Lipoprotein complexes and manufacturing and uses thereof

Abstract

To provide a lipoprotein complex and a lipoprotein population, and uses thereof for treating and / or preventing dyslipidemic disorders and / or conditions, as well as recombinant expression of apolipoproteins, purification of apolipoproteins, and production of lipoprotein complexes using a method based on thermal cycling.SOLUTION: Disclosed is a lipoprotein complex comprising an apolipoprotein fraction and a lipid fraction, where the lipid fraction consists essentially of 95 to 99 wt.% of neutral phospholipid and 1 to 5 wt.% of negatively charged phospholipid, and the ratio by weight of the apolipoprotein fraction to the phospholipid fraction is ranging from about 1:2.7 to about 1:3.SELECTED DRAWING: None

Description

[Technical field]

[0001] 1. CROSS REFERENCE TO RELATED APPLICATIONS This application is a joint venture of U.S. Provisional Patent Application No. 61 / 440,371, filed February 7, 2011. No. 61 / 452,630, filed March 14, 2011, and U.S. Provisional Patent Application No. No. 61 / 487,263, filed May 17, 2011 (all of which is incorporated herein by reference). 35 U.S.C. § 1 of the Act, the entire contents of which are incorporated herein by reference. Claim benefits under 19(e).

[0002] 2.Technical Field The present disclosure relates to lipoprotein complexes, pharmaceutical compositions containing the complexes, and methods for treating such complexes. A method for producing and purifying an apolipoprotein for use in a cardiac The present invention relates to a method for treating or preventing vascular diseases, disorders and / or conditions associated therewith. Provides a method for using coalescence. [Background technology]

[0003] 3.Background Overview Circulating cholesterol is a complex of lipids and protein components that transport lipids in the blood. They are carried by particles called plasma lipoproteins. There are four major classes of lipoproteins Particles circulate in the plasma and are involved in the fat transport system: chylomicrons, very low density lipoproteins Very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) Chylomicrons are short-lived products of intestinal fat absorption. VLDL and especially LDL is derived from the liver (where cholesterol is synthesized or obtained from dietary sources) and then transported to extrahepatic tissues. HDL is responsible for delivering cholesterol to tissues, e.g., arterial walls. role in lipid transport (RCT), specifically mediating the removal of cholesterol lipids from extrahepatic tissues to the liver HDL also acts as a marker for inflammation, oxidized lipids, and lipid metabolism. and plays a beneficial role in the transport of interleukins, which then play an important role in inflammation in the vascular wall. can be reduced.

[0004] Lipoprotein particles contain cholesterol (usually in the form of cholesteryl esters). The core is composed of phospholipids, non-esterified lipids, and triglycerides. surrounded by a surface envelope containing esterified cholesterol and apolipoproteins Apolipoproteins mediate lipid transport and some interact with enzymes involved in lipid metabolism. Can be used. ApoA-I, ApoA-II, ApoA-IV, ApoA-V, ApoB, ApoC-I, ApoC-II, ApoC-III, ApoD, ApoE, ApoJ and At least ten apolipoproteins have been identified, including ApoH and LCAT. (lecithin:cholesterol acyltransferase), CETP (cholesteryl estheryl lipid transfer protein), PLTP (phospholipid transfer protein) and PON (paraoxon Other proteins, such as ribosomal kinases, have also been found associated with lipoproteins.

[0005] Cardiovascular diseases such as coronary heart disease, coronary artery disease and atherosclerosis are caused by serum High cholesterol levels are closely related to atherosclerosis. Atherosclerosis is a slowly progressive disease characterized by the buildup of cholesterol in the walls of arteries. Compelling evidence suggests that lipids deposited in atherosclerotic lesions are derived primarily from plasma LDL. Thus, LDL is considered to be the "bad" cholesterol. In contrast, HDL serum levels are the opposite of coronary heart disease. In fact, high serum levels of HDL are considered a negative risk factor. Plasma HDL is not only protective against coronary artery disease, but actually prevents atherosclerosis. It has been hypothesized that this compound may induce regression of vascular endothelial plaques (e.g., Badimon et al., 1992, C Circulation 86 (Suppl. Ill): 86-94; Dansky and Fisher, 1999, Circulation 100: 17 62-63; Tangirala et al., 1999, Circulation 100(17): 1816-22; Fan et al., 1999, Atherosclerosis osis 147(l): 139-45; Deckert et al., 1999, Circulation 100(11): 1230-35; Boisvert et al. 1999, Arterioscler. Thromb. Vasc. Biol. l9(3):525-30; Benoit et al., 1999, Circulatio n 99(1): 105-10; Holvoet et al., 1998, J. Clin. Invest. 102(2):379-85; Duverger et al., 19 96, Circulation 94(4):713-17; Miyazaki et al., 1995, Arterioscler. Thromb. Vasc. Biol 15(11): 1882-88; Mezdour et al., 1995, Atherosclerosis 113(2):237-46; Liu et al., 1994, J. Lipid Res. 35(12):2263-67; Plump et al., 1994, Proc. Nat. Acad. Sci. USA 91(20):96 07-11; Paszty et al., 1994, J. Clin. Invest. 94(2):899-903; She et al., 1992, Chin. Med. J. (Engl). 105(5):369-73; Rubin et al., 1991, Nature 353(6341):265-67; ​​She et al., 1990, An n. NY Acad. Sci. 598:339-51; Ran, 1989, Chung Hua Ping Li Hsueh Tsa Chih (Zhongh (also translated as ua Bing Li Xue Za Zhi) 18(4):257-61; Quezado et al., 1995, J. Pharmacol Exp. Ther. 272(2):604-11; Duverger et al., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12): 1424-29; Kopfler et al., 1994, Circulation; 90(3): 1319-27; Miller et al., 1985, N ature 314(6006): 109-11; Ha et al., 1992, Biochim. Biophys. Acta 1125(2):223-29; Beit See, Z. et al., 1992, Prostaglandins Leukot. Essent. Fatty Acids 47(2): 149-52. As a result, HDL has become commonly known as "good" cholesterol. (See, e.g., Zhang et al., 2003 Circulation 108:661-663).

[0006] The “protective” role of HDL has been confirmed in several studies (e.g., Miller et al. , 1977, Lancet l(8019):965-68; Whayne et al., 1981, Atherosclerosis 39:411-19). In their study, elevated LDL levels were associated with increased cardiovascular risk, but High HDL levels are thought to confer cardiovascular protection. In vivo studies have shown that HDL Further demonstrating the protective role of L, HDL infusion in rabbits reduced cholesterol-induced arterial lesions. (Badimon et al., 1989, Lab. Invest. 60:455-61) and / or inhibit its regression. It has been shown that it can be induced (Badimon et al., 1990, J. Clin. Invest. 85: 1234-41).

[0007] Reverse cholesterol transport, HDL and apolipoprotein AI The reverse cholesterol transport (RCT) pathway eliminates cholesterol from many extrahepatic tissues RC functions as a cellular stimulator of the cellular endothelium and is important for maintaining the structure and function of many cells in the body. T is involved in three main steps: (a) cholesterol efflux, i.e., cholesterol is mobilized from various pools in peripheral cells; (b) the initial removal of cholesterol from the cytoplasm; (c) the re-entry of the effluxed cholesterol into the cell. Prevents cholesterol deficiency by the action of lecithin:cholesterol acyltransferase (LCAT) (c) cholesterol esterification by acetylcholine; and (d) delivery of HDL-cholesterol to liver cells for hydrolysis. Uptake of sterols and cholesteryl esters followed by recycling, storage, and excretion in the bile The bile acids are then converted to bile acids.

[0008] LCAT, a key enzyme in RCT, is produced by the liver and binds to the HDL fraction. LCAT converts cell-derived cholesterol to cholesteryl esters and circulates in plasma. These are sequestered in HDL and destined for elimination (Jonas 2000, Biochim. B (See Biophys. Acta 1529(1-3):245-56). Cholesteryl ester transfer protein The endothelial cell membrane protein (CETP) and phospholipid transfer protein (PLTP) are the major components of the circulating HDL population. CETP is a cholesteryl ester produced by LCAT. The lipoproteins that contain ApoB, such as VLDL and and LDL. PLTP provides lecithin to HDL. HDL triglycerides is catabolized by extracellular hepatic triglyceride lipase, and lipoprotein cholesterol is cleared by the liver via several mechanisms.

[0009] The functional characteristics of HDL particles are characterized by their major components, ApoA-I and ApoA-II. Mainly determined by apolipoprotein components: ApoC-I, ApoC-II in small amounts , ApoC-III, ApoD, ApoA-IV, ApoE and ApoJ also interact with HDL. HDL has been observed to bind to oxidized erythrocytes. Depending on the state of dering, in various different sizes and with different mixtures of the above components exists within.

[0010] Each HDL particle usually contains at least one molecule of ApoA and usually 2-4 molecules of ApoA. HDL particles also contain β-lactams, as described by Professor Gerd Assmann (see, for example, see Eckardstein et al., 1994, Curr Opin Lipidol. 5(6):404-16), cholesterol It may also contain only ApoE (γ-LpE particles), which is known to be responsible for role efflux. PoA-I is synthesized by the liver and small intestine as preproapolipoprotein AI. It is secreted as proapolipoprotein AI (proApoA-I) and is rapidly cleaved. This results in the production of the plasma form of ApoA-I, a single polypeptide chain of 243 amino acids ( Brewer et al., 1978, Biochem. Biophys. Res. Commun. 80:623-30). Injected preproApoA-I is also cleaved to plasma ApoA-I (Klon et al., 2000 , Biophys. J. 79(3): 1679-85; Segrest et al., 2000, Curr. Opin. Lipidol. 11(2): 105-1 5; Segrest et al., 1999, J. Biol. Chem. 274 (45):31755-58).

[0011] ApoA-I consists of 22 amino acids, 6–8 of which are α-helices or prolines. The repeat unit comprises functional repeats spaced apart by linker moieties, which are often It exists in an amphipathic helical conformation (Segrest et al., 1974, FEBS Lett. 38: 247-53). ), the main biological activities of ApoA-I, i.e., lipid binding and lecithin cholesterol acyltransferase (LCAT) activation.

[0012] ApoA-I exists in three types of stable complexes with lipids: pre-β-1 HDL , a small lipid-poor complex; called pre-β-2 HDL, a polar lipid (phospholipids and cholesterol)-containing flat, discoidal particles; and spherical or mature Spherical lipids containing both polar and non-polar lipids, called HDL (HDL3 and HDL2) Most HDL in the circulating population is composed of both ApoA-I and ApoA-II ( However, HDL that contains only ApoA-I is This fraction (the "AI-HDL fraction") appears to be more effective in RCTs. Epidemiological studies support the hypothesis that the ApoA-I-HDL fraction is anti-atherogenic ( Parra et al., 1992, Arterioscler. Thromb. 12:701-07; Decossin et al., 1997, Eur. J. Clin. Invest. 27:299-307).

[0013] HDL particles are a class of particles with different sizes, lipid and apolipoprotein compositions. They are made up of several populations, each of which is characterized by its hydration density, apolipoprotein composition, and The fraction can be separated according to characteristics such as molecular weight and charge characteristics. For example, the pre-β-HDL fraction can be separated according to molecular weight and charge characteristics. is characterized by a lower surface charge than mature α-HDL. Because of this charge difference, pre-β -HDL and mature α-HDL have different electrophoretic mobilities in agarose gels ( David et al., 1994, J. Biol. Chem. 269(12):8959-8965).

[0014] The metabolism of pre-β-HDL and mature α-HDL is also different. Pre-β-HDL has two metabolic pathways. Living: removed from plasma by the kidneys and catabolized or degraded preferentially by the liver Remodeling to medium-sized HDL (Lee et al., 2004, J. Lipid Res. 45(4):716-728 ).

[0015] The mechanism of cholesterol transfer from the cell surface (i.e., cholesterol efflux) is unknown. However, the lipid-poor complex pre-β-1 HDL is involved in peripheral tissue RCT. It is believed to be the preferred receptor for cholesterol transferred from , J. Biol. Chem. 269:22975-82; Bielicki et al., 1992, J. Lipid Res. 33: 1699-1709; Ro thblat et al., 1992, J. Lipid Res. 33: 1091-97; and Kawano et al., 1993, Biochemistry 32 :5025-28; Kawano et al., 1997, Biochemistry 36:9816-25). During this process of cholesterol mobilization, pre-β-1 HDL is converted to pre-β-2 H PLTP increases the rate of pre-β-2 HDL disk formation. However, there are no data to suggest a role for PLTP in RCTs. reacts preferentially with small discoidal (pre-β) and globular (i.e., mature) HDL The 2-acyl group of lecithin or other phospholipids is attached to the free hydroxyl residue of cholesterol. group to produce cholesteryl esters (retained in HDL) and lysolecithin. The LCAT reaction requires ApoA-I as an activator, i.e., ApoA-I is required for the reaction. oA-I is a natural cofactor for LCAT. Cholesterol sequestered in HDL The conversion of acetylcholine to its ester prevents cholesterol from re-entering the cell, with the net result means that cholesterol is removed from cells.

[0016] ApoAI-HDL fraction (i.e., containing ApoA-I and not ApoA-II) ) Cholesteryl esters in mature HDL particles are removed by the liver and converted to ApoA-I and than that derived from HDL containing both ApoA and II (AI / AII-HDL fraction). is processed into the bile more efficiently than leukocytes, which is due in part to the incorporation of ApoE into the hepatocyte membrane. This may be due to more efficient binding of AI-HDL. The existence of HDL receptors has been hypothesized. Scavenger receptor class B, type I (SR-BI) is also known as the HDL receptor. (Acton et al., 1996, Science 271:518-20; Xu et al., 1997, Lipid Res. 38:128 9-98). SR-BI is most abundant in steroidogenic tissues (e.g., adrenal gland) and liver. (Landschulz et al., 1996, J. Clin. Invest. 98:984-95; Rigotti et al., 1996, J. Biol. Chem. 271:33545-49). For a review of HDL receptors, see Broutin et al., 1988, Anal. . See Biol. Chem. 46: 16-23.

[0017] Early lipidation by ATP-binding cassette transporter AI regulates plasma HDL formation and cholesterol synthesis May be important for the ability of pre-β-HDL particles to cause teratogenic efflux (Lee and Parks, 2005, Curr. Opin. Lipidol. 16(1): 19-25). If this early lipidation were to occur, pre-β-HDL would be a more efficient cholesterol receptor. This allows ApoA-I to rapidly bind to naturally occurring plasma HDL particles. and higher availability of pre-β-HDL particles for cholesterol efflux bring about.

[0018] CETP may also play a role in RCT. These changes in VLDL and LDL may play a role in "remodeling" the HDL population. For example, in the absence of CETP, HDL becomes enlarged particles and does not disappear. (For a review of RCT and HDL, see Fielding and Fielding, 1995, J. Lipid Res. 36:211-28; Barrans et al., 1996, Biochem. Biophys. Acta 1300:73-85; Hirano et al., 1997 , Arterioscler. Thromb. Vasc. Biol. 17(6): 1053-59).

[0019] HDL also plays a role in the reverse transport of other lipids and non-polar molecules, as well as in detoxification, i.e. Role in transporting lipids from cells, organs, and tissues to the liver for catabolism and excretion Such lipids include sphingomyelin (SM), oxidized lipids, and and lysophosphatidylcholine. See, e.g., Robins and Fasulo (1997, J.C. Lin. Invest. 99:380-84) reported that HDL mediates the transport of plant sterols by the liver into bile secretion. has been shown to stimulate

[0020] ApoA-I, a major component of HDL, can bind to SM in vitro. When ApoA-I was reconstituted in vitro using BBSM, The maximum rate of assembly occurs at 28°C, which is close to the phase transition temperature of BBSM (Swaney, 1999). 83, J. Biol. Chem. 258(2), 1254-59). BBSM:A ratio of 7.5:1 or less (wt / wt) The poA-I ratio is 360:1 with 3 ApoA-I molecules per particle. Single reconstituted homogenous HDL particles with SM:ApoA-I molar ratio are formed. It was found in the electron microscope that the phospholipids of ApoA-I are located in a high phospholipid / protein ratio. Appears as a discoid complex similar to that obtained by recombination with acetylcholine However, a BBSM:ApoA-I ratio of 15:1 (wt / wt) resulted in a higher Larger diameter discoid complexes were formed with a lipid:protein molar ratio of 535:1. These complexes are formed by the ApoA-I complex formed with phosphatidylcholine. They are significantly larger, more stable, and more resistant to denaturation than the intact body.

[0021] Sphingomyelin (SM) is an early cholesterol receptor (pre-β-HDL and γ -Emerging ApoE-containing lipoproteins (ApoE-containing lipoproteins) are elevated, which indicates that SM is a mechanism by which these particles These results suggest that the ability of the IL-1 receptor to promote telomerase efflux may be enhanced (Dass and Je ssup, 2000, J. Pharm. Pharmacol. 52:731-61; Huang et al., 1994, Proc. Natl. Acad. Sci . USA 91: 1834-38; Fielding and Fielding 1995, J. Lipid Res. 36:211-28).

[0022] 3.3. Defensive Mechanism of HDL and ApoA-I Research into the defense mechanism of HDL has focused on apolipoprotein AI (Apo AI), the main component of HDL. High plasma levels of ApoA-I are associated with the absence or presence of coronary artery disease. (Maciejko et al., 1983, N. Engl. J. Med. 309:385-89; Sedlis et al., 1983, 6, Circulation 73:978-84).

[0023] Infusion of ApoA-I or HDL in experimental animals showed no significant biochemical changes. and reduces the extent and severity of atherosclerotic lesions. (1982, Arteriosclerosis 2:407a), Badimon et al. (1989, Lab. Invest . 60:455-61; 1989, J. Clin. Invest. 85: 1234-41) was significantly higher than HDL (d=1.063-1. 325g / ml) in cholesterol-fed rabbits, The degree of atherosclerotic lesions (45% reduction) and their cholesterol ester content (58 They also found that the HDL They also found that injections induced nearly 50% regression of established lesions. 7:523a) reported that HDL infusion improves the survival of patients with hereditary hypercholesterolemia who develop early arterial lesions. Significant changes in plasma lipoprotein composition in Watanabe rabbits with terolemia In these rabbits, the amount of HDL infused was related to the protective HDL and aortic hyperplasia. It may be more than twice the ratio to hard LDL.

[0024] The ability of HDL to prevent arterial disease in animal models was demonstrated by the in vitro This was further supported by the observation that ro can exhibit fibrinolytic activity (Saku et al., 1985 , Thromb. Res. 39: 1-8). Ronneberger (1987, Xth Int. Congr. Pharmacol., Sydney , 990) reported that ApoA-I increased fibrinolysis in beagle dogs and cynomolgus monkeys. Similar activity was also demonstrated in human plasma in vitro. Ronneberger reported that lipid deposition and vascular endothelial function in animals treated with ApoA-I were significantly reduced. A reduction in vascular plaque formation was confirmed.

[0025] In vitro studies have shown that complexes of ApoA-I with lecithin inhibit the proliferation and proliferation of cultured arteries. These results suggest that it may promote the efflux of free cholesterol from smooth muscle cells. (Stein et al., 1975, Biochem. Biophys. Acta, 380: 106-18). By this mechanism, HDL The proliferation of these cells can also be reduced (Yoshida et al., 1984, Exp. Mol Pathol. 41 :258-66).

[0026] Infusion therapy with HDL containing ApoA-I or ApoA-I mimetic peptides has also been shown to be Regulating plasma HDL levels by ABC1 transporter and its efficacy in treating cardiovascular diseases (e.g., Brewer et al., 2004, Arterioscler. Thromb. Vasc. (see Biol. 24: 1755-1760).

[0027] There are two naturally occurring human polymorphisms of ApoA-I in which arginine residues are replaced by cysteine. Apolipoprotein AI Milano (ApoA-I M ) in This substitution occurs at residue 173 in apolipoprotein AI Paris (ApoA-I P ), this substitution occurs at residue 151 (Franceschini et al., 1980, J. Clin. Inv. est. 66:892-900; Weisgraber et al., 1983, J. Biol. Chem. 258:2508-13; Bruckert et al., 199 7, Atherosclerosis 128: 121-28; Daum et al., 1999, J. Mol. Med. 77:614-22; Klon et al., 2000, J. 00, Biophys. J. 79(3): 1679-85). In addition, leucine was replaced by arginine at position 144. A further naturally occurring human polymorphism of ApoA-I has been isolated, which is thought to be involved in the Protein AI Zaragoza (ApoA-I z ) and is called severe low alpha lipoprotein Proteinemia and enhanced high-density lipoprotein (HDL) reverse cholesterol transport (Recalde et al., 2001, Atherosclerosis 154(3):613-623; Fiddyment et al., 2003, Atherosclerosis 154(3):613-623) 11, Protein Expr. Purif. 80(1): 110-116).

[0028] ApoA-I M or ApoA-I P Reconstituted disulfide-linked homodimers of HDL particles dissolve dimyristoylphosphatidylcholine (DMPC) emulsions Reconstitution with wild-type ApoA-I in the ability to promote cholesterol efflux Similar to HDL particles that have been isolated from mice (Calabresi et al., 1997b, Biochemistry 36: 12428-33; Franc eschini et al., 1999, Arterioscler. Thromb. Vasc. Biol. 19: 1257-62; Daum et al., 1999, J. Mol. Med. 77:614-22). For both mutations, heterozygous individuals have low levels of HDL. This, paradoxically, reduces the risk of atherosclerosis (Franceschini et al., 1980, J. Clin. Invest. 66:892-900; Weisgraber et al., 1983, J. Biol. Chem. 258:25 08-13; Bruckert et al., 1997, Atherosclerosis 128: 121-28). Reconstituted HDL particles can activate LCAT, but do not contain wild-type ApoA-I. The efficiency is low when compared with reconstituted HDL particles containing em. Biophys. Res. Commun. 232:345-49; Daum et al., 1999, J. Mol. Med. 77:614-22).

[0029] ApoA-I M The mutation is transmitted as an autosomal dominant trait; eight generations in a family Carriers of the gene have been identified (Gualandri et al., 1984, Am. J. Hum. Genet. 37: 1083-97). ApoA-I M The condition of carrier individuals is characterized by a marked reduction in HDL cholesterol levels. Despite this, carrier individuals do not appear to be at any increased risk for arterial disease. In fact, examination of family records has revealed that these subjects " (Sirtori et al., 2001, Circulation, 103: 1949-1954; Roma et al., , 1993, J. Clin. Invest. 91(4): 1445-520).

[0030] ApoA-I in carriers of the mutation M The possible mechanism of protective effect of is the single α -ApoA-I with loss of helices and increased exposure of hydrophobic residues M Mutant This is thought to be related to structural modifications (Franceschini et al., 1985, J. Biol. Chem. 260: 1632 -35) The loss of tightly packed α-helices is associated with a loss of function compared to normal ApoA-I. This results in increased molecular flexibility, allowing it to bind lipids more easily. The complex is more susceptible to denaturation, thus improving lipid delivery in the case of this mutant. This suggests that

[0031] Bielicki et al. (1997, Arterioscler. Thromb. Vasc. Biol. 17 (9): 1637-43) reported that Ap oA-I M shows limited ability to mobilize membrane cholesterol compared to wild-type ApoA-I Furthermore, it was demonstrated that ApoA-I M Nascent phospholipids formed by binding to membrane lipids HDL is more abundant than the larger 9 and 11 nm complexes formed by wild-type ApoA-I. Rather, the particles were predominantly 7.4 nm in size. These observations are consistent with the finding that the ApoA-I primary sequence Arg 173 →Cys 173 Replacement of cellular cholesterol mobilization and nascent HDL assembly This mutation blocks the normal process of cholesterol release from cells. Therefore, its anti-atherogenic properties are associated with a decrease in the efficiency of thrombin removal. You don't have to.

[0032] Arg 173 →Cys 173 The most striking structural change resulting from the substitution is in ApoA-I M (Bielicki et al., 1997, Arterioscler. Thromb. Vasc. Biol. 17 (9): 16 37-43). ApoA-I M forms homodimers with itself and heterodimers with ApoA-II. A study of blood fractions containing a mixture of apolipoproteins has shown that The presence of dimers and complexes in the circulation accounts for the increased elimination half-life of apolipoproteins. Such an increase in elimination half-life may be due to the As observed in clinical studies (Gregg et al., 1988, NATO ARW on Human Apolipoprotein Muta nts: From Gene Structure to Phenotypic Expression, Limone SG). Other studies are oA-I M Dimer (ApoA-I M / ApoA-I M ) in vitro in HDL particles These results suggest that the acetylcholinesterase inhibitor acts as an inhibitor in the interconversion of acetylcholine (Franceschini et al., 1990 , J. Biol. Chem. 265: 12224-31).

