α-1 and pre-βHDL is centrifuged at a density of 1.25 to produce an upper fraction containing α-1 and pre-βHDL and a bottom fraction containing plasma proteins. In one embodiment, centrifugation is performed over 24–48 hours. In step 176, the resulting upper fraction containing α-1 and pre-βHDL is isolated from the bottom fraction containing plasma proteins, producing a concentrated product containing α-1 and pre-βHDL.

[0161] At the end of the ultracentrifugation process, the α1 and pre-βHDL particles float to the top and are separated to obtain a solution containing high concentrations of α1 and pre-βHDL particles. The tube containing the concentrated α-1 and pre-βHDL particles can then be removed. The isolated lipoprotein fraction is separated from KBr by dialysis, gel filtration, or other suitable method.

[0162] Returning to Figure 1A, at 160, treated plasma containing a concentrated solution of pre-βHDL or a combination of α and pre-βHDL particles with reduced lipid content is separated from the solvent, appropriately processed, and then delivered to the patient. In the delivered solution, the concentrations of α and / or pre-βHDL are further increased. The treated plasma, containing HDL particles with reduced lipid content and increased pre-β concentration, is provided to the patient after dialysis with saline. In the case of affinity chromatography, dialysis also releases the entrapped desired substance (concentrated pre-βHDL particles). In the case of autologous plasma, if the patient's red blood cells have not yet been returned during plasmapheresis, they may be administered to the patient at some point during the procedure. In any embodiment, red blood cells may be returned to the patient after being combined with the isolated α and / or pre-βHDL particles. One route of administration is via the vascular system, preferably intravenously.

[0163] In another alternative embodiment, a two-step process is applied to completely isolate pre-βHDL particles. First, affinity chromatography is used to separate the solution containing both pre-β and αHDL particles. Then, ultracentrifugation is used to completely isolate the pre-βHDL particles.

[0164] The examples above are merely illustrative of the many applications of the system of the present invention. While only a few embodiments of the present invention have been described herein, it should be understood that many other specific forms may be implemented without departing from the spirit or scope of the invention. Therefore, these examples and embodiments should be considered illustrative rather than restrictive, and the present invention may be modified within the scope of the appended claims.

Claims

1. A method for preserving pre-β high-density lipoprotein, including the following steps: A step of obtaining a batch of defatted plasma containing the aforementioned pre-β high-density lipoprotein; A step of testing a portion of a batch of degreased plasma to characterize the pre-β high-density lipoprotein, thereby determining at least partially a first characteristic of the pre-β high-density lipoprotein, wherein the first characteristic includes a first concentration of the pre-β high-density lipoprotein in the degreased plasma; A step of preserving a batch of degreased plasma by freezing the degreased plasma; A step of preparing the stored defatted plasma after a certain period of time, wherein the certain period of time is in the range of one week to three years; and A step of testing the prepared defatted plasma, comprising: determining a second characteristic of the pre-β high-density lipoprotein from the prepared preserved defatted plasma using 2D gel electrophoresis, wherein the second characteristic includes a second concentration of the pre-β high-density lipoprotein in the prepared preserved defatted plasma; comparing the second characteristic of the pre-β high-density lipoprotein with the first characteristic of the pre-β high-density lipoprotein to determine the degree of degradation; and determining whether the prepared preserved defatted plasma is suitable for administration based on the second characteristic of the pre-β high-density lipoprotein, wherein if 80% or less of the pre-β high-density lipoprotein is degraded into Apo A1, the prepared preserved defatted plasma is deemed suitable for administration.

2. The method according to claim 1, further comprising changing the amount of pre-β high-density lipoprotein before the storage step to ensure that the concentration of the pre-β high-density lipoprotein is in the range of 1 mg / dl to 400 mg / dl.

3. The method according to claim 1, wherein the preservation step includes freezing the batch at a temperature below -30°C.

4. The method according to claim 1, wherein the preparation step includes thawing the stored defatted plasma in a temperature range of 2°C to 26°C.

5. The method according to claim 1, wherein the preservation step includes exposing a volume of 1 milliliter to 2 liters of degreased plasma to a temperature below -30°C for less than 20 minutes.

6. The method according to claim 1, further comprising adding a preservative to the delipidated plasma before storage.

7. The method according to claim 1, wherein the preparation step further comprises thawing the stored defatted plasma, and storing the thawed defatted plasma at a temperature in the range of 1°C to 6°C for 5 days or less.

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