[0033] 3.4. Current Treatments for Dyslipidemia and Related Disorders Dyslipidemia is characterized by elevated serum cholesterol and triglyceride levels and serum A disease associated with a decrease in the HDL:LDL ratio, and is known as hyperlipidemia, especially hypercholesterolemia. , coronary heart disease, coronary artery disease, vascular and perivascular diseases, and atherosclerotic arteries Cardiovascular diseases such as atherosclerosis and transient ischemic attacks caused by arterial insufficiency. or syndromes associated with atherosclerosis, such as intermittent claudication. To lower elevated serum cholesterol and triglycerides associated with the disorder. Several treatments are currently available; however, efficacy, side effects, and suitability for the patient population remain unclear. In terms of character, each has its own shortcomings and limitations.

[0034] Bile acid-binding resins are a class of drugs that block the recycling of bile acids from the intestine to the liver; e.g. For example, cholestyramine (Questran Light®, Bristol-Myers Squibb) Myers Squibb), colestipol hydrochloride (Colestid®, The Upjohn Company), and colesevelam hydrochloride (Welchol (registered trademark, Daiichi-Sankyo Company). When taken orally, These positively charged resins bind to negatively charged bile acids in the intestine. Since they are not absorbed from the blood, they are excreted carrying bile acids with them. Even the best use of such resins is sufficient to lower serum cholesterol levels by about 20%. It is associated with gastrointestinal side effects such as constipation and certain vitamin deficiencies. Other oral medications should be taken at least 1 hour before or 4–8 hours after ingestion of the resin to prevent binding to other drugs. must be taken six hours later; thus complicating the drug regimen for cardiac patients.

[0035] Statins inhibit the HMGCoA reductase, a key enzyme involved in the cholesterol biosynthesis pathway. It is a cholesterol-lowering drug that blocks cholesterol synthesis by inhibiting the enzyme that produces it. Statins, e.g., lovastatin (Mevacor®), simvastatin (Zocor®), pravastatin (Pravachol®), Rubastatin (Lescol®) and Atorvastatin (Lipitor®) Statins, which are sometimes used in conjunction with bile acid-binding resins, can increase serum cholesterol. Significantly reduced teratinocyte and LDL-serum levels and reduced coronary atherosclerosis Slows progression of the disease, but only moderately increases serum HDL cholesterol levels The mechanism of the LDL-lowering effect is a decrease in VLDL concentration and induction of cellular expression of LDL-receptors. It can induce both a decrease in the production of LDL and / or an increase in its catabolism. Side effects, such as impaired liver and kidney function, are associated with the use of these drugs (The Physicia ns Desk Reference, 56th ed., 2002, Medical Economics).

[0036] Niacin (nicotinic acid) is a water-soluble vitamin D3 inhibitor used as a dietary supplement and antihyperlipidemic agent. Niacin is a B-complex vitamin that reduces the production of VLDL and lowers LDL. In some cases it is used in conjunction with bile acid binding resins. Although nicotinamide can increase HDL when used in sufficient doses, such high doses When used in large doses, its usefulness is limited by serious side effects. Niacin is an extended-release form of niacin that has fewer side effects than pure niacin. Nicostatin® is a combination of niacin and lovastatin. It is a formulation that contains both valproate and valproate, combining the benefits of each drug.

[0037] Fibrates are used to treat various forms of hyperlipidemia (i.e. hypercholesterolemia) that may also be associated with hyperlipidemia. It is a class of lipid-lowering drugs used to treat Fibrates appear to reduce the VLDL fraction and modestly increase HDL. However, the effects of these drugs on serum cholesterol are variable. tetracycline (Atromid-S®), fenofibrate (Tricot®), Fibrates such as rifafibrate (R.T.M.) and bezafibrate (Bezalip®) , approved for use as an antihyperlipidemic agent, but not as a hypercholesterolemic agent. For example, clofibrate has been shown to reduce the VLDL fraction. Antihyperlipidemic, acting to lower serum triglycerides (via unknown mechanisms) It can lower serum cholesterol in certain patient subpopulations, but Biochemical responses to drugs vary, making it impossible to always predict which patients will have a favorable outcome. Atromid-S® is not effective in preventing coronary heart disease. The chemically and pharmacologic related drug gemfibrozil ( Lopid® reduces serum triglycerides and VLDL cholesterol moderately. The HDL cholesterol-HDL2 and HDL3 subfractions as well as Apo Moderately increase both AI and A-II (i.e., AI / AMT-HDL fraction) However, lipid responses are heterogeneous, especially among different patient populations. In male patients aged 40 to 55 years with no history or symptoms of coronary heart disease, Although prevention of cardiovascular events was observed, the extent to which these findings could be translated to other patient populations (e.g., women, It is unclear whether these findings can be extrapolated to older and younger men. However, no efficacy was observed in patients with established coronary heart disease. Serious adverse events include increased toxicity, such as cancer (especially gastrointestinal cancer), gallbladder disease, and non-coronary mortality. Significant side effects are associated with the use of fibrates.

[0038] Oral estrogen replacement therapy improves moderate hypercholesterolemia in postmenopausal women However, an increase in HDL is often accompanied by an increase in triglycerides. Estrogen therapy is, of course, limited to a specific patient population (postmenopausal women) and is associated with increased risk of malignant neoplasms. , gallbladder disease, thromboembolism, hepatocellular adenoma, elevated blood pressure, impaired glucose tolerance, and hypercalcemia. It is associated with serious side effects, including induction of

[0039] Other drugs useful for the treatment of hyperlipidemia include those that block or inhibit cholesterol absorption. However, ezetimibe (Zetia®; Merck) is an , inhibitors of ezetimibe have been shown to exhibit certain toxicities.

[0040] HDL and recombinant forms of ApoA-I complexed with phospholipids are non-polar or bipolar. Amphiphilic molecules, such as cholesterol and derivatives (oxysterols, oxidized sterols) , plant sterols, etc.), cholesterol esters, phospholipids and derivatives (oxidized phospholipids) A sink for lipids, triglycerides, oxidation products, and lipopolysaccharide (LPS) may serve as scavengers (see, e.g., Casas et al., 1995, J. Surg. Res. Nov 59(5):5 (See e.g., 44-52.) HDL also acts as a stimulator for TNF-α and other lymphokines. HDL can also serve as a scavenger. HDL also inhibits the activity of human serum paraoxonase, e.g. It may also serve as a carrier for PON-1, -2, and -3. Paraoxonase inhibits HDL It is an esterase associated with β-glucosamine, which is important in protecting cellular components against oxidation. Oxidation of LDL during chronic stress is directly related to the development of atherosclerosis (Aviram, 2000, Free Radic. Res. 33 Suppl:S85-97). is thought to play a role in susceptibility to atherosclerosis and cardiovascular disease. (Aviram, 1999, Mol. Med. Today 5(9):381-86). Human serum paraoxonase (PON-1) binds to high density lipoprotein (HDL). Its activity is related to atherosclerosis. PON-1 hydrolyzes organophosphates and promotes HDL and atherosclerosis. and against atherosclerosis by inhibiting the oxidation of low-density lipoprotein (LDL) (Aviram, 1999, Mol. Med. Today 5(9):381-86). This protection is achieved by hydrolyzing specific lipid peroxides in oxidized lipoproteins, called PON. These results suggest that the ability of PON-1 to preserve or enhance PON-1 activity may be related to Interventions that promote cardiovascular disease may help delay the onset of atherosclerosis and coronary heart disease. do.

[0041] HDL also acts as an antithrombotic and fibrinogen-lowering agent and in the treatment of hemorrhagic shock. (Cockerill et al., Published March 1, 2001) WO01 / 13939). HDL, and especially ApoA-I, are produced by sepsis. Facilitates the exchange of lipopolysaccharide into lipid particles containing ApoA-I, resulting in functional neutralization of lipopolysaccharide. (Wright et al., WO 97 / 06363, published Dec. 21, 1995) 534289; Wright et al., U.S. Patent No. 5,928,644 issued July 27, 1999 No. 24; Wright et al., U.S. Patent No. 5,932,536 issued August 3, 1999) .

[0042] There are a variety of methods available for producing lipoprotein complexes in vitro. US Patent Nos. 6,287,590 and 6,455,088 disclose the use of organic solvents ( Co-lyophilization and freeze-drying of apolipoprotein and lipid solutions in a solvent mixture A method is disclosed which requires the formation of charged lipoprotein complexes during hydration of the dried powder. Protein complexes can also be formed by detergent dialysis; for example, lipids, lipoproteins, A mixture of protein and detergent such as cholate is dialyzed and reconstituted to form the complex. (See, e.g., Jonas et al., 1986, Methods Enzymol. 128:553-82.) U.S. Pat. Example 1 of Published Application No. 2004 / 0067873 describes a lipid-based esterification process under conditions for forming micelles. The cellular dispersions were combined with cholate and then incubated with the apolipoprotein solution. The present invention discloses a cholate dispersion method in which the cholate is baited with cholic acid to form a complex. Cholate can be separated, for example, by dialysis, ultrafiltration or adsorption onto affinity beads or resins. The protein must be removed by adsorption. The present invention discloses the formation of lipoprotein complexes by subjecting a liquid mixture of glycerol and lipids to high pressure homogenization. However, proteins that are sensitive to high shear forces may be easily disrupted by exposure to high pressure homogenization. If so, activity may be lost.

[0043] Thus, currently available production methods rely on waste products of starting materials, such as protein hydrolysates. and / or require purification of the resulting product, such as removal of toxic factors; Furthermore, preparations of lipoprotein complexes are heterogeneous. and contain a mixture of complexes of varying size and composition. See US Pat. No. 5,876,968. Thus, efficient and preferably high purity To develop a new method for producing lipoprotein complexes, which results in more uniform complexes having Such processes have less risk of side effects caused by contaminants. , making production more economical on a large scale while producing a more uniform pharma- ceutically acceptable product. Make it possible.

[0044] In addition, however, the overall production yield is low and the recombinantly expressed protein Considering that proteolysis occurs in cultures of fibronectin, ApoA-I, Apo AI M , ApoA-I P and other variants, and therapeutic uses of reconstituted HDL - Patents.com The large amounts of apolipoproteins required for therapeutic administration and protein The cost of producing high-quality ELISA kits is currently limited (see, e.g., Mallory et al., 1987, J. Biol. Chem. 262( 9):4241-4247; see Schmidt et al., 1997, Protein Expression & Purification 10:226-236. For the treatment of cardiovascular disease, the dosage range is 1.5 to 4 g per injection. Early clinical trials suggest that the protein is the key to a complete cure. The number of injections required is unknown (e.g., Eriksson et al., 1999, Circulation 10 0(6):594-98; Carlson, 1995, Nutr. Metab. Cardiovasc. Dis. 5:85-91; Nanjee et al., 200 0, Arterioscler. Thromb. Vasc. Biol. 20(9):2148-55; Nanjee et al., 1999, Arterioscler Thromb. Vasc. Biol. 19(4):979-89; Nanjee et al., 1996, Arterioscler. Thromb. Vasc. Biol. 16(9): 1203-14).

[0045] Recombinant human ApoA-I has been expressed in heterologous hosts, but the yield of mature protein is Large scale synthesis is difficult, especially when coupled with purification methods that further reduce yields and result in impure products. This is insufficient for therapeutic applications.

[0046] Weinberg et al., 1988, J. Lipid Research 29:819-824, describes a method for determining the degree of cleavage by reversed-phase high-performance liquid chromatography. Apolipoproteins AI, A-II and A-IV purified from human plasma by and the isolation of its isoforms.

[0047] WO2009 / 025754 discloses the preparation of alpha-1-antitrypsin and ApoA from human plasma. The protein isolation and purification of -I are described.

[0048] Hunter et al., 2009, Biotechnol. Prog. 25(2):446-453, expressed recombinantly in E. coli. We describe the large-scale purification of the expressed ApoA-I Milano mutant.

[0049] Caparon et al., 2009, Biotechnol. And Bioeng. 105(2):239-249, reported that purified apolipoprotein To reduce the levels of two host cell proteins in the protein product, ApoA-I Milan from an E. coli host genetically engineered to lack the The expression and purification of o are described.

[0050] U.S. Patent No. 6,090,921 describes the isolation of ApoA using anion exchange chromatography. Apolipoprotein E (ApoE) from a fraction of human plasma containing AI and apolipoprotein E (ApoE) Purification of AI or ApoE is described.

[0051] Brewer et al., 1986, Meth. Enzymol. 128:223-246, reported the use of chromatographic techniques to detect HIV. describes the isolation and characterization of apolipoproteins from human blood.

[0052] Weisweiler et al., 1987, Clinica Chimica Acta 169:249-254, fast protein liquid chromatography The isolation of ApoA-I and ApoA-II from human HDL using chromatography was described. It is listed.

[0053] deSilva et al., 1990, J. Biol. Chem. 265(24): 14292-14297, report on the immunoaffinity chromosomes. of apolipoprotein J by fluoroscopy and reversed-phase high-performance liquid chromatography. The following is stated.

[0054] Establish the feasibility of lipoprotein therapy using various lipoprotein-based drugs Along with clinical studies, lipoproteins and lipoprotein complexes are currently being evaluated for clinical use. It is currently being developed (Tardif, 2010, Journal of Clinical Lipidology 4:399-404). The study evaluated autologous delipidated HDL (Waksman et al., 2010, J Am. Coll. Cardiol. 55:2 Another study showed that recombinant ApoA-I M and palmitoyl-oleoyl-PC (P The authors evaluated ETC-216, a complex with OPC (Nissen et al., 2003, JAMA 290:2292- CSL-111 is a plasma phosphatidylcholine complexed with soybean phosphatidylcholine (SBPC). The reconstituted human ApoA-I was purified from plasma (Tardif et al., 2007, JAMA 297:167 Current research drugs have shown efficacy in reducing atherosclerotic plaque. However, the effects are limited to increased transaminases or the formation of ApoA-I antibodies. (Nanjee et al., 1999, Arterioscler. Vasc. Thromb. Biol. 19:979-89 ; Nissen et al., 2003, JAMA 290:2292-2300; Spieker et al., 2002, Circulation 105:1399-140 2; Nieuwdorp et al., 2004, Diabetologia 51 : 1081-4; Drew et al., 2009, Circulation 119, 2 103-11; Shaw et al., 2008, Circ. Res. 103: 1084-91; Tardiff et al., 2007, JAMA 297: 1675-1 682; Waksman, 2008, Circulation 118:S 371; Cho, published September 25, 2007 No. 7,273,849B2). For example, the ERASE clinical trial (Tardiff et al., 200 7, JAMA 297: 1675-1682) compared two doses of CSL-111: 40 mg / kg and 80 The 80 mg / kg dose of ApoA-I was used. The 80 mg / kg dose group was not stopped due to liver toxicity. had to be increased (as indicated by severe transaminase elevations). Even in the dose group, some patients experience transaminase elevations. Summary of the Invention [Problem to be solved by the invention]

[0055] Therefore, lowering serum cholesterol, increasing HDL serum levels, dyslipidemia and / or or for the prevention and / or treatment of diseases, conditions and / or disorders associated with dyslipidemia. There is a need for safer agents that are more effective in treating Minimal increase in triglycerides, LDL-triglycerides or VLDL-triglycerides Lipoprotein preparations that induce only hypercalcemia (or no hypercalcemia) and commercial-scale It is anticipated that a robust manufacturing process will be developed that can be used to reliably produce these lipoprotein preparations. It is needed in the technical field. [Means for solving the problem]

[0056] 4. Overview The present disclosure relates to a method for treating dyslipidemia and diseases, disorders and / or conditions associated with dyslipidemia. Particularly suitable for treating and / or preventing the following protein fractions (e.g. apolipoproteins The present invention provides lipoprotein complexes, including a protein fraction and a lipid fraction, and populations thereof. having high purity and / or homogeneity and / or specificity as described herein A complex population containing lipids and proteins in a ratio of 1:1 has been shown to be effective in reducing the risk of side effects and improving the It has been found to have increased ability to transport cholesterol.

[0057] Lipoprotein complexes are divided into protein fractions (e.g., apolipoprotein fractions) and The protein fraction contains one or more lipid bonds. In a complex protein, such as an apolipoprotein, peptide, or lipoprotein complex Apolipoprotein peptides capable of mobilizing cholesterol when present in Such apolipoproteins and apolipoprotein analogs or mimetics include Non-limiting examples of peptides include preproapoliprotein, preproapoliprotein, ApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA oA-II, ApoA-II, preproApoA-IV, proApoA-IV, ApoA -IV, ApoA-V, pre-proApoE, proApoE, ApoE, pre-proApoA -I M , proApoA-I M , ApoA-I M , preproApoA-I P , proApoA -I P , ApoA-I P , preproApoA-I z , proApoA-I z and ApoA -I zApolipoproteins may be monomers, dimers, or trimers, or In certain embodiments, the apolipoprotein fraction is It consists essentially of a single isoform, most preferably ApoA-I. ApoA-I in the matrix complex is a protein corresponding to amino acids 25 to 267 of SEQ ID NO:1. In some cases, the sequence may have at least 90% or at least 95% sequence identity to Therefore, ApoA-I is a polypeptide that occupies the entire amino acid sequence of SEQ ID NO:1 at amino acid 25 (and position 1 of the mature protein). It further contains an aspartic acid at a position corresponding to full-length ApoA-I. At least 75%, at least 80%, at least 85%, at least 90% or at least 9 5% of ApoA-I is the correctly processed mature protein (i.e., signal and lacking the propeptide sequence) and may be oxidized, deamidated and / or truncated. It hasn't been done yet.

[0058] The present disclosure also provides a mammalian host cell genetically engineered to express ApoA-I, Cell cultures containing ApoA-I, and producing mature, biologically active ApoA-I The present invention also provides a method for the production of immature ApoA-I (proApoA-I) by protease digestion. The resulting truncated forms of ApoA-I and the large amounts of mature ApoA-I that are essentially free of both are produced. It has been discovered that mammalian host cells can be genetically engineered to express an AI. These results are surprising. First, the host cell for protein processing The main cellular machinery is overwhelmed by the overexpression of a foreign protein such as ApoA-I, resulting in the processing Second, the culture medium is expected to produce immature proteins that are not ligated. ApoA-I secreted into the soil is degraded by proteases and then released into the soil at low levels. Only truncated ApoA-I is observed in the culture medium. The cultures and methods for producing ApoA-I can be used to produce commercially relevant quantities of ApoA-I for therapeutic applications. It is particularly suitable for producing useful mature proteins.

[0059] As provided herein, the mammalian host cell preferably comprises a 25-28 nucleotide sequence of SEQ ID NO:1. At least 95%, at least 96%, at least 97%, at least 9 for 67th place 8%, at least 99%, or 100% identical to the amino acid sequence of the The protein is preferably or has an aspartic acid residue at the position corresponding to position 25 of SEQ ID NO:1. The host cell may optionally further secrete such proteins. In the example of is an 18 amino acid signal sequence (MKAAVLTLAVLFLTGSQA, SEQ ID NO:2 ) and / or a six amino acid propeptide sequence (RHFWQQ, SEQ ID NO: 3) In some examples, the host cell may contain at least 9 5%, at least 96%, at least 97%, at least 98%, at least 99%, or or engineered to express a protein containing an amino acid sequence that has 100% identity to the It is made.

[0060] Host cells include, but are not limited to, Chinese hamster ovary (e.g., CH O-K1 or CHO-S), VERO, BHK, BHK570, HeLa, COS- 1. Any mammalian strain, such as COS-7, MDCK, 293, 3T3, PC12 and W138. derived from an animal cell line or from a myeloma cell or cell line (e.g., mouse myeloma). The antibody may be derived from a human ovarian tumor cell or cell line.

[0061] The mammalian host cell can be transformed with an ApoA-I protein, e.g., a polypeptide comprising positions 25-267 of SEQ ID NO:1. and further comprising multiple copies of a nucleic acid encoding a protein comprising or consisting of For example, the mammalian host cell may contain at least about 5, 6, 7, 8 or more copies of the and up to about 10, 11, 12, 13 or 14 copies of the nucleic acid. In addition, promoters capable of expressing proteins at high levels in mammalian host cells are available. promoter, for example, a simian cytomegalovirus promoter or, more specifically, an immediate early promoter It can be operably linked to, for example, the lucytomegalovirus promoter.

[0062] The mammalian host cells are preferably cultured for at least about 0.5, 1, 2 or 3 days. g / L of ApoA-I and / or up to about 20 g / L of ApoA-I in culture, e.g. For example, up to 4, 5, 6, 7, 8, 9, 10, 12 or 15 g / L of Ap in the culture. This culture can produce oA-I. L, 2000L, 5000L, 10,000L, 25,000L or over 50,000L The culture volume may be any size within the range above. In various embodiments, the culture volume is 1 0L~50L, 50L~100L, 100L~150L, ​​150L~200L, 200L ~300L, 300L~500L, 500L~1000L, 1000L~1500L, 1 500L~2000L, 2000L~3000L, 3000L~5000L, 5000L ~7500L, 7500L~10,000L, 10,000L~20,000L, 20, The range may be from 000L to 40,000L, or from 30,000L to 50,000L. In some instances, the culture is a large scale culture, e.g., 15 L, 20 L, 25 L, 30 L. , 50L, 100L, 200L, 300L, 500L, 1000L, 5000L, 10, 000L, 15,000L, 20,000L, 25,000L, up to 50,000L Above.

[0063] The mammalian host cells of the present disclosure can be grown in culture. The mammalian host cell vector includes a mammalian host cell, comprising a plurality of mammalian host cells as described above or in Section 6.1.2 below. Further provided is an animal cell culture, the cell culture comprising one or more of the following characteristics: (a) a culture (optionally at least 10 liters, optionally at least 20 liters) , at least 30 liters, at least 50 liters, at least 100 liters, 30 0L, 500L, 1000L, 5000L, 10,000L, 15,000L, 20,0 00L, 25,000L, up to 50,000L large batch cultures or at least At least 10 liters, at least 20 liters, at least 30 liters, at least 50 liters Bottles of at least 100 liters, 300L, 500L, 1000L, 5000L, or up to 10,000 L of continuous culture) is 2. A method for the preparation of a nucleic acid sequence comprising: Mature Ap ≥ 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 g / L (b) at least 75%, at least 80% in the culture medium; At least 85%, at least 90%, at least 95%, at least 98%, or at least At least 99% of the protein is the ApoA-I protein lacking the signal sequence; c) at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the protein , the mature ApoA-I protein lacking the signal sequence and propeptide sequence; and (d) at least 75%, at least 80%, at least 85% of mature ApoA-I. , at least 90%, at least 95% are truncated, oxidized, or deamidated not present.

[0064] The present disclosure also provides methods for producing mature, biologically active ApoA-I. Generally, the method comprises administering to the subject a method as described above, or to a subject in need thereof, under conditions in which ApoA-I is expressed and secreted. The method includes culturing any of the mammalian host cells described in Section 6.1.2. Mature, biologically active ApoA-I is recovered from the supernatant of cultured mammalian host cells. The method may further comprise recovering and, optionally, purifying the product (see Section 3 below). (e.g., by the methods disclosed in Sections 6.1.3 and 6.1.4).

[0065] The ApoA-I obtained or obtainable by the above method may be further complexed with lipids. to form lipoprotein complexes as described herein and / or to be therapeutically effective. The pharmaceutical composition can be preferably incorporated in an amount of A phosphate buffer solution that may contain sucrose and / or mannitol.

[0066] ApoA-I was isolated using a novel combination of chromatography and filtration steps. The purification makes the protein particularly suitable for therapeutic use, reducing the risk of side effects sufficient to confer one or more attributes, such as reduced toxicity and low or no toxicity Very low levels of host cell proteins, host cell DNA, endotoxins and truncated forms It has further been discovered that it is possible to produce large amounts of pure ApoA-I containing protein. Preferably, the ApoA-I purified by the methods described herein is derived from a mammalian The ApoA antibody is recombinantly produced in the cells and secreted into the growth medium. A solution containing ApoA-I and an anion exchange matrix were placed in a 10-well plate, and the ApoA-I was then attached to the matrix. (b) contacting under non-binding conditions; and (b) removing the virus or virus particles. The ApoA-I-containing solution obtained in step (a) is passed through a membrane having a pore size sufficient to (c) filtering the first solution under conditions such that ApoA-I binds to the matrix. passing the filtrate from step (b) through a reverse phase chromatography column of (d) a first reversed-phase chromatography matrix using an increasing concentration gradient of organic solvent; (e) eluting a first ApoA-I-containing reverse phase eluate from the column; A second reversed-phase chromatography column was loaded under conditions such that -I binds to the matrix. passing the first ApoA-I reverse phase eluate from step (d); and and (f) a second reversed-phase chromatography matrix using an increasing concentration gradient of organic solvent. and eluting a second ApoA-I-containing reverse phase eluate from the column. The order in which the steps are performed is not important, and for example, In a typical embodiment, the sample is filtered through a membrane to remove viruses or viral particles. The step of determining whether or not the reaction is performed after step (f) rather than after step (a).

[0067] Also, the purification methods described herein have an ApoA-I concentration of at least 10 g / L. Also provided herein are substantially pure ApoA-I products obtained or obtainable thereby. Substantially pure ApoA-I products produced by the purification methods described herein. Preferably, the ApoA-I / ApoA-I antibody contains less than about 10 pg of host cell DNA per mg of ApoA-I. Less than approximately 100 ng of host cell protein per mg of AI and / or ApoA -I. The ApoA-I product contains less than 0.1 EU of endotoxin per mg of ApoA-I. 95% pure, at least 96% pure, at least 97% pure, at least 98% pure or may be at least 99% pure.

[0068] Additionally, substantially pure ApoA-I products can be prepared using the methods described herein that contain ApoA-I. It may be incorporated into either a pharmaceutical composition and / or a lipoprotein complex.

[0069] The lipid fractions are typically neutral, negatively charged, positively charged, or the like. The fatty acid chains on the phospholipids are preferably Or 12-26 or 16-26 carbons in length, ranging from saturated to monounsaturated. Examples of phospholipids include low molecular weight alkyl chain phospholipids. Protein, egg phosphatidylcholine, soy phosphatidylcholine, dipalmitoyl phosphatidylcholine lycoline, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine 1-Myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myri Ristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidyl Choline, 1-Stearoyl-2-Palmitoylphosphatidylcholine, Dioleoylphosphatidylcholine Phatidylcholine Dioleophosphatidylethanolamine, Dilauroyl Phosphatidyl Glycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanol Amino acids, phosphatidylinositol, phosphatidylglycerol, diphosphatidyl dimyristoyl phosphatidylglycerol, dipalmitoyl Phosphatidylglycerol, distearoylphosphatidylglycerol, dioleoyl Phosphatidylglycerol, Dimyristoylphosphatidic acid, Dipalmitoylphosphatidyl Phatidic acid, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine Phosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine Photidylserine, brain phosphatidylserine, brain sphingomyelin, egg sphingomyelin Phosphorus, milk sphingomyelin, palmitoyl sphingomyelin, phytosphingomyelin Myelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, di Palmitoylphosphatidylglycerol salt, phosphatidic acid, galactocerebroside , gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3)-D- Mannosyl-(1,3)diglyceride, aminophenylglycoside, 3-cholesteryl- 6'-(glycosylthio)hexyl ether glycolipid, and cholesterol and and its derivatives. Phospholipids including SM and palmitoyl sphingomyelin The fraction optionally includes, but is not limited to, lysophospholipids, palmitoylsphingolipids, Other than myelin, sphingomyelin, galactocerebroside, ganglioside, cerebroside such as glycides, glycerides, triglycerides, and cholesterol and their derivatives; It may contain small amounts of lipids of any type.

[0070] Most preferably, the lipid fraction comprises at least one neutral phospholipid and optionally one or contains multiple negatively charged phospholipids. In lipoprotein complexes that contain both neutral and negatively charged phospholipids are fatty acid chains having the same or different number of carbons and the same or different degree of saturation. In some instances, the neutral phospholipid and the negatively charged phospholipid may have the formula: , having the same acyl tail, e.g., C16:0, or palmitoyl, acyl chain.

[0071] The lipid components of the complex may include neutral and negatively charged phospholipids, or In the case of the lipid composition, the weight-to-weight (wt:wt) ratio of neutral phospholipids to negatively charged phospholipids is preferably Preferably, the ratio is about 99:1 to about 90:10, more preferably about 99:1 to about 95:5, and and most preferably in a wt:wt ratio ranging from about 98:2 to about 96:4. In this study, the ratio of neutral phospholipids to negatively charged phospholipids was approximately 97:3. The lipids are present in a weight-to-weight (wt:wt) ratio of positively charged phospholipids.

[0072] The neutral phospholipids may be natural or synthetic. Phospholipids are optionally saturated or monounsaturated lipids having a chain length of 16 to 26 carbon atoms. Sphingomyelin ("SM"), including fatty acids, such as palmitoyl sphingomyelin Phosphorus, phytosphingomyelin, diphytosphingomyelin, phosphosphingolipids , or glycosphingolipids. SM can be from any source. For example, For example, SM can be obtained from milk, eggs, brain, or made synthetically. In certain embodiments, the SM is obtained from chicken eggs ("egg SM"). In this case, SM is palmitoylsphingomyelin.

[0073] Any phospholipid that carries at least a partial negative charge at physiological pH is called a negatively charged phospholipid. The lipid may be selected from the group consisting of phosphatidylinositol, ... Negatively charged phosphatidylserine, phosphatidylglycerol and phosphatidic acid In certain embodiments, the negatively charged phospholipid is in the form of: 1,2-Dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol )], or DPPG, phosphatidylglycerol. Examples of suitable salts include ammonium and sodium salts.

[0074] The phospholipids used to prepare the conjugates of the present disclosure are preferably at least 95% pure. and / or 4 meq O / kg or less, more preferably 3 meq O / kg or less (e.g., 2 meq O / kg or less) The oxidation levels of oxygen and fatty acids are The initial reaction product is hydroperoxide. Using iodometric titration, The level of oxidation can be measured by assaying for the presence of peroxide.

[0075] The lipoprotein complexes of the present disclosure are preferably more completely isolated, as shown in the examples below. The ratio of apolipoproteins to lipids results in a uniform complex formation throughout the entire lipoprotein chain. Protein complexes are available at 1:80 to 1:120, 1:100 to 1:115, or 1:105 to 1:1 :110, wherein the apolipoprotein fraction is characterized by a molar ratio of the apolipoprotein fraction:lipid fraction in the range of In certain embodiments, apolipoproteins are expressed as ApoA-I equivalents. The molar ratio of protein fraction to lipid fraction is 1:80 to 1:90 (e.g., 1:82, 1:85, etc.). or 1:87), 1:90-1:100 (e.g., 1:95 or 1:98), 1:10 0 to 1:110 (e.g., 1:105 or 1:108).

[0076] In particular embodiments in which egg SM is used as the neutral lipid, apolipoprotein The weight ratio of the protein fraction to the lipid fraction is in the range of about 1:2.7 to about 1:3 (e.g., 1:2.7). be.

[0077] The lipoprotein complexes of the present disclosure can be used to deliver hydrophobic, lipophilic or non-polar active agents. For such applications, the lipid fraction may be used as a carrier for, but not limited to, However, it is not limited to the above. or a number of hydrophobic, lipophilic or non-polar active agents, or Specific examples of activators are described in Section 6.2. .

[0078] The present disclosure also provides a population of lipoprotein complexes. Typically, The populations each contain multiple lipoprotein complexes, each of which contains a protein fraction and a lipid fraction. Contains; The protein fraction may be prepared according to the methods described above and in Section 6.1 or Section 6.5.3. containing lipoproteins or lipoprotein analogs; most preferably, protein fractions was obtained or is obtainable by the method described in Section 6.1.2, and / or Lipoproteins (e.g., ApoA-I proteins) purified by the methods described in Section 6.1.4. Containing or consisting essentially of proteins; the lipid fraction comprises lipids as described above and in Section 6.2 or Section 6.5.2; Lipoprotein complexes are preferably purified by the thermal cycling method described in Section 6.5.4. It is manufactured by

[0079] Applicants have assessed the efficacy and safety profiles of lipoprotein complex populations individually and We have found several features that may contribute, or may combine, to the Most are 4nm to 15nm (e.g., 5nm to 12nm, 6nm to 15nm, or 8nm uniformity of size of complexes in the population, ranging from 0.1 nm to 10 nm); The purity of the apolipoprotein used to prepare the complex (e.g., oxidized, deaminated, lack of oxidized, truncated, and / or immature forms of apolipoproteins and / or lack of endotoxin and / or non-apolipoprotein (host) proteins. Absence of host cell proteins, etc., and / or host proteins that are often present in recombinant products principal cell DNA); The purity of the complex itself in the population (such as the solvent or detergent used to prepare the complex) absence of oxidized lipids; absence of deamidated, oxidized or truncated protein lack of protein; and / or small amounts of uncomplexed apolipoprotein and / or lipids or their lack) Includes.

[0080] Among these features are the homogeneity of the complex and the abundance of mature, unmodified apolipoproteins in the complex. The predominance of proteins is believed to increase efficacy. Body purity is affected by liver damage reflected by increased liver enzymes (e.g., transaminases) Furthermore, the present inventors have found that many apolipoproteins and generating a population of lipoprotein complexes by a method that results in the incorporation of The reduction in the amount of uncomplexed apolipoproteins also makes it possible to Beneficial in reducing the risk of an immunogenic response in a subject that may be caused by administration It is.

[0081] Thus, the present disclosure is characterized by one or more, or even all, of the following features: To provide a population of lipoprotein complexes comprising: (a) determining whether at least 75% by weight of lipoproteins in said population, typically ApoA-I, %, at least 80% by weight, at least 85% by weight, at least 90% by weight, or less at least 95% by weight is in the mature form; (b) lipoproteins in said population, typically 25% by weight or less of ApoA-I; 0% or less, 15% or less, 10% or less, or 5% or less by weight is in the immature form; (c) the population is comprised of a lipoprotein, typically ApoA-I, per milligram , 100 picograms or less, 50 picograms or less, 25 picograms or less, 10 picograms or less or containing less than 5 picograms of host cell DNA; (d) the population is comprised of a lipoprotein, typically ApoA-I, per milligram , 500 nanograms or less, 200 nanograms or less, 100 nanograms or less, 50 nanograms or less contains less than 100 mg of host cell protein, or 20 nanograms or less of host cell protein; (e) lipoproteins in the population, typically 25% by weight or less of ApoA-I; 0% by weight or less, 15% by weight or less, 10% by weight or less, or 5% by weight or less is truncated. in a morphological form; (f) the lipoprotein component comprises or consists of mature ApoA-I, 20% or more of each of methionine 112 and methionine 148 in the ApoA-I in the group 15% or less, 10% or less, 5% or less, 3% or less, 2% or less or 1% or less is oxidized are; (g) at least 80%, at least 85%, at least 90% of the lipoprotein complexes % or at least 95% are chromatographically resolved by gel permeation chromatography ("GPC") or dynamic photochromic chromatography (DPC) When measured by DLS, the size ranges from 4 nm to 15 nm, e.g., 6 nm A size of 1 to 15 nm, or a size of 8 to 12 nm, and even more preferably, a size of 5 nm to 1 in the form of particles with a size of 2 nm; (i) the population has, per milligram of lipoprotein, typically ApoA-I, Contains less than 1EU, less than 0.5EU, less than 0.3EU or less than 0.1EU of endotoxin ; (j) determining whether or not the amino acids in a lipoprotein in said population, typically ApoA-I, are identical to or different from those in said lipoprotein. Below 10%, below 5%, below 4%, below 3%, below 2%, or below 1% deamidated Has been; (k) 15% by weight or less, 10% by weight or less, 5% by weight or less of the lipid in the lipid fraction in the complex Hereinafter, less than 2% or 0% by weight is cholesterol; (l) The population contains no more than 200 ppm, 100 ppm, or 50 ppm of non-aqueous solvent. r; (m) the population does not contain detergents (e.g., cholate); (n) determining whether the population is a percentage of the population in a single peak during gel permeation chromatography; When measured by the percentage, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% uniform It is good to have; (o) the population is at least 80%, at least 85%, at least 90%, In compositions in which at least 95% or at least 97% of the protein is in complexed form be; (p) 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the lipids in the population Oxidized; and (q) 15% or less of the methionine and / or tryptophan residues in the population; % or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less is oxidized.

[0082] In certain embodiments, the population has a characteristic selected from the following group: Group I: characteristics (a), (b) and (c) above; Group II: characteristics (c), (d) and (i) above; Group III: characteristics (f), (j), (e), (p) and (q) above; Group IV: characteristics (g), (n) and (o) above; Group V: characteristics (l) and (m) above; and Group VI: characteristics (k) as above.

[0083] In certain embodiments, the population comprises one or more individuals independently selected from each of groups I and IV. or two characteristics; optionally, the population is characterized by a single characteristic from each of Groups I and IV. The population is further characterized by three independently selected characteristics. The population is characterized by one, two or three features independently selected from each of Group II. In addition, one or two features independently selected from Group V and / or one or two features independently selected from Group VI The one feature selected is characterized.

[0084] Certain lipid and protein components form multiple distinct but homogeneous lipoprotein complexes. Thus, the present disclosure also provides methods for producing apolipoproteins comprising different amounts of the apolipoprotein molecule. Compositions containing two, three or four populations of lipoprotein complexes are also provided (e.g., 3 or 4 ApoA-I molecules or ApoA-I equivalents). The composition comprises two populations of lipoprotein complexes, the first of which is lipoprotein complex complexes. Lipoproteins with two ApoA-I molecules or ApoA-I equivalents per protein complex The second population contained three or four ApoA- A lipoprotein complex having an ApoA-I molecule or ApoA-I equivalent, and optionally a third The population of ApoA-I or ApoA-I molecules was 4 or 3 per lipoprotein complex. Contains lipoprotein complexes with AI equivalents.

[0085] The composition comprising two or more populations of lipoprotein complexes preferably contains low levels of non-complex lipoproteins. The composition preferably contains lipoproteins and / or lipids. Quality 15% or less, 12% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less is in uncomplexed form, and and / or less than 15%, less than 12%, less than 10%, less than 9% of the lipoproteins in the composition below, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less or Less than 1% is in the uncomplexed form.

[0086] The present disclosure provides a method for producing a lipoprotein complex, the method comprising, inter alia: The suspension containing the uncomplexed lipids and the lipid-binding protein or peptide is then thermally cycled. By subjecting the lipoprotein complexes to ring conditions, the components are incubated at a fixed temperature. The results are favorable compared to other methods, such as by This is based on the discovery that the formation of a complex can be achieved.

[0087] The present disclosure relates to lipoprotein complexes, such as those described in Sections 6.5.1-6.5.4. The present invention provides a thermal cycling method for preparing lipid particles ("lipid particles") that are typically A suspension containing a lipid-binding protein or peptide ("protein component") and a lipid-binding protein or peptide ("protein component"). The solution is heated for multiple times until most of the protein components are incorporated into the lipoprotein complex. The method generally involves cycling the lipoprotein complexes until they are formed. The suspension is heated between a temperature in a first higher temperature range and a temperature in a second lower temperature range. The high and low temperature ranges of the thermal cycling process must be Based on the phase transition temperatures of the lipid and protein components of the protein complex. As may occur when using phospholipids or mixtures of phospholipids with fatty acid chains, the lipid If the component does not exhibit a defined or distinct phase transition, the high and low temperature ranges of thermal cycling The ranges may vary by at least about 20° C. and up to 40° C. or even more. For example, in some embodiments, the low and high temperature ranges are 20°C to 30°C, 20°C to 30°C, ~40℃, 20℃~50℃, 30℃~40℃, 30℃~50℃, 25℃~45℃, 35℃ ~55℃ difference.

[0088] For lipids, the phase transition is from a tightly packed, ordered structure known as the gel state to This involves a change to a looser, less ordered structure known as the liquid state. Protein complexes are typically referred to in the art as being based on the specific lipid or mixture of lipids used. By incubating lipid particles and apolipoproteins at temperatures close to the transition temperature, The phase transition temperature of the lipid component (which can be determined by calorimetric methods) + / - 12°C, more preferably + / - 10°C, is the "low" temperature range in the method of the present disclosure. In certain embodiments, the low temperature range is within + / - 3°C, + / - 5°C of the phase transition temperature of the lipid component. , or + / - 8°C. In one particular embodiment, the low temperature range is The phase transition temperature ranges from below 5°C or below 10°C to above 5°C.

[0089] For proteins, the phase transition temperature involves a change from tertiary to secondary structure. The phase transition temperature of the carbonaceous component is preferably within + / - 12°C, more preferably + / - 10°C, in the method of the present disclosure. In certain embodiments, the high temperature range is the "high" temperature range for the relative + / - 3°C, + / - 5°C, or + / - 8°C of the transition temperature. In this case, the low temperature range is from 10°C below the phase transition temperature of the protein component to 5°C above it. , below 10°C, or below 15°C.

[0090] The lipid component of the starting suspension, i.e., the suspension that has not yet been subjected to the thermal cycling method, Preferably, the lipid particles are complexed with, for example, a lipid-binding protein. The lipid components are generally constructed as described in Section 6.5.2 below. This is as it should be.

[0091] The protein component of the starting suspension is preferably a lipid-binding peptide that is not complexed with lipid. peptides and / or proteins or in the desired complex At least 5-fold higher than the peptide to lipid ratio (e.g., at least 5-fold, at least 1 0-fold or at least 20-fold higher) protein / peptide to lipid ratios The production of protein components is generally described in Sections 6.1 and 6.5.3 below. The protein component is preferably prepared according to the method described in Section 6.1.2. and / or purified according to the methods described in Section 6.1.3 or 6.1.4. It is manufactured.

[0092] In the method of the present disclosure, the suspension containing the protein component and the lipid component is typically , preferably starting from a high temperature range, until lipoprotein complexes are formed. The mixture is thermocycled between a warm range and a cold range. (see, for example, U.S. Pat. No. 6,393,343, the entire contents of which are incorporated herein by reference). (described in JP 2006-0217312 A1), lipid and protein components Substantially complete conjugation can be achieved after several cycles. Further details on suitable protein and lipid stoichiometries for the ELISA method are provided in Section 6.5.4. It is described in.

[0093] The conjugates produced by the method typically have protein components within a particular stoichiometry. Micelles, vesicles, globules or vesicles physically bound to lipid components with uniform size distributions are supramolecular assemblies shaped as discoid particles. The method of the invention advantageously comprises starting Provides substantially complete complexation of lipids and / or proteins in suspension and clotting Free lipids and / or free tungsten are observed by separation methods such as chromatography. The resulting composition is substantially free of protein. Thus, the disclosed method does not require a purification step. The reaction may be carried out in the absence of a solvent.

[0094] The lipid component in the starting suspension is typically in particulate form. The particles are preferably mainly composed of at least at least 45 nm, at least 50 nm, at least 55 nm or at least 60 nm The size is 65nm, 70nm, 75nm, and 80nm. Size, max. 100nm, max. 120nm, max. 150nm, max. 200nm, max. At most, it is 250 nm, at most, 300 nm, at most, 500 nm, for example, 45 nm to 10 0 nm or in the size range of 45 to 250 nm, more preferably in the size range of 50 nm to 90 nm. The size range is preferably 55 nm to 75 nm, and more preferably 60 nm to 80 nm. In a preferred embodiment, the lipid particles are composed primarily of egg sphingomyelin and In another preferred embodiment, the lipid particles are predominantly one or Several synthetic sphingomyelins (e.g., palmitoyl sphingomyelin or phytosphingomyelin) It is composed of sphingomyelin and has a size of 175 nm to 250 nm. In a preferred embodiment, the lipid particles are primarily composed of one or more synthetic lipids (e.g., It is composed of 250 In yet another preferred embodiment, the lipid particles have a size of from 1000 nm to 1000 nm. The lipids are primarily composed of one or more synthetic lipids (e.g., palmitoyl sphingomyelin or phytosphingomyelin). It is composed of sphingomyelin and has a size of 1000nm to 4000nm. The sizes reported herein are zeta (Z) average sizes as determined by dynamic light scattering. High pressure homogenization, e.g., microfluidization, advantageously produces lipid particles of suitable size. Other methods for forming particles are disclosed in Section 6.5.2 below and are Such methods can be used as an alternative method for homogenization of particles outside the preferred size range. When producing offspring, the particles can be subjected to size filtration to obtain particles of a suitable size. .

[0095] The methods for preparing lipoprotein complexes described herein produce lipoproteins that are homogeneous in size distribution. This advantageously produces complexes that are one and the same, avoiding the need for size fractionation. The method includes the steps of: At least 95%, at least 96%, at least 97%, at least 98% or at least 99%). Thus, the present disclosure includes lipoprotein complexes, e.g., gel immersion. At least one lipid-binding protein in the composition is present as determined by permeation chromatography. At least 95%, at least 96%, at least 97%, at least 98% or at least In certain embodiments, the present disclosure provides a composition that is 99% lipid complexed. , Zeta average size of 4nm to 20nm, e.g., Zeta average size of 4nm to 20nm , Zeta average size of 4nm~15nm, Zeta average size of 4nm~12nm, 5nm Zeta average size ~15nm, Zeta average size 5nm~12nm, 5nm~10nm m Zeta average size, 5nm~20nm Zeta average size, 6nm~15nm Zeta average size The zeta average size is between 8 nm and 12 nm, and the gel permeation crystals are preferably used. At least one lipid-binding protein in the composition is present as determined by chromatography. 95%, at least 96%, at least 97%, at least 98% or at least 99 The present invention provides a composition comprising a lipoprotein complex, wherein the lipoprotein complex comprises at least one lipoprotein having a lipid complex. By subjecting the lipid particles to thermal cycling with lipoproteins according to the method described above, Typically, a population of lipoprotein particles with sizes between 4 nm and 15 nm, e.g., between 6 nm and Lipo of 15nm size, 5nm~12nm size, or 8nm~12nm size A population of protein particles is obtained. The size of the lipid particles subjected to thermal cycling is 5 In a preferred embodiment, the lipid particles are mainly Another preferred embodiment is composed of egg sphingomyelin and has a size of 55 to 75 nm. In embodiments, the lipid particles are primarily composed of one or more synthetic sphingomyelins (e.g., , phytosphingomyelin) and are 175 nm to 250 nm in size.

[0096] One or more steps in the method for preparing lipoprotein complexes are carried out using an inert gas. By doing so, the apolipoprotein and / or It can reduce or prevent lipid oxidation, thereby reducing the risk of side effects such as liver damage. Suitable inert gases include nitrogen, helium, and argon. Gon is one example.

[0097] The lipoprotein complex obtained or obtainable by the above method is The compound is particularly suitable for therapeutic use since no further purification steps are required after it has been prepared.

[0098] The present disclosure also relates to populations of lipoprotein complexes, as well as lipoprotein complexes, as described herein. A protein complex or population thereof, optionally comprising one or more pharma- ceutically acceptable Pharmaceutical compositions are also provided that may include the carriers, excipients and / or diluents described herein. In some embodiments, the pharmaceutical compositions are packaged in a unit dosage suitable for administration. For example, in some embodiments, the composition is packaged in a sealed vial. Such a unit dose of lipoprotein complexes may be dried (e.g., lyophilized). The composition may be reconstituted with water, a physiological solution (such as saline) or a buffer; and Such compositions are suitable for administration by injection. one or more anticoagulants and / or anti-aggregating agents to facilitate reconstitution of the or designed to adjust the pH, osmolality and / or salinity of the reconstituted suspension. one or more buffers, isotonicity agents (e.g., sucrose and / or mannitol) Lipoprotein complexes may contain glycerol, sugar or salts (e.g. sodium chloride). The population of organisms and / or the pharmaceutical compositions described above are produced under conditions that minimize oxidation. This reduces the risk of side effects such as liver damage caused by oxidation products. For example, the pharmaceutical composition can be purged with an inert gas, such as nitrogen, helium, or argon. It can be manufactured under Gon.

[0099] For commercial applications, large-scale preparations of lipoprotein complexes and pharmaceutical compositions can be produced. Thus, the present disclosure also provides a method for the preparation of a medicament for the treatment of a disease comprising administering to a patient a therapeutically effective amount of at least about 3 mg / mL, at least About 4 mg / mL, or at least about 5 mg / mL, and up to about 10 mg / mL, about 15 mg / mL, or about 20 mg / mL, preferably about 8 mg / mL to about 12 mg / mL to achieve a lipid binding protein concentration in the range of about 8 mg / mL, most preferably about 8 mg / mL. At least 1 L, 2 L, 5 L or 10 L of lipoprotein complexes containing sufficient and preparations up to 15L, 20L, 30L, 50L or more (e.g., 5L to 30L, 10L In certain embodiments, preparations of 100 L to 250 L or 30 L to 50 L are also provided. The container has a volume of 15 L to 25 L and contains about 100 g to about 250 g of ApoA-I. In another particular embodiment, the preparation has a volume of 30 L to 50 L and contains about 240 g to about Contains 780g of ApoA-I.

[0100] The lipoprotein complexes described herein are intended to reduce lipid abnormalities in animals, and most preferably in humans. These conditions are useful for treating chronic disorders, including but not limited to: , hyperlipidemia, and especially hypercholesterolemia (heterozygous and homozygous familial hypercholesterolemia) cholesterolemia), and cardiovascular diseases such as atherosclerosis (atherosclerosis (including the treatment and prevention of arteriosclerosis) and, for example, stroke, ischemic stroke, transient ischemia Stroke, myocardial infarction, acute coronary syndrome, angina, intermittent claudication, critical limb ischemia, valvular stenosis, and The myriad clinical manifestations of atherosclerosis include atrial valvular sclerosis; restenosis (e.g., balloon Prevention of atherosclerotic plaques that develop as a result of medical procedures such as angioplasty or treatment); and other disorders such as endotoxemia, which often leads to septic shock. Some examples include:

[0101] The lipoprotein complexes and compositions described herein have been shown to be When administered at lower doses than those used for lipoprotein complexes, It has been found that 15 mg / kg- Doses ranging from 80 mg / kg were used. Spieker et al., 2002, Circulation 105: 1399-1402 (using a dose of 80 mg / kg); Nissen et al., 2003, JAMA 290:2292-2300 (15 mg / 40 mg / kg or 40 mg / kg; Tardif et al., 2007 JAMA 297:1675-1682 (Dose ranges from 0 mg / kg to 80 mg / kg are used). The lipoprotein complexes described above have been found to have reduced side effects. As shown, the lipoprotein complexes of the present disclosure inhibit cholesterol synthesis at doses as low as 2 mg / kg. In contrast, the lipoprotein complexes previously administered to human patients were efficiently transferred to the cytoplasm. has been shown to improve the levels of triglycerides, VLDL, and liver enzymes such as transaminases. (Nanjee et al., 1999, Arterioscler. Vasc. Thromb. Biol. 19:979-89). Additionally, the reduction in side effects is consistent with normal liver and / or kidney function. In normal functioning subjects, the maximum dose is approximately 15 mg / kg (absence of triglyceride elevation) ) or even increased dose to 45 mg / kg (lack of transaminase increase) Thus, the ability of the conjugates of the present disclosure to be administered without side effects is favorable. is assessed in individuals with normal liver function, normal renal function, or both.

[0102] Without being bound by theory, the inventors believe that the lipoprotein complexes of the present disclosure The benefits to the body include a more uniform size distribution of the complexes and a reduced amount of loss compared to previous treatments. Damaged proteins and / or lipids (e.g., oxidized proteins, deamidated The negative effects of the lipid fraction on the oxidized proteins and lipids were considered to be due to the The positively charged phospholipids are incorporated into the complexes and compositions described herein in a manner similar to that of conventional lipoproteins. It is believed to confer improved therapeutic properties over the conjugates. Small discoid pre-β HDL particles and their cholesterol are stored, recycled, and substituted. Large molecules are recognized by the liver to be digested (as bile acids) or eliminated (in the bile). One of the key differences between HDL and disk-like and / or spherical HDL is the charge of the particles. Small discoidal pre-β HDL particles are composed of negatively charged large discoidal and / or HDL particles have a lower negative surface charge than spherical HDL particles. The higher negative charge inhibits the granules from being absorbed by the liver. This may be one of the factors that induces the recognition of the particles by the kidney and thus avoids catabolism of the particles by the kidney. Furthermore, it has been shown that the kidney does not readily absorb charged particles (Hacker et al., 2003). (See Pharmacology: Principles and Practice, 2009, 183). Thus, in part Due to the presence of charged phospholipids, the negatively charged lipoprotein complexes described herein and the compositions remain in circulation longer than conventional lipoprotein complexes or The charge appears to affect the half-life of lipoproteins in a charge-dependent manner. As a result of the charge, they aggregate and fuse with existing HDL at a reduced rate and / or extent. In addition, their longer circulation (residence) time leads to cholesterol mobilization (cholesterol accumulation). (by giving the complex more time to react with LCAT) and esterification (by giving the complex more time to react with LCAT). This is expected to facilitate the synthesis of hydroxyl groups (by providing more time to catalyze the catalysis of the hydroxylation reaction). The charge also increases the rate of cholesterol capture and / or removal, thereby It may also facilitate the removal of larger amounts of cholesterol. The negatively charged lipoprotein complexes and compositions described herein are administered with reduced side effects. Fewer and less frequent complexes and / or compositions are required to achieve this, making it more suitable for use with conventional lipoproteins. It is expected that these drugs will provide therapeutic benefits beyond those offered by conventional therapeutic approaches.

[0103] Thus, the methods provided herein generally involve administering a therapeutically effective amount of a lipoprotein complex, A population of lipoprotein complexes or a pharmaceutical composition described herein may be administered to a subject to induce lipoprotein synthesis. The lipoprotein complexes are administered in amounts of 0.1 mg / kg / day or 0.2 mg / kg / day to treat or prevent lipid disorders. , at doses ranging from about 0.25 mg / kg ApoA-I equivalent to about 45 mg / kg, e.g. , from about 0.5 mg / kg to about 30 mg / kg or about 1 mg / kg of ApoA-I equivalent It can be administered in doses up to about 15 mg / kg of ApoA-I equivalent. In addition, triglycerides, VLDL-cholesterol and / or VLDL-triglycerides By selecting a dose that minimizes the increase in serine levels, the treatment can be tailored to the individual being treated. In certain embodiments, the dose is about 3 mg / kg, about 6 mg / kg, Or about 12 mg / kg.

[0104] The method may be carried out at intervals ranging from 5 to 14 days, or 6 to 12 days, for example, at intervals of 1 or 2 weeks. The method further comprises administering the lipoprotein complex at any of the intervals described above. At intervals of 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours, preferably at weekly intervals. The method may further include administering the lipoprotein complex up to 52 times. In an embodiment, the lipoprotein complexes are administered 6 times with one week intervals between each administration. For chronic conditions, more than 52 doses may be administered. proceeding with an initial induction phase during which the lipoprotein complex is administered more frequently; It is possible.

[0105] Administering the conjugate and / or pharmaceutical composition thereof parenterally, e.g., intravenously. Intravenous administration can be performed for about 1 to about 24 hours, about 1 to 4 hours, about 0.5 to 2 hours, Or it can be administered as an infusion over a period of about 1 hour.

[0106] The following examples show the triglyceride levels after administration of doses of 30 mg / kg and 45 mg / kg. This shows a slight increase in VLDL and cholesterol levels resulting from a high degree of cholesterol mobilization. These parameters were measured using a standard lipid panel. It is routinely measured in hospital laboratories and can therefore be controlled during treatment. Based on the examples, triglyceride and VLDL-cholesterol levels are determined in response to the patient's response to the drug. Dosage should be selected to minimize increases in sterol and VLDL-triglyceride levels. This will allow for a wide range of options, making personalized medicine possible.

[0107] The conjugates and / or compositions may be administered alone (as monotherapy) or The present invention relates to a method for treating and / or preventing dyslipidemia and / or its related conditions, diseases and / or disorders. They can be administered adjunctively with other therapeutic agents useful for preventing and / or treating The negatively charged lipoprotein complexes and compositions described herein can be administered adjunctively. Non-limiting examples of therapeutic agents that can be used include bile acid binding resins, HMG CoA reductase, Inhibitors (statins), niacin, resins, inhibitors of cholesterol absorption, platelet aggregation inhibitors agents, fibrates, anticoagulants, CETP inhibitors (e.g., anacetrapib and dalceto rapib), and / or PCSKG antibodies or ligands. [Brief description of the drawings]

[0108] 5. Brief description of the drawings [Figure 1]FIG. 1 shows the amino acid sequence of human preproapolipoprotein AI (SEQ ID NO:1; GenBank Accession No. AAB59514.1). Amino acids 1-18, shown in bold, correspond to the signal sequence of ApoA-I, and amino acids 19-24, underlined, correspond to the propeptide sequence. Both the signal sequence and the propeptide are cleaved in the cell to produce full-length mature human ApoA-I (amino acids 25-267). [Diagram 2] Figure 1 shows ApoA-I titers in 12-day fed-batch cultures of recombinant S-CHO cells expressing ApoA-I. Cells were cultured continuously by serial passage from generation 0 to generation 43. ApoA-I production was monitored by reverse-phase HPLC at generations 4, 8, 14, 19, 25, 21, 36 and 43. The amount of ApoA-I in the culture medium varied from 1259 mg / L to 1400 mg / L. [Figure 3A] Figure 3A shows the viable cell density in a 200 L 13-day culture of recombinant S-CHO cells expressing ApoA-I. The viable cell density peaked at 33.20x105 cells / mL on day 9 with 92.5% viability. Figure 3B shows the concentration of ApoA-I in the culture medium of a 13-day culture. The concentration of ApoA-I in the culture medium peaked at approximately 2 mg / mL on day 12. [Figure 3B] Figure 3A shows the viable cell density in a 200 L 13-day culture of recombinant S-CHO cells expressing ApoA-I. The viable cell density peaked at 33.20x105 cells / mL on day 9 with 92.5% viability. Figure 3B shows the concentration of ApoA-I in the culture medium of a 13-day culture. The concentration of ApoA-I in the culture medium peaked at approximately 2 mg / mL on day 12. [Figure 4] 1 shows an SDS polyacrylamide gel of ApoA-I purified by the methods described herein. The left hand lane shows molecular weight markers. The right hand lane shows purified ApoA-I having a molecular weight of approximately 28 kD. [Figure 5A]FIG. 5A shows HPLC chromatograms of proApoA-I / SM complexes with protein:lipid weight ratios of 1:2.5 (formulation A; FIG. 5A), 1:2.7 (formulation B; FIG. 5B), 1:3.1 (formulation C; FIG. 5C), and proApoA-I / SM / DPPC complexes with a protein:lipid weight ratio of 1:2.7 (formulation D; FIG. 5D). [Figure 5B] FIG. 5A shows HPLC chromatograms of proApoA-I / SM complexes with protein:lipid weight ratios of 1:2.5 (formulation A; FIG. 5A), 1:2.7 (formulation B; FIG. 5B), 1:3.1 (formulation C; FIG. 5C), and proApoA-I / SM / DPPC complexes with a protein:lipid weight ratio of 1:2.7 (formulation D; FIG. 5D). [Figure 5C] FIG. 5A shows HPLC chromatograms of proApoA-I / SM complexes with protein:lipid weight ratios of 1:2.5 (formulation A; FIG. 5A), 1:2.7 (formulation B; FIG. 5B), 1:3.1 (formulation C; FIG. 5C), and proApoA-I / SM / DPPC complexes with a protein:lipid weight ratio of 1:2.7 (formulation D; FIG. 5D). [Figure 5D] FIG. 5A shows HPLC chromatograms of proApoA-I / SM complexes with protein:lipid weight ratios of 1:2.5 (formulation A; FIG. 5A), 1:2.7 (formulation B; FIG. 5B), 1:3.1 (formulation C; FIG. 5C), and proApoA-I / SM / DPPC complexes with a protein:lipid weight ratio of 1:2.7 (formulation D; FIG. 5D). [Figure 6A] FIG. 13 shows HPLC chromatograms of lipoprotein complexes of Formulation D at 10, 20, 30 and 60 minutes, respectively. [Figure 6B] FIG. 13 shows HPLC chromatograms of lipoprotein complexes of Formulation D at 10, 20, 30 and 60 minutes, respectively. [Figure 6C] FIG. 13 shows HPLC chromatograms of lipoprotein complexes of Formulation D at 10, 20, 30 and 60 minutes, respectively. [Figure 6D] FIG. 13 shows HPLC chromatograms of lipoprotein complexes of Formulation D at 10, 20, 30 and 60 minutes, respectively. [Figure 7A]FIG. 13 shows HPLC chromatograms of lipoprotein complexes of Formulation B at 10, 20, 30 and 50 minutes, respectively. [Figure 7B] FIG. 13 shows HPLC chromatograms of lipoprotein complexes of Formulation B at 10, 20, 30 and 50 minutes, respectively. [Figure 7C] FIG. 13 shows HPLC chromatograms of lipoprotein complexes of Formulation B at 10, 20, 30 and 50 minutes, respectively. [Figure 7D] FIG. 13 shows HPLC chromatograms of lipoprotein complexes of Formulation B at 10, 20, 30 and 50 minutes, respectively. [Figure 8A] FIG. 1 shows HPLC chromatograms of lipoprotein complexes of Formulation F at 20, 40, 60 and 120 minutes, respectively. [Figure 8B] FIG. 1 shows HPLC chromatograms of lipoprotein complexes of Formulation F at 20, 40, 60 and 120 minutes, respectively. [Figure 8C] FIG. 1 shows HPLC chromatograms of lipoprotein complexes of Formulation F at 20, 40, 60 and 120 minutes, respectively. [Figure 8D] FIG. 1 shows HPLC chromatograms of lipoprotein complexes of Formulation F at 20, 40, 60 and 120 minutes, respectively. [Figure 9] FIG. 1 shows pre-β HDL complex formation over time illustrating the formation of lipoprotein complexes with a lipoprotein:total phospholipids wt:wt ratio of 1:2.7, including proApoA-I and SM (Formulation B), proApoA-I, SM, DPPC and DPPG with a SM:DPPC:DPPG wt:wt ratio of 48:48:4 (Formulation F), and proApoA-I, SM, DPPC and DPPG with a SM:DPPC:DPPG wt:wt ratio of 73:23:4 (Formulation G). [Figure 10A] FIG. 1 shows HPLC chromatograms of formulations E, H, I and J, respectively. [Figure 10B] FIG. 1 shows HPLC chromatograms of formulations E, H, I and J, respectively. [Figure 10C] FIG. 1 shows HPLC chromatograms of formulations E, H, I and J, respectively. [Figure 10D] FIG. 1 shows HPLC chromatograms of formulations E, H, I and J, respectively. [Figure 11] FIG. 1 is a schematic diagram of an exemplary process for making lipoprotein complexes. [Figure 12] FIG. 1 illustrates an exemplary thermal cycling apparatus used for non-commercial scale thermal cycling runs. [Figure 13A] 13A-E show gel permeation chromatograms of the SM / DPPG / ApoA-I protein complex using an increasing number of thermal cycles. Components were thermal cycled between 57° C. and 37° C. for 5 min at each temperature for a total of 30 min (FIG. 13A), 60 min (FIG. 13B), 120 min (FIG. 13C), 180 min (FIG. 13D), or 210 min (FIG. 13E). [Figure 13B] 13A-E show gel permeation chromatograms of the SM / DPPG / ApoA-I protein complex using an increasing number of thermal cycles. Components were thermal cycled between 57° C. and 37° C. for 5 min at each temperature for a total of 30 min (FIG. 13A), 60 min (FIG. 13B), 120 min (FIG. 13C), 180 min (FIG. 13D), or 210 min (FIG. 13E). [Figure 13C] 13A-E show gel permeation chromatograms of the SM / DPPG / ApoA-I protein complex using an increasing number of thermal cycles. Components were thermal cycled between 57° C. and 37° C. for 5 min at each temperature for a total of 30 min (FIG. 13A), 60 min (FIG. 13B), 120 min (FIG. 13C), 180 min (FIG. 13D), or 210 min (FIG. 13E). [Figure 13D] 13A-E show gel permeation chromatograms of the SM / DPPG / ApoA-I protein complex using an increasing number of thermal cycles. Components were thermal cycled between 57° C. and 37° C. for 5 min at each temperature for a total of 30 min (FIG. 13A), 60 min (FIG. 13B), 120 min (FIG. 13C), 180 min (FIG. 13D), or 210 min (FIG. 13E). [Figure 13E] 13A-E show gel permeation chromatograms of the SM / DPPG / ApoA-I protein complex using an increasing number of thermal cycles. Components were thermal cycled between 57° C. and 37° C. for 5 min at each temperature for a total of 30 min (FIG. 13A), 60 min (FIG. 13B), 120 min (FIG. 13C), 180 min (FIG. 13D), or 210 min (FIG. 13E). [Figure 14] FIG. 1 shows a gel permeation chromatogram of the SM / ApoA-I complex. [Figure 15] FIG. 1 shows a gel permeation chromatogram of the N-palmitoyl-4-hydroxysphinganine-1-phosphocholine (a form of plant SM or phytosphingomyelin) / DPPG / ApoA-I complex. [Figure 16] FIG. 1 shows a gel permeation chromatogram of a synthetic palmitoyl SM / DPPG / ApoA-I complex. [Figure 17] FIG. 1 shows a gel permeation chromatogram of the phytosphingomyelin / DPPG / ApoA-I complex. [Figure 18] FIG. 1 shows a gel permeation chromatogram of the SM / DPPC / DPPG / ApoA-I peptide complex. [Figure 19A] Figure 19 shows the characterization of lipid particles by a dynamic light scattering system using a Malvern Instruments Zetasizer (Malvern Instruments Inc.). In Figure 19A, the Z average is 84.49 nm ("84 nm particles"); in Figure 19B, the Z average is 76.76 nm ("77 nm particles"); in Figure 19C, the Z average is 66.21 nm ("66 nm particles"); and in Figure 19D, the Z average is 59.50 nm ("60 nm particles"). [Figure 19B]Figure 19 shows the characterization of lipid particles by a dynamic light scattering system using a Malvern Instruments Zetasizer (Malvern Instruments Inc.). In Figure 19A, the Z average is 84.49 nm ("84 nm particles"); in Figure 19B, the Z average is 76.76 nm ("77 nm particles"); in Figure 19C, the Z average is 66.21 nm ("66 nm particles"); and in Figure 19D, the Z average is 59.50 nm ("60 nm particles"). [Figure 19C] Figure 19 shows the characterization of lipid particles by a dynamic light scattering system using a Malvern Instruments Zetasizer (Malvern Instruments Inc.). In Figure 19A, the Z average is 84.49 nm ("84 nm particles"); in Figure 19B, the Z average is 76.76 nm ("77 nm particles"); in Figure 19C, the Z average is 66.21 nm ("66 nm particles"); and in Figure 19D, the Z average is 59.50 nm ("60 nm particles"). [Figure 19D] Figure 19 shows the characterization of lipid particles by a dynamic light scattering system using a Malvern Instruments Zetasizer (Malvern Instruments Inc.). In Figure 19A, the Z average is 84.49 nm ("84 nm particles"); in Figure 19B, the Z average is 76.76 nm ("77 nm particles"); in Figure 19C, the Z average is 66.21 nm ("66 nm particles"); and in Figure 19D, the Z average is 59.50 nm ("60 nm particles"). [Figure 20A] 20A-D show gel permeation chromatograms of the complexes after five thermal cycles with 84 nm (FIG. 20A), 77 nm (FIG. 20B), 66 nm (FIG. 20C) and 60 nm (FIG. 20D) lipid particles. [Figure 20B] 20A-D show gel permeation chromatograms of the complexes after five thermal cycles with 84 nm (FIG. 20A), 77 nm (FIG. 20B), 66 nm (FIG. 20C) and 60 nm (FIG. 20D) lipid particles. [Figure 20C]20A-D show gel permeation chromatograms of the complexes after five thermal cycles with 84 nm (FIG. 20A), 77 nm (FIG. 20B), 66 nm (FIG. 20C) and 60 nm (FIG. 20D) lipid particles. [Figure 20D] 20A-D show gel permeation chromatograms of the complexes after five thermal cycles with 84 nm (FIG. 20A), 77 nm (FIG. 20B), 66 nm (FIG. 20C) and 60 nm (FIG. 20D) lipid particles. [Figure 21A] 21A and 21B show gel permeation chromatograms of the complexes after six thermal cycles starting from 450 nm (FIG. 21A) and 40 nm (FIG. 21B) lipid particles. [Figure 21B] 21A and 21B show gel permeation chromatograms of the complexes after six thermal cycles starting from 450 nm (FIG. 21A) and 40 nm (FIG. 21B) lipid particles. [Figure 22] Figure 1 shows gel permeation chromatograms of the complex after six thermal cycles starting with 65 nm lipid particles, with the first cycle beginning at a "cold" temperature of 37°C. [Figure 23] FIG. 1 is a schematic diagram of an exemplary embodiment for making a pharmaceutical composition comprising lipoprotein complexes, comprising formulating lipoprotein complexes produced by the methods of the present disclosure into a commercially useful pharmaceutical composition. [Figure 24] Figure 1 shows the increase in plasma VLDL-total cholesterol levels after injection of lipoprotein complexes according to formulation B and formulation H. Lipoprotein complexes according to formulation H (■) and formulation B (▼) were injected into fasted rabbits at a dose of 5 mg / kg. The increase in plasma VLDL-total cholesterol levels was determined by subtracting the baseline values ​​for the three groups, which ranged from 0.03 to 0.3 g / L. [Diagram 25] Figure 1 shows the increase in plasma triglyceride levels after injection of lipoprotein complexes according to formulation B and formulation H. Lipoprotein complexes according to formulation H (■) and formulation B (▲) were injected into fasted rabbits at a dose of 5 mg / kg. The increase in plasma triglyceride levels was determined by subtracting the baseline values ​​for the three groups, which ranged from 0.31 to 0.71 g / L. [Figure 26A] Figure 26A shows the increase in plasma total cholesterol (Fig. 26A), triglycerides (Fig. 26B), phospholipids (Fig. 26C), and ApoA-I (Fig. 26D) following injection of 5 or 20 mg / kg, and in rabbits 20 mg / kg, of ApoA-I / egg SM complex (●,▲) or ApoA-I / synthetic SM complex (◆,▼) compared with diluent (■). Baseline values ​​ranged for the various plasma lipids measured as follows: 0.28-0.4 g / L for plasma cholesterol, 0.23-0.29 g / L for plasma triglycerides, and 0.45-0.61 g / L for plasma phospholipids. [Figure 26B] Figure 26A shows the increase in plasma total cholesterol (Fig. 26A), triglycerides (Fig. 26B), phospholipids (Fig. 26C), and ApoA-I (Fig. 26D) following injection of 5 or 20 mg / kg, and in rabbits 20 mg / kg, of ApoA-I / egg SM complex (●,▲) or ApoA-I / synthetic SM complex (◆,▼) compared with diluent (■). Baseline values ​​ranged for the various plasma lipids measured as follows: 0.28-0.4 g / L for plasma cholesterol, 0.23-0.29 g / L for plasma triglycerides, and 0.45-0.61 g / L for plasma phospholipids. [Figure 26C] Figure 26A shows the increase in plasma total cholesterol (Fig. 26A), triglycerides (Fig. 26B), phospholipids (Fig. 26C), and ApoA-I (Fig. 26D) following injection of 5 or 20 mg / kg, and in rabbits 20 mg / kg, of ApoA-I / egg SM complex (●,▲) or ApoA-I / synthetic SM complex (◆,▼) compared with diluent (■). Baseline values ​​ranged for the various plasma lipids measured as follows: 0.28-0.4 g / L for plasma cholesterol, 0.23-0.29 g / L for plasma triglycerides, and 0.45-0.61 g / L for plasma phospholipids. [Figure 26D]Figure 26A shows the increase in plasma total cholesterol (Fig. 26A), triglycerides (Fig. 26B), phospholipids (Fig. 26C), and ApoA-I (Fig. 26D) following injection of 5 or 20 mg / kg, and in rabbits 20 mg / kg, of ApoA-I / egg SM complex (●,▲) or ApoA-I / synthetic SM complex (◆,▼) compared with diluent (■). Baseline values ​​ranged for the various plasma lipids measured as follows: 0.28-0.4 g / L for plasma cholesterol, 0.23-0.29 g / L for plasma triglycerides, and 0.45-0.61 g / L for plasma phospholipids. [Figure 27A] Figure 27 shows the increase in plasma HDL-total cholesterol (Figure 27A), LDL-total cholesterol (Figure 27B), and VLDL-total cholesterol (Figure 27C) following infusion in rabbits of 5 mg / kg ApoA-I / egg SM complex (●) and ApoA-I / synthetic SM complex (◆) compared with diluent (■). Baseline values ​​ranged as follows: 0.20-0.31 g / L for plasma HDL-total cholesterol, 0.06-0.09 g / L for plasma LDL-total cholesterol, and 0.007-0.011 g / L for plasma VLDL-total cholesterol. [Figure 27B] Figure 27 shows the increase in plasma HDL-total cholesterol (Figure 27A), LDL-total cholesterol (Figure 27B), and VLDL-total cholesterol (Figure 27C) following infusion in rabbits of 5 mg / kg ApoA-I / egg SM complex (●) and ApoA-I / synthetic SM complex (◆) compared with diluent (■). Baseline values ​​ranged as follows: 0.20-0.31 g / L for plasma HDL-total cholesterol, 0.06-0.09 g / L for plasma LDL-total cholesterol, and 0.007-0.011 g / L for plasma VLDL-total cholesterol. [Figure 27C]Figure 27 shows the increase in plasma HDL-total cholesterol (Figure 27A), LDL-total cholesterol (Figure 27B), and VLDL-total cholesterol (Figure 27C) following infusion in rabbits of 5 mg / kg ApoA-I / egg SM complex (●) and ApoA-I / synthetic SM complex (◆) compared with diluent (■). Baseline values ​​ranged as follows: 0.20-0.31 g / L for plasma HDL-total cholesterol, 0.06-0.09 g / L for plasma LDL-total cholesterol, and 0.007-0.011 g / L for plasma VLDL-total cholesterol. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0109] 6. Detailed Description The present disclosure provides lipoprotein complexes, as well as methods for making lipoprotein complexes, The complexes, as well as populations and compositions (e.g., pharmaceutical compositions) thereof, are provided. In particular, the present invention relates to a method for treating dyslipidemia and / or diseases, disorders and / or conditions associated with dyslipidemia. are useful for the treatment and / or prevention of conditions. The lipoprotein complex is preferably divided into two major fractions in a defined weight or molar ratio. , apolipoprotein fraction and phospholipid fraction, preferably containing a specific amount of neutral phosphorus It comprises a lipid and optionally one or more negatively charged phospholipids.

[0110] 6.1. Protein Fractions The present disclosure provides lipoprotein complexes comprising protein fractions. Further provided are methods for producing lipoprotein complexes. The duration of the reaction is not critical to success in the methods of the invention. Virtually any lipid-binding protein that provides a therapeutic benefit, such as apolipoproteins and or its derivatives or analogs may be included in the conjugate. Polypoproteins (e.g., ApoA-I) activate LCAT, or lipids It "mimics" its activity in that it can form discoid particles when bound to Any α-helical peptide or peptide analogue, or any other type of molecule that , which can be contained in lipoprotein complexes and are referred to by the term "lipid-binding protein". Included.

[0111] Lipid-binding proteins The present disclosure provides recombinantly produced lipoprotein complexes for use in, for example, making lipoprotein complexes. The recombinantly produced proteins are further provided with methods for purifying the recombinantly produced proteins. A preferred protein is an apolipoprotein. A preferred protein is an apolipoprotein, preferably in its mature form. , apolipoproteins ApoA-I, ApoA-II, ApoA-IV, ApoA-V and and ApoE. Lipid binding proteins also have active polymorphisms, isoforms, Variants and mutants as well as truncated forms of said apolipoproteins. most commonly apolipoprotein AI Milano (ApoA-I M ), Apollo Polyprotein AI Paris (ApoA-I P ) and apolipoprotein AI Za ragoza (ApoA-I Z ) Apolipoproteins containing cysteine ​​residues Mutants are also known and can be used (see, e.g., U.S. Patent Application Publication No. 2003 / 013363). (See US Pat. No. 5,372,372.) Apolipoproteins exist in monomeric or dimeric forms. and the dimer may be a homodimer or a heterodimer. AI (Duverger et al., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12): 1424-29), ApoA-I M (Franceschini et al., 1985, J. Biol. Chem. 260: 1632-35), ApoA- I P (Daum et al., 1999, J. Mol. Med. 77:614-22), ApoA-II (Shelness et al., 1985, J. Biol. Chem. 260(14):8637-46; Shelness et al., 1984, J. Biol. Chem. 259(15):9929-35 ), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem. 201(2):373-83), Apo E (McLean et al., 1983, J. Biol. Chem. 258(14):8993-9000), ApoJ and ApoH Homo- and heterodimers (where feasible) can be used. Proteins retain some biological activity when apolipoproteins are included in the complex. As long as the nucleic acid has an element that facilitates its isolation, such as a His tag, or is designed for other purposes, The amino acid sequence may include residues corresponding to other elements defined above.

[0112] Such apolipoproteins may be purified from animal sources (and in particular from human sources) or or recombinantly produced as known in the art. Cheung et al., 1987, J. Lipid Res. 28(8):913-29 See also U.S. Patent Nos. 5,059,528, 5,128,318, and 6, ,617,134; U.S. Patent Application Publication No. 20002 / 0156007, No. 2004 / Nos. 0067873, 2004 / 0077541, and 2004 / 0266660 No. 5,333,636; and PCT Publication Nos. WO / 2008 / 104890 and WO / 2007 See also US Pat. No. 6,234,666, for example, Sections 6.1.3 and 6.1.4 below. Other purification methods are possible, such as those described in.

[0113] Peptides and peptide analogs corresponding to apolipoproteins, as well as the peptides and peptide analogs described herein, Apolipoproteins suitable for use as apolipoproteins in the complexes and compositions described herein are AI, ApoA-I M Mimics the activity of ApoA-II, ApoA-IV and ApoE Non-limiting examples of agonists that are suitable for use in the treatment of rheumatoid arthritis include those described in U.S. Pat. Nos. 6,004,925, 6,037,323 ... Nos. 5,813,633 and 6,046,166 (issued to Dasseux et al.), U.S. Pat. No. 40,688 (issued to Tso), U.S. Patent Application Publication No. 2004 / 02666 71, No. 2004 / 0254120, No. 2003 / 0171277 and No. 200 No. 3 / 0045460 (to Fogelman), U.S. Patent Application Publication No. 2003 / 00878 No. 19 (to Bielicki) and PCT Publication No. WO / 2010 / 093918 (to Dasseux et al.), the disclosures of which are hereby incorporated by reference in their entireties. These peptides and peptide analogs may be synthesized using L-amino acids or D-amino acids. They may be composed of amino acids or mixtures of L- and D-amino acids. Also, one or more non-peptide isosteres, such as one or more known peptide / amide isosteres. Such "peptides and / or peptides" may contain a peptide or amide bond. "Mimetic" apolipoproteins are described, for example, in U.S. Pat. No. 6,004,925, U.S. Pat. The present invention relates to a method for producing a phenolic compound having a phenolic group, the method comprising the steps of: The peptides may be synthesized or prepared using any technique for peptide synthesis.

[0114] The complexes may contain a single type of lipid-binding protein, which may be derived from the same or different species. The lipid-binding protein may comprise a mixture of two or more different lipid-binding proteins. However, lipoprotein complexes may be used to avoid inducing an immune response to the therapy. is preferably derived from the animal species to be treated or has a similar amino acid sequence to the therapeutic Thus, the present invention provides a method for the treatment of human patients, comprising administering to a human subject a lipid-binding protein corresponding to the animal species to which the lipid-binding protein is administered. Preferably, lipid-binding proteins of human origin are used in the conjugates of the present disclosure. The use of anti-inflammatory mimetic apolipoproteins can also reduce or avoid immune responses. This can be done.

[0115] In certain preferred embodiments, the lipid binding protein is a mature human ApoA-I protein. A protein having an amino acid sequence with at least 95% sequence identity to the For example, a protein having an amino acid sequence corresponding to positions 25 to 267 of SEQ ID NO: 1. In certain embodiments, the mature human ApoA-I protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. At least 96%, at least 97%, or at least 98% for positions 5 through 267; or In some embodiments, the amino acid sequence has at least 99% sequence identity. In this regard, the mature human ApoA-I protein has a sequence corresponding to position 1 (i.e., position 25 of SEQ ID NO:1). In certain embodiments, the amino acid sequence has an aspartic acid at the position corresponding to The mature human ApoA-I protein is a sequence corresponding to amino acids 25 to 267 of SEQ ID NO:1. In a preferred embodiment, the ApoA-I protein is a mammalian host. Main cells, most preferably Chinese hamster ovary ("CHO") cells, as described in the following subsection. ) cells.

[0116] 6.1.2. Recombinant Expression of Apolipoproteins The present disclosure provides recombinant expression methods for producing lipid binding proteins, such as ApoA-I, and related nucleic acids, mammalian host cells, and cell cultures. The conjugated proteins can be purified and / or incorporated into lipoprotein complexes as described herein. You can put it in.

[0117] Generally, for recombinant production, a polypeptide encoding a lipid binding protein or peptide is The nucleotide sequence is inserted into a suitable expression vehicle, i.e., a vector capable of transcription and transcription of the inserted coding sequence. and the necessary elements for replication and translation, or, in the case of RNA viral vectors, replication and translation The expression vector is inserted into a vector that contains the necessary elements for translation. Viruses, adeno-associated viruses, herpes viruses, retroviruses or lentiviruses The expression vehicle can then be derived from a virus, such as a virion, or a recombinant vector. is transfected into a suitable target cell that expresses the peptide. include, but are not limited to, bacterial species, mammalian or insect species such as baculovirus systems. Host cell lines (see, e.g., Luckow et al., Bio / Technology, 6, 47 (1988)), and and established cell lines, e.g., 293, COS-7, C127, 3T3, CHO, HeL a, BHK, etc. Then, depending on the expression system used, the expressed peptide The recombinant proteins and peptides are isolated by procedures well established in the art. Methods for purification are well known in the art (e.g., Sambrook et al., 1989, Molecular Cl. oning A Laboratory Manual, Cold Spring Harbor Laboratory, NY; and Ausubel et al. , 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY (each of which is incorporated by reference in its entirety) (See ).

[0118] If ApoA-I is a lipid-binding protein, then the ApoA-I protein is ApoA- In some embodiments, the recombinant nucleotide sequence encoding I is expressed from a recombinant nucleotide sequence encoding I. The nucleotide sequence encoding ApoA-I is human. Non-limiting examples of peptide sequences are described in U.S. Patent Nos. 5,876,968; 5,643,757; and and 5,990,081, and WO 96 / 37608, The disclosure of which is incorporated herein by reference in its entirety. The nucleotide sequence preferably includes a signal sequence for secretion of ApoA-I from the host cell ( For example, amino acids 1-18 of SEQ ID NO:1) and / or a proprotein sequence (e.g., operably linked to amino acids 19-25 of SEQ ID NO:1; It also encodes the amino acid sequence of the protein, which is suitable for directing the secretion of ApoA-I. The null sequence may be heterologous to ApoA-I, e.g., a human albumin signaling site. It may be a human IL-2 signal peptide or a human IL-2 signal peptide, or it may be ApoA-I may be of the same kind.

[0119] Preferably, the nucleotide sequence is a mature human ApoA-I polypeptide, e.g., the sequence At least 95% and at least 96% of the amino acid sequence corresponding to positions 25 to 267 of No. 1 , an amino acid sequence that is at least 97%, at least 98% or at least 99% identical and optionally, the amino acid sequence includes an asparagus at position 25. In a preferred embodiment, the nucleotide sequence comprises the amino acid sequence of SEQ ID NO:1. The nucleotide sequence is also contained in the GenBank Accession No. Numbers NP_000030, AAB59514, P02647, CAA30377, AAA 51746 or AAH05380.1. At least 95%, at least 96%, at least 97%, at least 98% 100% or at least 99% identical amino acid sequence Often, and optionally, at a position corresponding to the first amino acid of the mature human ApoA-I protein Contains aspartic acid.

[0120] A polynucleotide encoding ApoA-I is cloned for expression in a recombinant host cell. Preferred host cells are mammalian host cells, e.g., Although not required, Chinese hamster ovary cells (e.g., CHO-K1; ATC C Number CCL61; CHO-S (GIBCO Life Technologies I N.C., Rockville, MD, Catalog No. 11619012), VERO Cells , BHK (ATCC number CRL1632), BHK570 (ATCC number CRL1031 4), HeLa cells, COS-1 (ATCC number CRL1650), COS-7 (ATC C number CRL1651), MDCK cells, 293 cells (ATCC number CRL1573; Gr aham et al., J. Gen. Virol. 36:59-72, 1977), 3T3 cells, myeloma cells (especially mouse In a particular embodiment, CHO-S Mammalian cells, such as cells (Invitrogen™, Carlsbad Calif.) , adapted for growth in serum-free medium. Further suitable cell lines are known in the art. American Type Culture Collection, Man These are available from public depositories such as Assas, Va.

[0121] For recombinant expression of ApoA-I, a polynucleotide encoding ApoA-I can be One or more regulatory sequences, e.g., those that regulate the expression of ApoA-I in a host cell of interest. The control sequence is operably linked to a promoter or terminator that is -I coding sequence may be native or foreign, ApoA-I may also be native or foreign to the host cell in which it is expressed. The control sequence may be, but is not limited to, a promoter, a ribosomal Binding site, leader, polyadenylation sequence, propeptide sequence, signal peptide sequence In some embodiments, the control sequence includes a transcriptional sequence, a transcription terminator, and a transcriptional terminator. It contains a promoter, ribosome binding sites, and transcriptional and translational stop signals. The control sequence is a sequence that is a nucleotide sequence that encodes ApoA-I. One or more restriction sites to introduce specific restriction sites that facilitate ligation to the The insulating layer may include an anchor.

[0122] The promoters driving recombinant expression of ApoA-I can be constitutive, regulated or The promoter sequence may be a promoter that is specific to the host cell. Encodes an extracellular or intracellular polypeptide that is endogenous or heterologous to the cell. The promoters of various strengths can be isolated, identified and manipulated. Methods for producing the same are available in the art or can be readily adapted from the art. For example, Nevoigt et al. (2006) Appl. Environ. Microbiol. 72:5266-5273 ( (the disclosure of which is incorporated herein by reference in its entirety).

[0123] One or more of the control sequences can be derived from viral origins. In one embodiment, the promoter is a polyoma or adenovirus major late promoter. In another embodiment, the promoter is derived from the SV40 viral origin of replication. Simian virus 40 (SV40) (Fiers et al., 1999) can be obtained as a fragment containing 978, Nature, 273: 113-120), or derived from cytomegalovirus, e.g. The luc cytomegalovirus immediate early promoter (see U.S. Pat. No. 4,956,288) Other suitable promoters include those derived from metallothionein genes. (See U.S. Pat. Nos. 4,579,821 and 4,601,978. I want to be illuminated.

[0124] Also provided herein are recombinant ApoA-I expression vectors. The present invention relates to a method for the preparation of a recombinant DNA molecule for the expression of a heterologous ApoA-I in a recombinant host cell. The vector may be any vector that can be manipulated by the The expression vector can be integrated into the chromosome of a recombinant host cell and can express ApoA- one or more control sequences useful for the production of I In another embodiment, the expression vector comprises an extrachromosomally replicating D NA molecules, e.g., low copy number (e.g., about 1 to about 10 copies per genome equivalent) or They are found either in high copy number (e.g., greater than about 10 copies per genome equivalent) or in In various embodiments, the expression vector is a linear or closed circular plasmid. Conferring antibiotic resistance (e.g., ampicillin, kanamycin) to recombinant host organisms containing vectors Selection factors, such as genes that confer resistance to cyclosporine, chloramphenicol, or tetracycline In particular embodiments, the DNA constructs, vectors and polynucleotides The leutides are suitable for expression of ApoA-I in mammalian cells. Vectors for expression of ApoA-I in cells contain an origin of replication, a promoter, or a vector compatible with the host cell system. and any necessary ribosome binding sites, RNA splice sites, polyadenylation sites In some embodiments, the nucleic acid sequence may include a transcription terminator sequence, as well as a transcription site. The origin of production is heterologous to the host cell, e.g., of viral origin (e.g., S. In another embodiment, the origin of replication is Provided by the host cell chromosome replication machinery.

[0125] Methods, reagents and tools for introducing foreign DNA into mammalian host cells are known in the art. Known techniques include, but are not limited to, calcium phosphate mediated transfection. (Wigler et al., 1978, Cell 14:725; Corsaro et al., 1981, Somatic Cell Genetics 7:603; Gra ham et al., 1973, Virology 52:456), electroporation (Neumann et al., 1982, EMBO J. 1:841-5), DEAE-dextran-mediated transfection (Ausubel et al. (eds.) , Short Protocols in Molecular Biology, 3rd Edition (John Wiley & Sons 1995), and Liposome-mediated transfection (Hawley-Nelson et al., 1993, Focus 15:73; Ciccar one et al., 1993, Focus 15:80).

[0126] For high yield production, stable expression of ApoA-I is preferred. For example, exogenous expression of ApoA-I in a host cell is preferred. After introduction of the native DNA, the host cells are grown in rich medium for 1-2 days and then switched to selective medium. Rather than using expression vectors that contain viral origins of replication, The host cells are then cultured with an A gene that is controlled by suitable expression control elements and a selectable marker. The vector can be transformed with a nucleotide sequence that includes the poA-I coding sequence. The selectable marker in the vector confers resistance to the selection and allows cells to The vector is stably integrated into the cells, grown to form foci, and then cloned. The vector can be transformed into a cell line and propagated in the cell line. Several selection systems can be used, and the selection is limited. Although not a known inhibitor, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 1 1 : 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adenine phosphorylation The tk gene was used to express the tk oxidase enzyme (Lowy et al., 1980, Cell 22: 817). - , hgprt - or aprt - It can also be used in cells. dhfr, which confers resistance to sert (Wigler et al., 1980, Natl. Acad. Sci. USA 77: 3 567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); against mycophenolic acid gpt, which confers resistance to 2072); neo (Colberre-Garapin), which confers resistance to the aminoglycoside G-418 et al., 1981, J. Mol. Biol. 150: 1); and / or conferring resistance to hygromycin As a basis for selection, the gene expression vector hyg (Santerre et al., 1984, Gene 30: 147) was used. Antimetabolite resistance can be used.

[0127] Stable, high-yield expression is achieved using retroviral vectors that integrate into the host cell genome. The present invention can also be achieved (see, for example, U.S. Patent Application Publication No. 2008 / 0286779 and and 2004 / 0235173). Alternatively, see, for example, WO 1994 2. The method of claim 1, further comprising: A gene activation method that requires activating and amplifying the expression of the oA-I gene. By using this method, stable and high-yield expression of ApoA-I can be achieved. A copy of the vector (containing the ApoA-I coding sequence and one or more regulatory elements) is Increasing the number of ApoA-I vectors can facilitate high-yield expression of ApoA-I. The mammalian host cell in which oA-I is expressed may comprise at least 2, at least 3, at least 4 or has an ApoA-I gene copy index of at least 5. For example, the mammalian host cell in which ApoA-I is expressed may be at least 6, at least 7, or at least 9. have an ApoA-I gene copy index of at least 8, at least 9, or at least 10 do.

[0128] In certain embodiments, the mammalian cells are cultured at a concentration of at least 0.5 g / L, at least 1 g / L, at least 1.5g / L, at least 2g / L, at least 2.5g / L, at least At least 3g / L, at least 3.5g / L, and in some cases, at most 4g / L, at most Ap in amounts of 4.5g / L, up to 5g / L, up to 5.5g / L, or up to 6g / L The mammalian host cell is preferably adapted to produce oA-I in culture. At least about 0.5, 1, 2, or 3 g / L of ApoA-I and / or at least about 1, 2, or 3 g / L of ApoA-I in culture. ApoA-I at a maximum of about 20 g / L, e.g., up to 4, 5, 6, 7, 8, 9, ApoA-I can be produced at 10, 12 or 15 g / L.

[0129] In certain embodiments, the mammalian cells are adapted for growth in serum-free medium. In these embodiments, the ApoA-I is secreted from the cell. In this case, ApoA-I is not secreted from the cell.

[0130] The mammalian host cells provided herein can be used to produce ApoA-I. Generally, the method comprises culturing a mammalian cell line as described herein under conditions in which ApoA-I is expressed. The method further comprises culturing the mammalian host cell. This may include recovering and optionally purifying the mature ApoA-I.

[0131] Culture conditions, such as culture medium, temperature, and pH, are determined based on the mammalian host cells and the selected The culture format (shake flask, bioreactor, roller bottle, etc.) can be adapted to suit the needs of the culture. Mammalian cells can be grown in large-scale batch cultures, continuous or semi-continuous cultures. It is possible.

[0132] Also provided are mammalian host cells that produce multiple ApoA-Is as described herein. Also provided herein are mammalian cell cultures. In some embodiments, mammalian cell cultures Nutrients should be at least 0.5g / L, at least 1g / L, at least 1.5g / L, At least 2g / L, at least 2.5g / L, at least 3g / L, at least 3.5g / L L, and possibly up to 4g / L, up to 4.5g / L, up to 5g / L, up to Contains 5.5 g / L, or up to 6 g / L ApoA-I. Cultures are approximately 150 mL Approximately 500mL range, 1L, 10L, 15L, 50L, 100L, 200L, 250L, 300L, 350L, 400L, 500L, 750L, 1000L, 1500L, 200 0L, 2500L, 3000L, 5000L, 7500L, 10000L, 15000L The capacity may be any scale, such as 20,000 L, 25,000 L, 50,000 L or more. In some instances, the culture is a large-scale culture, e.g., 15 L, 20 L, 25 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 11 L, 12 L, 13 L, 14 L, 15 L, 16 L, 17 L, 18 L, 19 L, 20 L, 25 L, 26 L, 0L, 50L, 100L, 200L, 300L, 500L, 1000L, 5000L, 1 0000L, 15000L, 20000L, 25000L, up to 50000L or more do.

[0133] 6.1.3. Purification of apolipoproteins The present disclosure provides methods and methods for producing lipoprotein complexes and compositions thereof as described herein. The present invention relates to a method for obtaining a highly purified apolipoprotein, the method comprising the steps of: including but not limited to ApoA-I, -II, -III or -IV; ApoB4 8 and ApoB100; ApoC-I, -II, -III or -IV; ApoD; A The present invention can be applied to any apolipoprotein, such as ApoE, ApoH, or ApoJ. More specifically, the present disclosure relates to methods for obtaining highly purified ApoA-I. In some embodiments, ApoA-I is prepared using a method such as, but not limited to, Genba nk Accession Nos. NP_000030, AAB59514, P02647, CAA30377 AAA51746 and AAH05380.1. In certain embodiments, ApoA-I is a human protein comprising the polypeptides described in Section 6.1.2. In another embodiment, the method of the present disclosure is used to detect: Non-human animals (e.g., U.S. Patent Application Publication No. 2004 / 0077541), e.g., bovine , horses, sheep, monkeys, baboons, goats, rabbits, dogs, hedgehogs, badgers, mice, rats, cats, guinea pigs, hamsters, ducks, chickens, salmon and eels (Brouille tte et al., 2001, Biochim. Biophys. Acta. 1531 :4-46; Yu et al., 1991, Cell Struct. Funct. 16(4):347-55; Chen and Albers, 1983, Biochim Biophys Acta. 753(l):40-6; Luo et al. , 1989, J Lipid Res. 30(11): 1735-46;Blaton et al., 1977, Biochemistry 16:2157-63;S Parrow et al., 1995, J. Lipid Res. 36(3):485-95; Beaubatie et al., 1986, J. Lipid Res. 27: 140-49;Januzzi et al., 1992, Genomics 14(4): 1081-8;Goulinet and Chapman, 1993, J. Lipid Res. 34(6):943-59; Collet et al., 1997, J Lipid Res. 38(4):634-44; and Fra Purified ApoA-I from IgG4-derived IgG4 (NK and Marcel, 2000, J. Lipid Res. 41(6):853-72) was used. It is possible.

[0134] Examples of ApoA-I proteins that can be purified by the methods disclosed herein are: Examples include, but are not limited to, the preproapolipoprotein form of ApoA-I; Pro- and mature forms of ApoA-I, as well as active polymorphs, isoforms, variants and and mutant and truncated forms, e.g., ApoA-I M , ApoA-I Z , and ApoA-I P ApoA-I M R173C in ApoA-I Molecular variants (e.g., Parolini et al., 2003, J Biol Chem. 278(7):4740-6; Calabres i et al., 1999, Biochemistry 38: 16307-14; and Calabresi et al., 1997, Biochemistry 36: (See 12428-33.) ApoA-I P is the R151C molecular variant of ApoA-I. (See, e.g., Daum et al., 1999, J Mol Med. 77(8):614-22). ApoA-I Z is the L144R molecular variant of ApoA-I (Recalde et al., 2001, Atherosclerosis 154(3):613-623; see Fiddyment et al., 2011, Protein Expr. Purif. 80(1):110-116. Apolipoprotein AI mutants containing cysteine ​​residues are also known. , and may be purified by the methods described herein (see, e.g., U.S. Patent Application Publication No. 2003 / 013363). For use in the methods described herein, see US Pat. No. 5,399,343. ApoA-I may be in a monomeric, homodimeric, or heterodimeric form. For example, pro- and mature ApoA-I homo- and heterodimers can be prepared. In particular, ApoA-I (Duverger et al., 1996, Arterioscler Thromb Vasc Biol. 16(12): 1424-29), ApoA-I M (Franceschini et al., 1985, J Biol Chem. 260: 1632-35), and ApoA-IP (Daum et al., 1999, J Mol Med. 77:614-22).

[0135] The purification methods described herein can be carried out at any scale convenient to one of skill in the art. can.

[0136] In some embodiments, the ApoE purified by the methods described herein. The AI ​​protein is at least 75% identical to amino acids 25 to 267 of SEQ ID NO:1, At least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical 1. At least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, They have an amino acid sequence that is at least 99% identical or at least 100% identical.

[0137] The apolipoproteins may be derived from any source, such as plasma, or may be prokaryotic or eukaryotic. In certain embodiments, the apoE-binding domain may be derived from recombinant expression in a nuclear cell. The lipoprotein is ApoA-I, e.g., human ApoA-I. In this study, ApoA-I is expressed in the cytoplasm or periplasm of prokaryotic or eukaryotic host cells. In these embodiments, the cells are disrupted to release ApoA-I into the supernatant. After purification, the ApoA-I is purified. Methods for cell disruption are well known in the art. Exemplary methods for this include, but are not limited to, enzymatic methods, ultrasonic methods, detergent methods, In certain preferred embodiments, ApoA-I is delivered to mammals by immunoprecipitation or mechanical methods. The vector is expressed in mammalian cells, preferably CHO cells, and secreted into the growth medium. In one embodiment, ApoA-I is purified from the clarified cell-free medium. The specific features described herein in connection with ApoA-I, but which can be readily ascertained by one of skill in the art, are described in detail herein. Protein properties (e.g., molecular weight, isoelectric point, Stokes radius, hydrophobicity, multimeric state, etc.) Depending on the purification conditions, the purification conditions may be adjusted to other apolipoproteins, as well as non-human ApoA-I, ApoA-II, and ApoA-III. AI polymorphisms, isoforms, variants, mutants and truncated forms or other apolipoproteins. It is understood that.

[0138] When ApoA-I is prepared from plasma, it can be prepared according to, but not limited to, the method of Cohn et al., 1946, J. Am. Chem. Soc. 68:459-475 ("Cohn process") or Oncley et al., 1949, J. Am. hem. Soc. 71:541-550 ("Cohn-Oncley process") ApoA-I is separated from the plasma by any known method, such as a low-temperature fractionation process. Other methods for isolating apolipoproteins from plasma include the Ki Cohn and Cohn-Oncl, such as the Stler-Nitschmann process A variation of the ey process is shown in (Nitschmann et al., 1954, Helv. Chim. Acta 37:866-873 (see Kistler et al., 1962, Vox Sang. 7:414-424).

[0139] In these embodiments, the apolipoproteins are activated by cold alcohol, e.g., ethanol. It is obtained by precipitation from plasma with alcohol. The alcohol may be a C1-C6 straight or branched chain alcohol, such as methanol, n-Propanol, isopropanol, n-butanol, sec-butanol, isobutanol In various embodiments, the protein Apolipoproteins are extracted from plasma using agents other than alcohol that reduce the solubility of the proteins. Such drugs include, but are not limited to, ether, ammonium sulfate, 7-ethoxyacridine-3,9-diamine (Rivanol) and The precipitated protein can be precipitated using a method including, but not limited to, polyethylene glycol. However, the supernatant may be separated by any method known in the art, such as precipitation, centrifugation, and filtration. It can be separated.

[0140] ApoA-I can be recovered from any fraction of plasma containing proteins. In some embodiments, the filtrate is purified by precipitation with about 8% (w / w) ethanol. ApoA-I is recovered from the serum fraction of human plasma in which the amount of fibrinogen has been reduced. In an embodiment, other serum proteins are removed by precipitation with about 25% (w / w) ethanol. Human plasma with reduced concentrations of globulins (e.g., β-globulin and γ-gamma globulin) In yet another embodiment, the apolipoprotein is recovered from the serum fraction of ethanol. The results were obtained by increasing the concentration of ethanol from about 38% (w / w) to about 42% (w / w). Apolipoproteins are recovered as precipitates from the human serum. was obtained from human serum by increasing the ethanol concentration to approximately 40% (w / w). The apolipoproteins are then recovered as a precipitate (Cohn's fraction IV). The precipitate may be precipitated by any method known in the art, including, but not limited to, centrifugation and filtration. The ApoA-I can be recovered from the serum fraction.

[0141] In some embodiments, the temperature of the plasma fraction from which the apolipoproteins are recovered is It is sufficiently low so as to prevent denaturation of the protein. The temperature of the ApoA-I fraction is in the range of about -10°C to about 0°C, for example, about -8°C to about -2°C. In various embodiments, the pH of the plasma fraction from which ApoA-I is recovered is In these embodiments, the ApoA-I-containing protein is in a range that prevents the degradation of the protein. The pH of the fraction is in the range of about 5 to about 7, for example, about 5.5 to about 6.5.

[0142] 6.1.4. Improved Lipoprotein Purification Process Applicants have produced lipoproteins that are mature, intact, and substantially free of contaminants. We have further discovered an improved purification process which is described below and illustrated in the Examples. The purification methods described herein can be carried out at any scale convenient to one of skill in the art. Cut.

[0143] The method includes, but is not limited to, ApoA-I, -II, -III or -I V; ApoB48 and ApoB100; ApoC-I, -II, -III or -IV Applies to any apolipoprotein, such as ApoD; ApoE; ApoH; ApoJ More specifically, the present disclosure relates to a method for obtaining highly purified ApoA-I. In some embodiments, the ApoA-I is, but is not limited to, Genbank accession numbers NP_000030, AAB59514, P02647, C A sequence selected from the sequences set forth in AA30377, AAA51746 and AAH05380.1. In certain embodiments, ApoA-I is a human protein having a sequence represented by the formula: In other embodiments, the protein of the present disclosure is a human protein as described above in Section 6.1.2. The methods can be used to culture non-human animals (see, e.g., U.S. Patent Application Publication No. 2004 / 0077541). see for example, cattle, horses, sheep, monkeys, baboons, goats, rabbits, dogs, hares, Mice, badgers, mice, rats, cats, guinea pigs, hamsters, ducks, chickens , salmon and eels (Brouillette et al., 2001, Biochim. Biophys. Acta. 1531:4-46; Yu et al., 1991, Cell Struct. Funct. 16(4):347-55; Chen and Albers, 1983, Biochim Biop hys Acta. 753(l):40-6;Luo et al., 1989, J Lipid Res. 30(11): 1735-46;Blaton et al., 1977 , Biochemistry 16:2157-63; Sparrow et al., 1995, J Lipid Res. 36(3):485-95; Beaubatie et al., 1986, J. Lipid Res. 27: 140-49;Januzzi et al., 1992, Genomics 14(4): 1081-8;Gou Linet and Chapman, 1993, J. Lipid Res. 34(6):943-59; Collet et al., 1997, J. Lipid Re s. 38(4):634-44; and Frank and Marcel, 2000, J. Lipid Res. 41(6):853-72). The ApoA-I obtained from the above can be purified.

[0144] Examples of ApoA-I proteins that can be purified by the methods disclosed herein are: Examples include, but are not limited to, the preproapolipoprotein form of ApoA-I; Pro- and mature forms of ApoA-I, as well as active polymorphs, isoforms, variants and and mutant and truncated forms, e.g., ApoA-I M , ApoA-I Z and ApoA-I P Apolipoprotein A, which contains a cysteine ​​residue. -I mutants are also known and can be purified by the methods described herein (e.g., See, for example, U.S. Patent Application Publication No. 2003 / 0181372. The ApoA-I for use in the method of the present invention may be a monomer, homodimer, or heterodimer. For example, the pro- and mature ApoA-I forms can be prepared in the form Monomers and heterodimers include, in particular, ApoA-I (Duverger et al., 1996, Arterioscler Thromb Vasc Biol. 16(12): 1424-29), ApoA-I M (Franceschini et al., 1985, J Bio l Chem. 260: 1632-35), and ApoA-I P (Daum et al., 1999, J Mol Med. 77:614-22 ) are mentioned.

[0145] In some embodiments, the ApoE purified by the methods described herein. The AI ​​protein is at least 75% identical to amino acids 25 to 267 of SEQ ID NO:1, At least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical 1. At least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, They have an amino acid sequence that is at least 99% identical or at least 100% identical.

[0146] Apolipoproteins can be expressed in any manner, such as plasma or recombinantly expressed in prokaryotic or eukaryotic cells. In certain embodiments, the apolipoprotein may be derived from , ApoA-I, e.g., human ApoA-I. The AI ​​is expressed in the cytoplasm or periplasm of a prokaryotic or eukaryotic host cell. In one embodiment, the cells are disrupted to release ApoA-I into the supernatant, and then ApoA -I. Methods for disrupting cells are well known in the art. Exemplary methods for disrupting cells include: These include, but are not limited to, enzymatic, ultrasonic, detergent, and mechanical methods. In certain preferred embodiments, ApoA-I is produced in mammalian cells, preferably is expressed in CHO cells and secreted into the growth medium. ApoA-I is purified from the clarified cell-free medium. The purification method is related to human ApoA-I. Although described in detail herein as specific proteins, which can be readily ascertained by one of ordinary skill in the art, Depending on the protein properties (e.g., molecular weight, isoelectric point, Stokes radius, hydrophobicity, polymeric state, etc.), The purification conditions were then adjusted to those for other apolipoproteins, as well as non-human ApoA-I and ApoA-I. Polymorphisms, isoforms, variants, mutants and truncated forms or other It is understood that adaptation to apolipoproteins is within the skill of the art. It is understood.

[0147] In general, the purification method comprises: (a) combining an ApoA-I containing solution with an anion exchange matrix; (b) contacting the virus under conditions in which ApoA-I does not bind to the matrix; through a membrane having a pore size sufficient to remove germs or virus particles in step (a). (c) filtering the resulting ApoA-I-containing solution; The filtrate from step (b) is subjected to a first reverse phase chromatograph under conditions such that the filtrate binds to (d) passing the first eluate through a column of increasing concentrations of organic solvent. eluting a first ApoA-I-containing reversed-phase eluate from the reversed-phase chromatography matrix; (e) subjecting ApoA-I to binding to the matrix; The first ApoA-I reversed-phase eluate from step (d) was then loaded onto a second reversed-phase chromatography column. and (f) passing the second solution through a gradient of increasing concentrations of organic solvent. A second ApoA-I-containing reversed-phase eluate is eluted from the reversed-phase chromatography matrix. The order in which the steps are performed is not important. As such, various permutations in the order of the steps are possible, some of which are described below. will be done.

[0148] In a particular embodiment, the ApoA-I containing solution is anionized in step (a). The protein solution is conditioned before contacting it with the ion-exchange matrix. to the extent that ApoA-I does not bind to the anion exchange matrix in step (a) (i.e., the protein has no net negative charge and step (a) is negative In these embodiments, the present invention is implemented to adjust the The pH of the ApoA-I-containing solution is about 5 to about 7, preferably about 5.0 to about 5.6. In a particular embodiment, the pH is about 5.1 to about 5.5. The pH is about 5.3. The pH is adjusted by adding a suitable acid ( For example, hydrochloric acid) or a base (for example, sodium hydroxide) can be added. In some embodiments, the ApoA-I containing solution can be In another embodiment, the conditioning step is If no, the ApoA-I containing solution is optionally filtered prior to step (a), Cells and cell debris are removed.

[0149] In some embodiments, a solution containing ApoA-I and an anion exchange matrix are The contacting step (a) may include passing a protein solution through a chromatography column. In these embodiments, the distance is about 10 cm to about 50 cm, preferably Preferably, the bed height is about 10 cm to about 30 cm, and more preferably about 20 The column is packed to a bed height of about 10 g per liter. about 50 g, for example, about 10 g to about 30 g, for example, about 25 g to about 35 g of ApoA-I A protein solution containing the following is loaded onto the column: The protein solution containing up to about 32 g of ApoA-I per column is loaded onto the column. In an embodiment, step (a) is carried out in a batch mode, i.e., by adding an anion exchange matrix. Add the matrix to the protein solution in the flask to allow the contaminants to bind to the matrix. After mixing for a sufficient period of time, the protein solution is separated, for example, by filtration or centrifugation. In certain embodiments, the method is performed by separating the matrix material from the The protein solution is filtered to remove any particles in the solution, and then it is placed on an anion exchange matrix. and bring it into contact with.

[0150] Anion exchange matrices for use in step (a) of the methods described herein The anion exchange matrix may be any anion exchange matrix known in the art. The ion exchange matrix may be, but is not limited to, Q-Sepharose F. F, Q-Spherosil, DEAE-Sepharose FF, Q-Cellul Examples of such cellulose acetate polymers include cellulose acetate, DEAE-cellulose, DEAE-cellulose, and Q-spherodex. In certain embodiments, the anion exchange matrix is ​​Q-Sepharose FF. (GE Healthcare). In certain embodiments, the Before contacting the protein solution with the anion exchange matrix, ApoA-I was allowed to react with the matrix. In a buffer having a pH within the preferred ranges discussed above such that the Before step (a), equilibrate the anion exchange matrix. Buffers useful for equilibrating and for carrying out step (a) are well known to those skilled in the art. In a particular embodiment, the buffer is TAMP A (20 mM phosphate buffer). sodium, pH 5.3).

[0151] In various embodiments, step (a) comprises binding to an anion exchange matrix, This allows separation of ApoA-I from the matrix-bound ApoA-I under the above pH and salt conditions. Proteins other than ApoA-I (e.g., host cell proteins), host cell DNA, In some embodiments, the ApoA-I is purified with respect to A and endotoxin. In the case of a 200 mg / mL ... In either case, 85%, or at least 90%, or more, is recovered from the anion exchange step.

[0152] In various embodiments, the purification method comprises the steps of: capturing viruses and viral particles; The ApoA-I lysate from step (a) is then filtered using a filter having a pore size sufficient for the (b) filtering the liquid, optionally to remove viruses or viral particles. and (b) filtering the mixture through a membrane to obtain a final product, rather than after (a). In a particular embodiment, the anion exchange matrix is ​​subjected to step (f) followed by step (f). The pH of the eluate from the virus filtration medium is adjusted to 0.1 by the addition of sodium hydroxide or other suitable base. The ApoA-I containing solution from step (a) is mixed with about 7 ml of water prior to the step (b). In a particular embodiment, the ApoA-I-containing solution is adjusted to a pH of about 0.8 to about 8.2. The filter used in step (b) is used to remove the virus. Any filter having a suitable pore size for trapping, for example, a pore size of about 15 nm to about 75 nm, may be used. In certain embodiments, the pore size of the filter is about 20 nm. (For example, Planova 20N, Asahi Kasei Medical). The flow rate of the protein solution through the filter determines the properties of the solution (e.g., its viscosity, particle concentration, etc.). Typical flow rates for virus filtration are about 12 .5L / h / m2, but the flow rate is higher to maintain a filter pressure below 1 bar. The filtrate from step (b) may be adjusted to a lower or higher concentration. In certain embodiments, the recovery of ApoA-I from the viral filtration step is at least 80% of the amount of ApoA-I in the anion exchange eluate from step (a). , at least 85%, at least 90%, at least 95%, or at least 98% It is.

[0153] In certain embodiments, the purification methods described herein further comprise the step of: under buffer and salt conditions that allow binding of apolipoprotein AI to the matrix. Step (c) after step (b) of passing the mixture through a first reverse phase chromatography column In these embodiments, ApoA-I is treated with a gradient of increasing concentrations of organic solvent. The sequences are used to identify host cell DNA, host cell proteins, endotoxins and truncated forms of Purify with respect to the form of the polymer. C18 Hydrophobic ligand-grafted cross-linked agarose-based media known in the art Reversed phase chromatography can be performed using a variety of matrix variants. Commercially available hydrophobic matrices useful in the methods described herein include, but are not limited to, However, Butyl Sepharose-FF, Octyl Sepharose ose-FF, Dianon HP20ss, C18 Hypersil and Sour ce 30RPC. In a particular embodiment, the enzyme used in step (c) The matrix used is Source 30RPC (GE Healthcare). In certain embodiments, the reverse phase chromatography column is about 10 cm to about 50 cm, e.g. For example, the bed height is about 10 cm to about 30 cm. The lithography column has a bed height of about 25 cm.

[0154] In some embodiments, the ApoA-I filtrate from the viral filtration step (b) at a concentration of about 1 to about 20 g of ApoA-I per liter, for example, about 1.5 g to about At a concentration of about 5 g of ApoA-I, and more preferably about 2.5 g to about 3.5 g of ApoA -I. In a particular embodiment, the eluate from step (b) is loaded onto the reverse phase column at a concentration of -I. The ApoA-I filtrate is loaded onto a reverse phase column at a concentration of approximately 3.4 g per liter. The reversed-phase column was equilibrated before loading ApoA-I, and the protein was allowed to react with the column upon loading. The buffer conditions that can be used to ensure binding to the enzyme are well within the skill of the art. Preferably, the column equilibration buffer maintains the pH of the column at about 9.5. In certain embodiments, the equilibration buffer is a strong buffer that can be reduced by The buffer is TAMP D (20 mM ammonium carbonate). , the ApoA-I-containing filtrate from step (b) (pH of about 8.0) was stirred at 0.5° C. for 1 min. Column at a flow rate of 5 cm to about 5.0 cm, for example, about 2.0 cm to about 4.0 cm per minute. In certain embodiments, the ApoA-I containing filtrate is loaded onto the column at a rate of about 2 Load onto the column at a flow rate of .8 cm.

[0155] After binding of ApoA-I to the reverse phase matrix in step (c), buffer A gradient of increasing concentrations of organic solvent in TAMP D buffer, e.g., from about 35% to about Proteins are eluted in step (d) by exposure to 50% acetonitrile. In some embodiments, the incubation time is about 60 to about 90 minutes, for example, about 70 minutes or more. A linear gradient of about 35% to about 50% acetonitrile over a period of about 80 minutes is used. ApoA-I can be eluted from the column. In certain embodiments, a linear gradient This is followed by an isocratic elution using 50% acetonitrile for approximately 10 minutes. The exact conditions for eluting ApoA-I from the rum can be readily ascertained by one skilled in the art. In various embodiments, about 60%, e.g., about 65%, about 70%, About 75% or about 80% or more of ApoA-I is present in the column eluate in step (d). exists.

[0156] In certain embodiments, the purification methods described herein further comprise, after step (d), , host cell proteins and truncated forms of Ap derived from the full-length protein To further remove oA-I, a second reversed-phase chromatography column was used in step (d (e) passing an apolipoprotein AI reverse phase eluate from Preferably, the reversed phase eluate from step (d) is subjected to purification by subjecting apolipoprotein AI to maturation. and loading the second reversed-phase column under conditions that allow binding to the glycerol in step (e). The reversed phase matrix used is of the same type as that used in step (d). It may be a matrix or may be a matrix of a different kind. In an embodiment, the reversed phase matrix used in step (e) is C18. Rica matrix, e.g., Daisogel SP-300-BIO C18 matrix The slides were then cut with a 300 Å cutter slide (300 Å, 10 μm; Daiso Co., Ltd.). Equilibrate the C18 column to ensure that proteins bind to the column upon loading. Buffer conditions that can be used to achieve this can be readily ascertained by one of skill in the art. The column equilibration buffer is a strong buffer that can lower the pH of the column to about 9.5. In a particular embodiment, the equilibration buffer is TAMP E(10 0 mM ammonium carbonate). In various embodiments, the purification methods described herein The reversed-phase column used in step (e) of the method can be from about 10 cm to about 50 cm, e.g. In certain embodiments, the bed height is about 10 cm to about 30 cm. The reversed phase column used in e) has a bed height of about 25 cm.

[0157] In various embodiments, the ApoA-I eluate from step (d) is diluted to 1 liter For example, about 1 g to about 10 g of ApoA-I is present at a concentration of about 0.5 g to about 30 g of ApoA-I per 100 g of ApoA-I. and more preferably at a concentration of about 4 g to about 5 g of ApoA-I. 8 reverse phase column. In certain embodiments, the Apo from step (d) is The AI ​​eluate was loaded onto a C18 column at a concentration of approximately 4.7 g ApoA-I per liter. Preferably, after equilibration, the ApoA-I-containing eluate from step (d) is About 0.5 cm to about 5.0 cm per minute, for example, about 1.0 cm to about 3.0 cm per minute In certain embodiments, the ApoA-I containing filtrate is loaded onto the column at a flow rate of 100 rpm. , and loaded onto the column at a flow rate of approximately 2.1 cm per minute.

[0158] After binding of ApoA-I to the reverse phase matrix in step (e), buffer A gradient of increasing concentrations of organic solvent in E buffer, e.g., from about 40% to about 100% TAMP in E buffer. 50% acetonitrile is used to elute the protein in step (f). In some embodiments, a linear gradient of about 40% to about 50% acetonitrile is used to obtain a 20% acetonitrile solution. From the column for a period of 40 to about 80 minutes, for example, about 50 minutes, about 60 minutes, or about 70 minutes. In a particular embodiment, ApoA-I is eluted from the TAMP for about 60 minutes. A linear gradient of approximately 40% to approximately 50% acetonitrile in E buffer was used to perform reversed-phase matrix chromatography. In a particular embodiment, the ApoA-I is eluted from the column by a linear gradient followed by a 50% Isocratic elution is performed using acetonitrile for about 15 minutes. The exact conditions for eluting the AI ​​can be readily ascertained by one of skill in the art. In the above, about 60%, for example, about 65%, about 70%, about 75% or Approximately 80% or more of the ApoA-I is recovered in the column eluate in step (f).

[0159] In certain embodiments, the organic solvent is obtained in step (f) of the method described herein. The solvent is removed from the eluate containing ApoA-I. The solvent removal may be performed by, but is not limited to, Concentrating the ApoA-I-containing eluate obtained in step (f) and dissolving the concentrate in an aqueous buffer. This can be accomplished by any method known in the art, such as by diafiltration into a buffer. In certain embodiments, the eluate from step (f) can be purified by the steps of step (f). Compared to the volume of the eluate from the reverse phase column in The eluate is concentrated 5-fold, about 4-fold, about 4.5-fold, or about 5-fold. After about 2.5-fold concentration, about 10, 15 or 20, preferably 15 volumes of a suitable aqueous buffer solution are added. Suitable aqueous buffers are known in the art. A preferred buffer is TAMP C (3 mM sodium phosphate, pH 8.0).

[0160] In some embodiments, the order of the chromatography columns is reversed.

[0161] Optionally, after concentration and buffer exchange, the aqueous ApoA-I solution is subjected to negative mode (i.e., under conditions where ApoA-I does not bind to the anion exchange matrix) Further purification by ion exchange chromatography to remove residual DNA and other negatively charged (g) removing any residual contaminants, such as host cell proteins. In an embodiment, the anion exchange step is carried out in batch mode. In some cases, the anion exchange step is carried out by column chromatography. Suitable anion exchange matrices for use in column or formalin chromatography Examples of the gels include, but are not limited to, Q-Sepharose-FF or Step Any of the anion exchange matrices discussed above for use in (a) may be used. In certain embodiments, anion exchange membranes, e.g., membranes with high surface area and strong cationic Membranes with ionic charges, such as Sartobind Q or Mustang QA The anion exchange step is carried out by passing a solution of poA-I through the anion exchange step. , using Mustang Q anion exchange membrane (Pall Life Sciences). In certain embodiments, the anion exchange step is carried out using any suitable acid. Prior to this anion exchange step, the pH of the aqueous ApoA-I solution is adjusted to about 5.5 or about 6.0. or to about 6.5. In certain embodiments, dilute phosphoric acid is used to reduce the aqueous ApoA-I The pH of the solution is lowered to about 6.0. In a preferred embodiment, the ApoA-I solution is , and passed through a Mustang Q cartridge at approximately 12.5 L / m2 / h.

[0162] In some embodiments, the anion exchange membrane filtrate is concentrated and, optionally, diluted for storage. or by complexation with lipids as described in Section 6.5.1 below and / or Further processing of ApoA-I, such as formulation into pharmaceutical compositions, as described in Section 6.6 of the present invention, is also possible. Diafilter to exchange the solvent to one suitable for cutting. Suitable buffers for further processing of ApoA-I are readily available to those skilled in the art. In certain embodiments, purified ApoA-I is incubated with TAMP C buffer. The membrane is then replaced with a new membrane that allows the buffer to pass through but not the protein. , any of the above, as long as it has a molecular weight cutoff that is below the molecular weight of full-length mature ApoA-I. Ultrafiltration membranes can be used in this step. In certain embodiments, 10,0 Polyethersulfone membrane with a nominal molecular weight cutoff of 0.00 (e.g., Filtron Omega series). Preferably, the ApoA-I concentration in the solution after ultrafiltration is , at least 10g / L, at least 12g / L, at least 15g / L, at least 2 0g / L, at least 25g / L, at least 30g / L, at least 35g / L, at least at least 40 g / L, at least 45 g / L or at least 50 g / L.

[0163] 6.1.5. Apolipoprotein Products The present disclosure also provides substantially pure mature full-length apolipoproteins. The term "substantially pure" as used herein refers to a protein that is at least 95% pure. In certain embodiments, a substantially pure protein is at least 96%, at least At least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.99% or 100% pure. In certain embodiments, the purified The substantially pure apolipoprotein product produced by the method is photochromatic against a white background. When visually examined with a light source, the particles appear transparent to slightly opalescent and contain no visible particles. In various embodiments, the method described in Section 6.1.4 above is used to prepare a solution of color. The substantially pure apolipoprotein obtained or obtainable is described in further detail below. In order to detect the presence of small or undetectable amounts of one or more host cell DNAs, apolipoproteins Non-protein proteins (e.g., host cell proteins), endotoxins, residual solvents, and Contains low bioburden (i.e., low numbers of microorganisms on or in the sample) The purity of the apolipoprotein product may include, but is not limited to, the presence or absence of the N-terminal Edman sequence. Sequence determination, MALDI-MS, gel electrophoresis, HPLC and / or immunoassays. It may be determined by any method known in the art.

[0164] In various embodiments, the substantially pure apolipoprotein obtained by the methods described herein is The lipoprotein product is the full-length mature human A1 having a mass of approximately 28.1 kilodaltons. The mass of ApoA-I in the product is, but is not limited to, MA It can be determined by any method known in the art, such as LDI-MS. In an embodiment, at least 75% of the ApoA-I protein in the product is At least 80%, at least 85%, at least 90%, at least 91%, at least 92% , at least 93%, at least 94%, at least 95%, at least 96%, at least At least 97%, at least 98%, or at least 99% of the total ApoA-I was mature full-length (e.g., For example, ApoA-I comprising amino acids 25 to 267 of SEQ ID NO: 1. In some cases, the substantially pure ApoA-I product is about 15% by weight or less, about 10% by weight or less, About 5% by weight or less, about 4% by weight or less, about 3% by weight or less, about 2% by weight or less, or about 1% by weight or less The following N-terminally extended ApoA-I isoforms (eg, proApoA-I): As will be appreciated by those of skill in the art, any N-terminally extended ApoA-I in the product may be administered. Upon administration, ApoA-I is rapidly converted to mature ApoA-I in the blood. The poA-I product is about 25% by weight or less, about 20% by weight or less, about 15% by weight or less, about 10% by weight or less Weight% or less, about 5% by weight or less, about 4% by weight or less, about 3% by weight or less, about 2% by weight or less, about 1 % by weight or less, about 0.75% by weight or less, about 0.5% by weight or less, about 0.25% by weight or less, The truncated form of ApoA-I is about 0.1% or less by weight. The amount of extended or expanded ApoA-I can be determined, for example, by N-terminal Edman sequencing and / or or MALDI-MS and / or relative intensity to the total intensity of all bands, if present SDS-PAGE gel analysis to determine the ratio of the intensities of the purified ApoA-I band regions In various embodiments, ApoA can be determined by electrophoresis and scanning of the gel. -I product is about 20% by weight or less, about 10% by weight or less, about 5% by weight or less, about 4% by weight or less , about 3% by weight or less, about 2% by weight or less, about 1% by weight or less, about 0.75% by weight or less, about 0.5 % by weight or less, about 0.25% by weight or less, or about 0.1% by weight or less of the oxidized form of ApoA- I, especially, Met 112 Rank and / or Met 148 Contains ApoA-I oxidized at position nothing.

[0165] In certain embodiments, the substantially pure α-aminobutyric acid produced by the methods described herein is Polypoproteins are less than about 100 ppm (e.g., ng / mg), e.g., less than about 75 ppm Less than m, Less than about 50 ppm, Less than about 40 ppm, Less than about 30 ppm, Less than about 20 ppm, or less than about 10 ppm of host cell protein. The substantially pure apolipoprotein product contains less than about 20 ppm of host cell protein. More preferably, the apolipoprotein product comprises less than about 10 ppm of the host cell. The presence and amount of host cell proteins in the apolipoprotein sample is The apolipoprotein can be determined by any method known in the art. For example, when recombinantly produced in mammalian cells, a commercially available ELISA kit (e.g., Cy The host cell tandem was transformed with the GFP-specific GFP-specific GFP gene using Kit F015 from GNUs Technologies. Proteins can be detected and their levels quantified.

[0166] In some embodiments, the substantially pure apolipoprotein purified as described herein is The apolipoprotein product is less than about 50 pg / mg of apolipoprotein, e.g., less than about 40 pg / mg of apolipoprotein. Less than g / mg, less than about 30 pg / mg, less than about 20 pg / mg, less than about 10 pg / mg, or less than about 5 pg / mg apolipoprotein. In a preferred embodiment, the substantially pure apolipoprotein product is obtained by eluting 1 mg of apolipoprotein. The apolipoprotein sample contains less than about 10 pg of host cell protein per apolipoprotein. The presence and amount of host cell DNA in the sample can be determined by real-time PCR or, preferably, quantitative PCR. Using anti-SSB antibody (Glycotype Biotechnology) by CR Determined by any method known in the art, such as detection of complexes with single-stranded binding proteins. It is possible.

[0167] In certain embodiments, the substantially pure α-aminobutyric acid produced by the methods described herein is Apolipoprotein products contain less than about 0.5 EU per mg of apolipoprotein, e.g. For example, less than about 0.4 EU, less than about 0.3 EU, less than about 0.2 EU or less than about 0.1 EU. Preferably, the substantially pure apolipoproteins described herein contain a certain amount of endotoxin. The product contains less than about 0.1 EU of endotoxin per mg of apolipoprotein. Detection and quantification of endotoxins can be performed using, for example, Limulus Ame for gram-negative bacterial endotoxins. LAL quantitative test (Cambrex; sensitivity 0.125EU) This can be accomplished by any method known in the art using a 100% ethanolamine solution (100 / mL).

[0168] The substantially pure apolipoprotein products described herein have low bioburden. The term "bioburden" refers to the amount of aerobic bacteria, anaerobic bacteria, yeast and In various embodiments, the purified substance as described herein is The bioburden of the essentially pure apolipoprotein product is approximately 1 CFU per mL. Bioburden testing can be performed according to any known method, for example, the European Pharma This can be done according to the acopoeia Chapter 2.6.12.B and USB Chapter 61 uniform methods. Cut.

[0169] The substantially pure apolipoprotein products described herein contain small amounts of residual solvent. In certain embodiments, the residual solvent is less than about 10 mg / L of apolipoprotein. , less than about 50 ppm, less than about 45 ppm, less than about 40 ppm, less than about 35 ppm, less than about 30 Less than ppm, less than about 25 ppm, less than about 20 ppm, less than about 15 ppm or less than about 10 ppm m. Preferably, the residual solvent is present in an amount less than 10 mg / L of apolipoprotein A. The amount of residual solvent is not limited. However, it can be assayed by any method known in the art, such as GC-MS and HPLC. It is possible to do so.

[0170] Preferably, the apolipoprotein is ApoA-I (e.g., amino acid 2 of SEQ ID NO:1). In some embodiments, the mature human ApoA-I comprises ApoA-I 5 to 267. The oA-I protein contains an aspartate amino acid sequence at position 1 (i.e., the position corresponding to position 25 of SEQ ID NO:1). It has an amino acid sequence containing arginic acid.

[0171] 6.2. Lipid fraction The lipoprotein complexes and compositions of the present disclosure include a lipid fraction. In various embodiments, the lipid or lipids include saturated and The lipid fraction may be a mixture of saturated and / or unsaturated lipids and natural or synthetic lipids. Contains at least one phospholipid.

[0172] Suitable lipids that may be present in the lipid fraction include, but are not limited to, small molecule lipids. Low alkyl chain phospholipids, egg phosphatidylcholine, soybean phosphatidylcholine, dipalmitoyl Dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, Phosphatidylcholine 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl Lumitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoyl 1-Stearoyl-2-palmitoylphosphatidylcholine, 1-Stearoyl-2-palmitoylphosphatidylcholine , Dioleoylphosphatidylcholine Dioleophosphatidylethanolamine, Dioleoylphosphatidylcholine Phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, Sphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol diphosphatidylglycerol, e.g. dimyristoylphosphatidylglycerol , dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol Cerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid , dipalmitoyl phosphatidic acid, dimyristoyl phosphatidylethanolamine, Dipalmitoylphosphatidylethanolamine, Dimyristoylphosphatidylserine , dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin palmitoyl sphingomyelin, dipalmitoyl sphingomyelin, egg sphingomyelin Sphingomyelin, milk sphingomyelin, phytosphingomyelin, distearoyl Phingomyelin, dipalmitoyl phosphatidylglycerol salt, phosphatidic acid, Galactocerebroside, ganglioside, cerebroside, dilaurylphosphatidylcholine , (1,3)-D-mannosyl-(1,3) diglyceride, aminophenyl glycoside , 3-cholesteryl-6'-(glycosylthio)hexyl ether glycolipid, and cholesteryl Synthetic lipids, such as synthetic palmitoyl sulphate, N-palmitoyl-4-hydroxysphinganine-1-phosphocholine (a form of phytosphingomyelin) can be used to minimize lipid oxidation. The phospholipid fraction containing palmitoylsphingomyelin may be limited in some cases. Not so, but lysophospholipids, sphingomyelins other than palmitoylsphingomyelin , galactocerebrosides, gangliosides, cerebrosides, glycerides, triglycerides and small amounts of any type of lipid, such as cholesterol and its derivatives. stomach.

[0173] In a preferred embodiment, the lipid fraction is composed of two types of phospholipids: sphingomyelin ( SM contains phospholipids and negatively charged phospholipids. SM has a net charge of about 0 at physiological pH. The identity of the SM used is not critical to success. Thus, as used herein, the expression "SM" refers to any substance that is derived or taken from a natural source. Like the isolated sphingomyelin and natural SM, it was not affected by hydrolysis by LCAT. SM includes analogs and derivatives of natural SM that are not affected by lecithin. SM is structurally very similar to lecithin. It is a similar phospholipid, but unlike lecithin, it does not have a glycerol backbone and therefore is It does not have an ester bond connecting the selenium chain. Rather, SM has a ceramide backbone and an amino acid. A carboxyl bond connects the acyl chains. SM is not a substrate for LCAT and is generally It cannot be hydrolyzed. However, it may act as an inhibitor of LCAT. Alternatively, LCAT activity can be reduced by diluting the concentration of the substrate phospholipid. Because SM cannot be hydrolyzed, it remains in the circulation longer. As a result, the negatively charged lipoprotein complexes described herein provide a longer lasting physiological It has a more specific effect (cholesterol transfer) than the apolipoprotein complexes that do not contain SM. It is expected that the enzyme will be able to pick up more lipids, especially cholesterol. is required for therapy compared to that required for SM-free lipoprotein complexes. This can result in less frequent or lower doses being administered.

[0174] SM can be obtained from virtually any source. For example, SM can be obtained from milk, eggs, Alternatively, SM analogs or derivatives may be used. Non-limiting examples of useful SM analogs and derivatives include, but are not limited to, palmitoyltransferase (SMT), ... Irradiol sphingomyelin, N-palmitoyl-4-hydroxysphinganine-1-phosphingamylein Phospholin (a form of phytosphingomyelin), palmitoylsphingomyelin, sphingomyelin Thearylsphingomyelin, D-erythro-N-16:0-sphingomyelin and and its dihydro isomer, D-erythro-N-16:0-dihydro-sphingomyelin. Synthetic SM, such as synthetic palmitoyl sphingomyelin or N-palmitoyl sphingomyelin. Toyl-4-hydroxysphinganine-1-phosphocholine (phytosphingomyelin ) to produce fewer contaminants and / or oxidation products than sphingolipids of animal origin. In this way, a more uniform composite containing

[0175] Exemplary sphingomyelins include palmitoylsphingomyelins. elin and phytosphingomyelin are shown below.

[0176] [ka]

[0177] Sphingomyelin isolated from natural sources is synthesized by cleaving certain saturated or unsaturated acyl chains. For example, milk sphingomyelin (Av anti Phospholipid, Alabaster, Ala.) is a long saturated amine. Characterized by an acyl chain (i.e., an acyl chain having 20 or more carbon atoms). Egg sphingomyelin contains short saturated acyl chains (i.e., less than 20 carbon atoms). For example, only about 20% of milk sphingomyelin is characterized by C1 Approximately 80% of egg sphingomyelin contains 6:0 (16 carbon, saturated) acyl chains. Using solvent extraction, the composition of milk sphingomyelin was compared with that of egg sphingomyelin. can be enriched to have an acyl chain composition comparable to that of sphingomyelin or vice versa.

[0178] The SM may be semi-synthetic so that it has specific acyl chains. For example, milk Sphingomyelin is first purified from milk and then modified to remove certain acyl chains, e.g., C1 The 6:0 acyl chain can be cleaved and replaced with a different acyl chain. SM can also be used, e.g. For example, on January 15, 1993, The paper, "Synthesis of D-erythro-sphingomyelinase," was published. No. 5,220,043 to Dong et al., entitled "Methods of Fluorescence Imaging in Polymeric Materials," Weis, 1999, Chem. Phys. . See Lipids 102(l-2):3-12.

[0179] Selectively vary the length and saturation level of the acyl chains containing semi-synthetic or synthetic SMs The acyl chain may be saturated or unsaturated and may contain from about 6 to about 24 carbon atoms. Each chain may contain the same number of carbon atoms, or alternatively, Each chain may contain a different number of carbon atoms. Synthetic or synthetic SMs are mixed acyl groups in which one chain is saturated and one chain is unsaturated. In such mixed acyl chain SMs, the chains may be of the same or different lengths. In other embodiments, the acyl chains of the semi-synthetic or synthetic SM may both be saturated. Either both are saturated or both are unsaturated. Again, the chains may have the same or different numbers of carbon atoms. In some embodiments, the acyl group may include a semi-synthetic or synthetic SM. Both chains are identical. In certain embodiments, the chains are, for example, oleic acid, palladium, oleic acid ... In another embodiment, the acyl chains correspond to those of naturally occurring fatty acids such as glycerol or stearic acid. In another specific embodiment, SM having a saturated or unsaturated functional chain is used. In these, both acyl chains are saturated and contain 6 to 24 carbon atoms. Non-limiting examples of acyl chains present in commonly occurring fatty acids that may be included in synthetic SMs are as follows: Provided in Table 1.

[0180] [Table 1]

[0181] In a preferred embodiment, the SM is palmitoyl SM, e.g., a C16:0 acyl chain. or a synthetic palmitoyl SM having palmitoyl SM as the main component. It's SM.

[0182] In certain embodiments, a functional SM, such as phytosphingomyelin, is used. can be.

[0183] The lipid fraction preferably comprises negatively charged phospholipids. A "charged phospholipid" is a phospholipid that has a net negative charge at physiological pH. The phospholipids can be a single type of negatively charged phospholipid or two or more different negatively charged phospholipids. In some embodiments, the mixture may include a mixture of charged phospholipids. The main substance is a negatively charged glycerophospholipid. The identity of the charged phospholipid is crucial for success. Specific examples of suitable negatively charged phospholipids include, but are not limited to, However, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glyceryl) lol)], phosphatidylglycerol, phosphatidylinositol, phosphatidyl In some embodiments, negative Charged phospholipids consist of one or more phosphatidylinositols, phosphatidylceramides, phosphorus, phosphatidylglycerol and / or phosphatidic acid. In the form of the negatively charged phospholipid, 1,2-dipalmitoyl-sn-glycero- 3-[phospho-rac-(1-glycerol)], or DPPG.

[0184] Similar to SM, negatively charged phospholipids can be obtained from natural sources or chemically synthesized. In an embodiment using a synthetic negatively charged phospholipid, As discussed above in relation to SM, selectively altering the identity of the acyl chains Some embodiments of the negatively charged lipoprotein complexes described herein can In some embodiments, both acyl chains on the negatively charged phospholipid are identical. In this state, the acyl chains on the SM and negatively charged phospholipids are all identical. In an embodiment, all of the negatively charged phospholipids and / or SMs are C16:0 or or C16:1 acyl chain. In certain embodiments, the fatty acid moiety of SM is primarily , C16:1 palmitoyl. In one particular embodiment, the charged phospholipid and / or the acyl chain of the SM matches the acyl chain of palmitic acid.

[0185] The phospholipids used are preferably at least 95% pure and / or oxidized. Lipids obtained from natural sources preferably contain less Contains polyunsaturated fatty acid moieties and / or fatty acid moieties that are less susceptible to oxidation. The level of oxidation is expressed as milliequivalents of isolated iodine per kg of sample (meq O / k The peroxide value is determined using an iodometric titration method, which provides a peroxide value expressed in grams (abbreviated as g). For example, Gray, JI, Measurement of Lipid Oxidation: A Review, Journal of l of the American Oil Chemists Society, Vol. 55, p. 539-545 (1978);Heaton, F.W. and Uri N., Improved Iodometric Methods for the Determination of Lipid Peroxidation. es, Journal of the Science of food and Agriculture, vol 9. P, 781-786 (1958). Preferably, the level of oxidation, or peroxide levels, is low, e.g., 5me < q O / kg, < 4 meq O / kg, < 3 meq O / kg, or < 2 meq O / kg.

[0186] The lipid fraction containing SM and palmitoylsphingomyelin may be optionally enriched in small amounts. The lipids may include, but are not limited to, lysophospholipids, galactocerebris lipids, and the like. glycerides, triglycerides, and cholesterol. Virtually any type of lipid can be used, including oleyl cobalt and its derivatives.

[0187] If included, such optional lipids typically comprise less than about 15 wt.% of the lipid fraction. In some instances, more optional lipids may be included. In some embodiments, the optional lipid is less than about 10 wt%, about 5 wt% In some embodiments, the lipid fraction comprises less than about 2 wt.%. Contains no optional fats.

[0188] In certain embodiments, the phospholipid fraction is in the range of 90:10 to 99:1, more preferably or egg SM in a weight ratio (SM:negatively charged phospholipid) ranging from 95:5 to 98:2. or palmitoyl SM or phytosphingomyelin and DPPG. In an embodiment, the weight ratio is 97:3.

[0189] The lipoprotein complexes of the present disclosure can be used to treat a variety of conditions, including hydrophobic, lipophilic, and / or immunological disorders, for a variety of therapeutic or diagnostic applications. Alternatively, it may be used as a carrier for delivering non-polar active agents. For this purpose, the lipid fraction may contain, but is not limited to, fatty acids, drugs, nucleic acids, vitamins, and and / or nutrients, Suitable hydrophobic, lipophilic or non-polar active agents are not limited by therapeutic class. For example, analgesics, anti-inflammatory agents, antihelminthic agents, antiarrhythmic agents, antibacterial agents, Antiviral, anticoagulant, antidepressant, antidiabetic, antiepileptic, antifungal, antigout, anti Antihypertensives, antimalarials, antimigraine drugs, antimuscarinic drugs, anti-neoplastic drugs, erectile dysfunction improving drugs, Immunosuppressants, antiprotozoal agents, antithyroid agents, tranquilizers, sedatives, hypnotics, neuroleptics, beta-blockers Discontinuation drugs, cardiac stimulants, corticosteroids, diuretics, anti-Parkinson's drugs, gastrointestinal drugs, histamine Receptor antagonists, keratolytic agents, lipid regulating agents, antianginal agents, cox-2 inhibitors, leukotrienes Antibody inhibitors, macrolides, muscle relaxants, nutritional supplements, nucleic acids (e.g., small interfering RNA), opioids Opioid analgesics, protease inhibitors, sex hormones, stimulants, muscle relaxants, anti-osteoporosis agents, Anti-obesity agents, cognitive function enhancers, anti-urinary incontinence agents, nutritional oils, anti-benign prostatic hyperplasia agents, essential fatty acids, Essential fatty acids, and mixtures thereof.

[0190] Specific non-limiting examples of suitable hydrophobic, lipophilic or non-polar active agents include acetretin, albetin, Indazole, albuterol, aminoglutethimide, amiodarone, amlodipine, phetamine, amphotericin B, atorvastatin, atovaquone, azithromycin, Baclofen, beclomethasone, benzepryl, benzonatate, betamethasone, bical Tanidin, budesonide, bupropion, busulfan, butenafine, calcifediol , calcipotriene, calcitriol, camptothecin, candesartan, capsaicin carbamezepine, carotene, celecoxib, cerivastatin, cetirizine, chlorf Pheniramine, cholecalciferol, cilostazol, cimetidine, cinaridine, cipro Floxacin, cisapride, clarithromycin, clemastine, clomiphene, clomiphene pramine, clopidogrel, codeine, coenzyme Q10, cyclobenzaprine, cyclo Rosporin, danazol, dantrolene, dexchlorpheniramine, diclofenac, Dicoumarol, digoxin, dehydroepiandrosterone, dihydroergotamine, di Hydrotachysterol, dirithromycin, donezepil, efavirenz, eposartan , ergocalciferol, ergotamine, essential fatty acid source, etodolac, etoposide, fuc Amotidine, fenofibrate, fentanyl, fexofenadine, finasteride , fluconazole, flurbiprofen, fluvastatin, fosphenytoin, fluvastatin Liptan, Furazolidone, Gabapentin, Gemfibrozil, Glibenclamide, Glypi Zide, glyburide, glimepiride, griseofulvin, halofantrine, ibuprofen irbesartan, irinotecan, isosorbide dinitrate, isotretinoin, itraconazole Nazole, Ivermectin, Ketoconazole, Ketorolac, Lamotrigine, Lansopra zole, leflunomide, lisinopril, loperamide, loratadine, lovastatin, L-thion Loxin, Lutein, Lycopene, Medroxyprogesterone, Mifepristone, Meflo quinolones, megestrol acetate, methadone, methoxsalen, metronidazole, miconazole , Midazolam, Miglitol, Minoxidil, Mitoxantrone, Montelukast, Nab Meton, nalbuphine, naratriptan, nelfinavir, nifedipine, nisolidine nilutamide, nitrofurantoin, nizatidine, omeprazole, oplerkin, Estradiol, oxaprozin, paclitaxel, paracalcitol, paroxetine , pentazocine, pioglitazone, pizofetin, pravastatin, prednisolone, Robukol, progesterone, pseudoephedrine, pyridostigmine, rabeprazole ru, raloxifene, rofecoxib, repaglinide, rifabutin, rifapentine, Rimexolone, ritanovir, rizatriptan, rosiglitazone, saquinavir, sertraliz sildenafil citrate, simvastatin, sirolimus, spironolactone methotrexate, sumatriptan, tacrine, tacrolimus, tamoxifen, tamsulosin, Lugretin, tazarotene, telmisartan, teniposide, terbinafine, terazosin, Tetrahydrocannabinol, tiagabine, ticlopidine, tirofibran, tizanidine, Topiramate, topotecan, toremifene, tramadol, tretinoin, troglitazo valsartan, venlafaxine, vertepotin, trovafloxacin, ubidecarenone, Rufin, Vigabatrin, Vitamin A, Vitamin D, Vitamin E, Vitamin K, Zafil These are lukast, zileuton, zolmitriptan, zolpidem, and zopiclone. Salts, isomers and derivatives, as well as mixtures of the drugs, may also be used.

[0191] Lipoprotein Complexes The present disclosure relates to a method for the preparation of a medicament for use in a pharmaceutical composition comprising the steps of: The present invention provides a lipoprotein complex comprising a protein fraction and a lipid fraction.

[0192] Generally, the protein fractions are apolipoproteins that provide therapeutic and / or prophylactic benefits. One or more lipid-binding proteins, such as proteins and / or derivatives or analogs thereof The complex may contain a single type of lipid-binding protein or multiple proteins of the same or different types. The lipid-binding protein may include a mixture of two or more different lipid-binding proteins that may be derived from different species. Suitable lipid binding proteins are described above in Section 6.1. The polyprotein complex is preferably a polypeptide that is capable of binding to the antibody in a manner that avoids inducing an immune response against the therapy. In addition, a lipid derived from or identical in amino acid sequence to the animal species to be treated is used. Thus, for the treatment of human patients, lipid-binding proteins of human origin are required. Peptide mimetic apolipoproteins are preferably used in the conjugates of the present disclosure. The use of substances can also reduce or avoid the immune response.

[0193] High purity (e.g., mature, untruncated, unoxidized, deoxyribonucleic acid, It is not amidated, is not contaminated with endotoxins, and / or is not contaminated with other proteins. The use of apolipoproteins (which are often not contaminated with nucleic acids) is a promising treatment for lipoprotein complexes. This is believed to enhance the therapeutic efficacy and / or safety of the protein. The quality fraction may be, in some cases, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or about 0% oxidized methionine-112 or methionine-148, and / or having less than 15%, less than 10%, less than 5%, or about 0% deaminated amino acids. at least 75%, at least 80%, at least 85%, at least 90%, at least 9 5%, at least 96%, at least 97%, at least 98%, at least 99% mature The apolipoprotein may comprise a full-length ApoA-I as described herein. Preferably, the apolipoprotein can be purified according to any of the methods described in Section 6. It can be prepared as described in section 1.4.

[0194] In certain embodiments, the protein fraction is ApoA-I, e.g., subunit 6.1.5. The present invention relates to a method for producing a substantially pure, mature, full-length ApoA-I comprising the steps of: It consists of:

[0195] The lipid fraction may be saturated, unsaturated, natural and synthetic lipids and / or phospholipids. Suitable lipids include, but are not limited to, one or more lipids having low molecular weight. Low alkyl chain phospholipids, egg phosphatidylcholine, soybean phosphatidylcholine, dipalmitoyl Dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, Phosphatidylcholine 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl Lumitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoyl 1-Stearoyl-2-palmitoylphosphatidylcholine, 1-Stearoyl-2-palmitoylphosphatidylcholine , Dioleoylphosphatidylcholine Dioleophosphatidylethanolamine, Dioleoylphosphatidylcholine Phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, Sphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol diphosphatidylglycerol, e.g. dimyristoylphosphatidylglycerol , dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol Cerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid , dipalmitoyl phosphatidic acid, dimyristoyl phosphatidylethanolamine, Dipalmitoylphosphatidylethanolamine, Dimyristoylphosphatidylserine , dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin sphingomyelin, egg sphingomyelin, milk sphingomyelin, phytosphingomyelin, pa Palmitoyl sphingomyelin, dipalmitoyl sphingomyelin, distearoyl Phingomyelin, dipalmitoyl phosphatidylglycerol salt, phosphatidic acid, Galactocerebroside, ganglioside, cerebroside, dilaurylphosphatidylcholine , (1,3)-D-mannosyl-(1,3) diglyceride, aminophenyl glycoside , 3-cholesteryl-6'-(glycosylthio)hexyl ether glycolipid, and cholesteryl terol and its derivatives. SM and palmitoylsphingomyelin The phospholipid fraction containing, optionally, but not limited to, lysophospholipids, palmitoylphospholipids, Sphingomyelin other than tolylsphingomyelin, galactocerebroside, ganglioside Osides, cerebrosides, glycerides, triglycerides, and cholesterol and It may also contain small amounts of any type of lipid, such as derivatives of synthetic palmitoyl sphingomyelin. Sphinganine or N-palmitoyl-4-hydroxysphinganine-1-phosphocholine (F Synthetic lipids such as sphingomyelin (sphingomyelin) are preferred. Further lipids are described in section 6.2 above. Preferably, the lipoprotein complex comprises sphingomyelin. .

[0196] Optionally, the lipoprotein complexes of the present disclosure can be used in a variety of therapeutic or diagnostic applications, including: Such as, but not limited to, fatty acids, drugs, nucleic acids, vitamins, and / or nutrients. The hydrophobic, lipophilic or non-polar active agents can be loaded. Suitable agents are those listed above. This is described in Section 6.2.

[0197] Lipoprotein complexes can be made using any of the methods described herein. Preferably, the complex is prepared as described in Sections 6.5.1 to 6.5.4.

[0198] The lipid and protein fractions of the negatively charged lipoprotein complexes described herein The molar ratio may vary and may depend on, among other factors, the apolipoprotein containing protein fraction. The identity of the protein, the identity and amount of the charged phospholipids comprising the lipid fraction, and the amount of the charged phospholipids The desired size of the apolipoprotein complex depends on the apolipoprotein, such as ApoA-I. The biological activity of is thought to be mediated by amphipathic helices containing apolipoproteins. Therefore, the apolipoprotein fraction with a lipid:apolipoprotein molar ratio was calculated as ApoA-I protein. It is convenient to express the helix in terms of protein equivalents. Therefore, it is generally accepted that ApoA-I contains 6–10 amphipathic helices. Other apolipoproteins are classified according to the number of amphipathic helices they contain. For example, the ApoA-I equivalent based on the disulfide bond can be expressed as a unit. ApoA-I exists as a cross-linked dimer M ApoA-I M Each molecule of ApoA- It contains twice as many amphipathic helices as one molecule of ApoA-I and can therefore be expressed as 2 ApoA-I equivalents. Conversely, peptide apolipoproteins containing a single amphipathic helix can Because the molecule contains 1 / 10 to 1 / 6 the number of amphipathic helices of the ApoA-I molecule, / 10~1 / 6 ApoA-I equivalent. Generally, the lipoprotein complex The lipid:ApoA-I equivalent molar ratio (defined herein as "Ri") is approximately 105:1 In some embodiments, Ri is in the range of about 108:1. For phospholipids, a MW of approximately 650-800 can be used to obtain weight ratios.

[0199] In some embodiments, the lipid:ApoA-I equivalent molar ratio ("RSM") is: In particular, the range is from about 80:1 to about 110:1, for example, from about 80:1 to about 100:1. In the present invention, the RSM for lipoprotein complexes may be about 82:1.

[0200] Various apolipoproteins and / or lipoproteins, including negatively charged lipoprotein complexes The lipid molecules are ionized with a stable isotope (e.g., 13 C. 15 N, 2 H, etc.); Radioactive I Sotopes (e.g. 14 C. 3 H, 125 I, etc.); fluorophores; chemiluminescent agents; and The antibody can be labeled with any detectable marker known in the art, such as an enzymatic marker. Cut.

[0201] In a preferred embodiment, the lipoprotein complex is preferably a mature full-length Apo. The protein fractions that were AI and those that were neutral phospholipids, sphingomyelin (SM) and negatively and a lipid fraction containing charged phospholipids.

[0202] Composition and phase relationship of negatively charged phospholipids with SM containing lipid fraction of lipoprotein complexes. It was found that the amount of the complex affects the homogeneity and stability of the composition containing the complex. As exemplified in the previous section, the lipid fraction is a complex composed of SM and negatively charged phospholipids. The compositions containing the conjugates were more homogeneous in lipid fraction than similar compositions containing DPPC in addition to SM. Yes, it is stable.

[0203] Thus, the complexes of the present disclosure that contain SM and negatively charged lipids preferably Formed in the absence of lecithin to improve homogeneity and stability. Once a homogeneous complex containing the carboxyl group and the negatively charged lipid is formed, additional lipid such as lecithin can be added. Quality can be built in.

[0204] If included, optional lipids typically comprise less than about 15% of the lipid fraction. However, in some instances, more optional lipids may be included. In some embodiments, the optional lipid is less than about 10 wt%, less than about 5 wt%, or In some embodiments, the negatively charged lipoprotein The lipid fraction of the protein complex does not include optional lipids.

[0205] In certain embodiments, the phospholipid fraction is in the range of 90:10 to 99:1, more preferably or in the range of 95:5 to 98:2, for example, a weight ratio of 97:3 (SM: negatively charged phosphorus lipids) containing egg SM or palmitoyl SM or phyto SM and DPPG.

[0206] Some apolipoproteins are synthesized in vivo from certain lipoprotein complexes. (This is the case for the apolipoprotein ApoA-I.) During this process of exchange, apolipoproteins typically migrate along with one or more Because of this property, the negatively charged lipoproteins described herein The lipid complex "seeds" endogenous HDL with negatively charged phospholipids, thereby promoting its transport to the kidney. It is expected that the negative charge will convert them into alpha particles, which are more resistant to removal by the negative charge. Administration of positively charged lipoprotein complexes and compositions described herein increases serum levels of HDL. Increase levels and / or alter the half-life of endogenous HDL and endogenous HDL metabolism. This is expected to result in alterations in cholesterol metabolism and reverse lipid transport. It is expected.

[0207] As illustrated in the Examples section, the weight ratio of ApoA-I:SM and DPPG phospholipids Compositions containing a complex having a weight:weight ratio of about 1:2.7 are more effective than similar compositions having other weight:weight ratios. Thus, the present disclosure provides a method for preparing a complex having a protein:lipid weight ratio of 100% or more. The lipoprotein composition is optimized for the formation of a homogenous population. The ratios are given for the complexes of ApoA-I, SM and DPPG, as well as for the complexes of similar molecular weights. For the complex of components, the range is 1:2.6 to 1:3, optimally 1:2.7. In certain embodiments, the ratio of the ApoA-I protein fraction to the lipid fraction is typically is in the range of about 1:2.7 to about 1:3, with 1:2.7 being preferred. This corresponds to a molar ratio of ApoA-I to phospholipids in the range of 1:140. The molar ratio of protein to lipid is about 1:90 to about 1:120, about 1:100 to about 1:140 or about 1:95 to about 1:125. In the ratios determined, the ApoA-I protein fraction and the SM and DPPG lipid fractions were, respectively, When assayed by column chromatography and gel electrophoresis, it was found to be similar to native HDL. Substantially uniform complexes are formed having the same size and charge characteristics.

[0208] Controlling the size of negatively charged lipoprotein complexes by varying Ri That is, the smaller Ri is, the smaller the disk is. Small discs typically have Ri in the range of about 200:1 to 100:1, but smaller Diameter discs typically have an Ri in the range of about 100:1 to 30:1.

[0209] In some specific embodiments, the negatively charged lipoprotein complexes are 2-4 ApoA-I equivalents (e.g., 2-4 molecules of ApoA-I, 1-2 molecules of ApoA-I M two 1 molecule of negatively charged phospholipid and a large circular disk containing 400 molecules of SM. In the present study, the negatively charged lipoprotein complexes consist of 2-4 ApoA-I equivalents; 1-10, More preferably, 3 to 6 molecules of negatively charged phospholipid; and 90 to 225 molecules, more preferably or small disc-shaped disks containing 100 to 210 SM molecules.

[0210] 6.3.1. Complex and particle size measurements Composition of Lipoprotein Complexes and Their Sizes and Preparation of Lipoprotein Complexes The lipid particles used in the method are determined using a variety of techniques known in the art. It is possible.

[0211] The protein and lipid concentrations of the lipoprotein complex in solution may be determined, but are not limited to, However, protein and phospholipid assays, chromatographic methods, e.g., HPLC, GC with various detectors such as gel filtration, mass spectrometry, UV or diode array, fluorescence It can be measured by any method known in the art, such as light, elastic light scattering, etc. Lipid and protein intensities were analyzed using the same chromatographic techniques and peptide mapping. cloning, SDS-page gel electrophoresis, N- and C-terminal sequencing of ApoA-I, and lipid oxidation can also be determined by standard assays for determining lipid oxidation.

[0212] Lipoprotein complexes and lipid particles used in the preparation of lipoprotein complexes The particles may be in the size ranges described herein. The size of the protein complex and the protein complex size can be determined using methods known in the art. Exemplary methods include dynamic light scattering and gel permeation chromatography.

[0213] Dynamic Light Scattering (DLS), also known as photon correlation spectroscopy, is a method for measuring the dynamics of light in solution by Brownian motion. It measures the shift in wavelength of a light beam that strikes a particle moving at a speed of 1000 Hz. When illuminated, the moving particles scatter light, and the resulting intensity variations in the scattered light are used to calculate The globular size distribution in the solution can be calculated by the Zetasizer Nano Series User Manual. ual, MAN0317 Issue 2.1 (July 2004). DLS is a method for measuring particle volume and number distributions. Based on this, the intensity distribution and average of particles in the solution can be calculated. DLS techniques are used to determine the lipids used to make the lipoprotein complexes. The size of the particles, as well as the size of the lipoprotein complex itself, can be determined. A suitable DLS instrument is the Zetasizer from Malvern Instruments. Nano.

[0214] In addition, gel permeation chromatography (GPC) was used to identify protein-containing complexes. Gel permeation chromatography can also determine the size of molecules based on their size. Typically, lipid-specific analytes are separated by comparison with a calibration curve. Compare the elution profile of the protein complex with that of known standards or reference samples The size of the complex can be determined by this method. Protein, albumin, ferritin, and vitamin B 12 Non-protein standards such as Current Protocols in Molecular Biology (1998), Section I V, 10.9.1-10.9.2.

[0215] Lipid particles useful in preparing lipoprotein complexes of the present disclosure may be characterized by their ability to be measured by DLS. If measured (e.g., using intensity-based measurements), at least 45 nm, at least 5 0 nm, at least 55 nm, at least 60 nm in size. The particles are sized at 65 nm, 70 nm, and 80 nm as measured by DLS. m, up to 90nm, up to 100nm, up to 120nm, up to 150nm, up to 200nm, up to 250nm, up to 300nm, or up to 500nm in size That's fine.

[0216] The lipoprotein complexes of the present disclosure have a molecular weight of 4n when measured by the techniques described herein. m~15nm, 6nm~15nm, 4nm~12nm, 5~12nm, 6nm~12nm , 8 nm to 12 nm, or 8 nm to 10 nm in size.

[0217] 6.4. Lipoprotein Complex Populations The disclosure further provides a population of lipoprotein complexes as described herein. The population comprises: For example, the protein fraction and lipid fraction, respectively, as described in Section 6.3 above. The present applicants have discovered that lipoprotein complexes, which include a plurality of lipoprotein complexes as described herein, These drugs are believed to contribute individually or in combination to the efficacy and safety profile of the combined population. The lipoprotein complex populations described herein are characterized by several features that may be useful in determining whether the lipoprotein complexes are in a soluble form. Any number of features may be included alone or in combination.

[0218] First, the homogeneity of the lipoprotein complexes in the population, i.e., the One or more of the complexes in the population, as indicated by one or more distinct peaks of the complex. The predominance of individual lipoprotein complexes and the prevalence of mature unmodified apolipoproteins It is believed that the predominance of lipoprotein complexes increases efficacy. The population of protein complexes includes an apolipoprotein, e.g., ApoA-I, or The population may include a protein fraction consisting essentially of the protein fraction and a lipid fraction, the population being gel. The field is measured by the percentage of the population in a single peak in permeation chromatography. In all cases, at least 80%, at least 85%, at least 90%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% homogeneous do.

[0219] In some embodiments, the size of the lipoprotein complexes in the population is between 4 nm and 15 nm, for example, in the range of 5 nm to 12 nm, 6 nm to 15 nm, or 8 nm to 10 nm It may be an area.

[0220] The apolipoproteins in the population, e.g., ApoA-I, are mature, preferably full-length. (non-truncated) ApoA-I, and the population may be at least 75 wt. %, at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 95% by weight, at least 96% by weight, at least 97% by weight, at least 98% by weight, At least 99% by weight of mature, preferably full-length (not truncated) Ap In some embodiments, the population may comprise 25% by weight or less of oA-I. 0% by weight or less, 15% by weight or less, 10% by weight or less, or 5% by weight or less of immature, if Incompletely processed ApoA-I and / or 20% by weight or less, 15-fold % by weight or less, 10% by weight or less, or 5% by weight or less of truncated ApoA-I. include.

[0221] Second, the purity of the lipoprotein complex, also known as the apolipoprotein and The purity of lipids and the relative absence of contaminants in the lipoprotein complexes were determined by the activity of liver enzymes. Risk of side effects such as liver damage, reflected by increased levels of serotonin (e.g., transaminases) The purity of apolipoproteins is thought to be improved by oxidation and / or deamidation. The relative lack of cyclization can be measured by the relative lack of cyclization. The population of polyprotein complexes was 20% or less, 15% or less, 10% or less, 5% or less, and 4% or less. 3% or less, 2% or less, 1% or less of oxidized methionine, in particular, methionine-112 or is methionine-148, or 15% or less, 10% or less, 5% or less, 4% or less, 3% or less , low levels of oxidized apolipoproteins, such as oxidized tryptophan (less than 2% or less than 1%) The population of lipoprotein complexes may also have a low percentage of deamidated lipoproteins. modified amino acids, such as 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less or less, or 1% or less deaminated amino acids.

[0222] It is also desirable to control the purity of lipids in the lipoprotein complex. In some embodiments, the percentage of the population is 5% or less, 4% or less, 3% or less, 2% or less, or Less than 1% of lipids are oxidized.

[0223] Another measure of the purity of the complex and its population is the amount of apolipoprotein produced. or contaminants resulting from the process of purification or the process of producing the lipoprotein complex itself. Thus, apolipoproteins are a reduced or absent part of a host cell, e.g. When purified from mammalian host cells, the population of lipoprotein complexes is preferably In certain embodiments, the population is free of cellular DNA or proteins. Per milligram of protein, 500 nanograms or less, 200 nanograms or less, 100 Subnanograms, 50 nanograms, or 20 nanograms of host cell protein protein, and / or lipoproteins, typically per milligram of ApoA-I. 100 picograms or less, 50 picograms or less, 25 picograms or less, 10 picograms or less or 5 picograms of host cell DNA.

[0224] Other contaminants that may occur and should be avoided are particularly present in cell culture and plasma samples. It is used to produce and / or purify endotoxins and lipoprotein complexes that may be present in the Solvents and detergents that may be present depending on the process being carried out. is at most about 1 EU per milligram of lipoprotein, e.g., ApoA-I, The lipoprotein may contain 0.5 EU, about 0.3 EU, or about 0.1 EU of endotoxin. The protein complex populations were below 200 ppm, below 250 ppm, below 100 ppm, In certain embodiments, the population may be limited to the inclusion of any detergent, e.g. For example, it does not contain cholate.

[0225] Additionally, most of the apolipoprotein starting material can be purified using the methods disclosed herein. into a complex, limiting the amount of uncomplexed apolipoprotein present in the population. The reduction in the amount of uncomplexed apolipoproteins indicates that they respond better to exposure to foreign proteins. Lipoprotein complexes are beneficial in reducing the risk of immunogenic responses caused by dew. The population of proteins is at least 80%, at least 85%, at least 90%, or at least at least 95%, at least 97%, at least 98%, at least 99% or 100% may be in a lipoprotein complex, i.e., in a complex form. The lipoprotein complex population is composed of at least 80%, at least 85%, or at least at least 90%, at least 95%, at least 97%, at least 98%, at least 9 It may be in a composition where 9% or 100% is in lipoprotein complexes.

[0226] Optionally, the population has a lipid fraction of 15% or less, 10% or less, 5% or less, by lipid weight. Complexes containing 4% or less, 3% or less, 2% or less, 1% or less, or 0% cholesterol It may include.

[0227] Certain lipid and protein components form multiple distinct but homogeneous lipoprotein complexes. Thus, the present disclosure also provides methods for producing apolipoproteins with different amounts of apolipoprotein molecules (e.g., Lipoprotein complexes containing 1, 3 or 4 ApoA-I molecules or ApoA-I equivalents Compositions comprising two, three or four populations of the combination are also provided. In an exemplary embodiment, The compositions each comprise a first population of two ApoA-I molecules per lipoprotein complex. or a lipoprotein complex having Apo-I equivalents, and the second population comprises lipoprotein complexes having Apo-I equivalents. Lipoproteins with 3 or 4 ApoA-I molecules or ApoA-I equivalents per lipid complex and optionally a third population containing up to four per lipoprotein complex. lipoprotein complexes containing one or three ApoA-I molecules or equivalents It contains two lipoprotein complex populations:

[0228] The composition comprising two or more populations of lipoprotein complexes preferably contains low levels of non-complex lipoproteins. The composition preferably contains lipoproteins and / or lipids. Quality 15% or less, 12% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less is in uncomplexed form; and / or 15% or less, 12% or less, 10% or less, 9% or less of the lipoproteins in the composition Below, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, if Approximately 1% or less is in the uncomplexed form.

[0229] It is also useful for commercial applications, such as large-scale production of lipoprotein complexes for therapeutic purposes. Large-scale preparations of lipoprotein complexes, or populations thereof, which are particularly useful, are also provided herein. The preparations contemplated herein are provided in the form of lipoprotein complexes, as described herein. Concentrations of negatively charged lipoprotein complexes include, for example, populations of negatively charged lipoprotein complexes.

[0230] The preparations are suitable for the manufacture of compositions, e.g., pharmaceutical compositions and dosage forms, on a commercial scale. The volume, amount and resulting concentration of lipoprotein complexes are provided as typical. The volume of the preparation is about 5 L to about 50 L or more, for example, about 10 L to about 40 L, about 15 L to about 30 L, 5L, about 15L to about 30L, about 20L to about 40L, about 20L to about 30L, about 25L to about 4 The preparations range from about 5 L, about 6 L, about 7 L, about 8 L, Approximately 9L, approximately 10L, approximately 11L, approximately 12L, approximately 13L, approximately 14L, approximately 15L, approximately 16L, approximately 17L, approx. 18L, approx. 19L, approx. 20L, approx. 25L, approx. 30L, approx. 35L, approx. 40L, approx. In a preferred embodiment, the preparation may have a volume of about 45 L, or about 50 L. , has a capacity of approximately 20L.

[0231] The preparations may further comprise from about 5 mg / mL to about 15 mg / mL, from 5 mg / mL to about 10 mg / mL L, about 10 mg / mL to about 15 mg / mL, or about 8 mg / mL to about 12 mg / mL A sufficient amount of lipoprotein to achieve an apolipoprotein concentration in the range of Depending on the volume of the preparation, the amount ranges from about 25 g to about 350 g, and the apolipoprotein in the preparation, e.g., ApoA-I In certain embodiments, the preparation contains about 8 mg / mL of ApoA- Contains I.

[0232] In certain embodiments, the preparation has a volume of 15 L to 25 L and a volume of about 100 g to about In another specific embodiment, the preparation contains 30 L of ApoA-I. It has a volume of about 50 L and contains about 240 g to about 780 g of ApoA-I.

[0233] 6.5. Methods for preparing lipoprotein complexes 6.5.1. Thermal Cycling-Based Method for Producing Lipoprotein Complexes Using the methods described herein using thermal cycling of protein and lipid components, It has been discovered that lipoprotein complexes can be produced that have advantages over other methods. In the thermal cycling methods provided herein, the protein component and the lipid The bulk of the protein component (e.g., at least 60%, at least 70%, At least 80%, or at least 90%, are complexed with lipid components to form lipoproteins The reaction is then subjected to thermal cycling until the complex is formed. Advantages of the method of the present invention compared to other methods for the production of protein complexes include: Fewer or no by-products (e.g., uncomplexed proteins) are produced, or the product obtained is Manufacturing impurities present in the composition (e.g., detergents or surfactants, degraded proteins, High complexation resulting in a substantially homogenous and pure final product free of oxidized components These include efficient purification and avoidance of the need for costly and wasteful purification steps. The method is efficient and produces little to no waste of starting materials. The process is easy to install and has low equipment costs. Their ability to do so also makes them environmentally friendly.

[0234] Preferably, one of the complexes is prepared in order to minimize oxidation of the protein and lipid components. More than one or all steps (e.g., homogenization of lipid components) can be carried out under inert gas (e.g., The reaction is carried out under a blanket of nitrogen, argon or helium.

[0235] 6.5.2. Lipid composition The thermal cycling method of the present disclosure can be used to selectively cycle saturated, unsaturated, natural and various lipids, such as synthetic lipids and / or phospholipids, alone or in combination. It can be used.

[0236] Multilamellar vesicles ("MLVs"), small unilamellar vesicles ("SUVs"), large unilamellar vesicles ("LUVs") The protein component can be prepared using any method that produces lipid particles, such as UV (UV), micelles, or dispersions. Lipids can be prepared for thermal cycling using minutes.

[0237] A variety of techniques are known for producing lipid particles. The thermal cycling methods disclosed herein have been used to fabricate fluorophores using a variety of protocols. The particles used in the present invention are preferably in the size range of 45 to 80 nm, and most preferably The size ranges from 55nm to 75nm.

[0238] High pressure homogenization, e.g., microfluidization, can be used to obtain lipid particles of suitable size. The equalization pressure is preferably at least 1,000 bar, at least 1 ,200bar, at least 1,400bar, at least 1,600bar, at least At least 1,800 bar, and most preferably at least 2,000 bar, e.g. At least 2,200 bar, at least 2,400 bar, at least 2,600 bar , at least 2,800 bar, at least 3,000 bar, at least 3,200 bar ar, at least 3,400 bar, at least 3,600 bar, at least 3,80 0 bar, or at least 4,000 bar. The curing pressure may range between any pair of the above values, for example, 1,600 to 3,200 bar; 800~2,800bar;1,900~2,500bar;2,000~2,500b ar;2,000~3,000bar;2,400~3,800bar;2,800~3 , 400 bar, etc. 1 bar is 100 kPa, 1,000,000 dynes / square. Barley, 0.987 atm (atmosphere), 14.5038 psi, 2 Equal to 9.53 inHg and 750.06 torr.

[0239] In one preferred homogenization method, the lipid emulsion is homogenized using a Microfluidizer. Model 110Y (Microfluidics Inc, Newton, Mass. s) into a supply vessel. The unit is immersed in a bath to maintain the process temperature during homogenization (e.g., Maintain the temperature at room temperature (55°C, 58°C, 62°C, etc.) and flush with an inert gas such as argon before use. After priming, the emulsion is pressured across the interaction head. Pass the mixture through a homogenizer for 5-20 minutes at a gradient with continuous recycle. Homogenization of the lipid component in the absence of the protein component is achieved by the high shear used in homogenization techniques. Avoid the destruction of quality components.

[0240] Other methods may be suitably used, provided that particles of suitable size can be obtained. For example, hydration of lipids with aqueous solutions can result in the formation of lipid dispersions and multilamellar vesicles ("MLVs"). MLVs can result in the simultaneous formation of multiple lipid bilayers surrounding a central aqueous core. These types of particles are larger than small unilamellar vesicles (SUVs) and The MLVs may be dissolved in chloroform in a round bottom flask. Solubilize the lipids and evaporate the chloroform until the lipids form a thin layer on the walls of the flask. An aqueous solution is added to rehydrate the lipid layer. Rotate or vortex the vesicles formed in the flask. Deamer et al., 1999 83, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (Bangham et al., 1965, J. Mol. Biol. 13:238. This method has also been used to generate unilamellar vesicles. Johnson et al., 1971, Biochim. Biophys. Acta 233:820.

[0241] Small unilamellar vesicles (SUVs) are particles that contain a single lipid bilayer enveloping an aqueous core. Depending on the method used to generate SUVs, they range from 25 to 110 nm in diameter. The first SUVs were prepared from phospholipid preparations in chloroform under nitrogen. The mixture was dried and the aqueous layer was added to give lipid concentrations in the millimolar range, and the solution was incubated at 45°C. The lipids were prepared by sonication until clear. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York. SUVs prepared in this manner are 25-5 mm in diameter. This results in particles in the 0 nm range.

[0242] Another method for creating SUVs is to mix an ethanol / lipid solution with the aqueous solution to be encapsulated. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel De Kker, Inc. New York (citing Batzri et al., 1973, Biochim. Biophys. Acta 298: 1015) The SUVs produced by this method range in size from 30 to 110 nm in diameter.

[0243] The multilamellar vesicles were also passed through a French Press four times at 20,000 psi. By using this method, SUVs can be manufactured. The SUVs manufactured have a diameter of 30 to 50 mm. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekk er, Inc. New York (citing Barenholz et al., 1979, FEBS Letters 99:210).

[0244] Aqueous preparation of multilamellar and unilamellar phospholipid vesicles by high pressure phospholipids through small pore membranes They can also be formed by extrusion of materials (Hope et al., 1996, Chemistry and Physics of Lipids, 1998). ids, 40:89-107).

[0245] Large unilamellar vesicles are S in that they are a single lipid bilayer surrounding a central aqueous core. Although similar to UVs, LUVs are much larger than SUVs. Depending on the method used to prepare them, LUVs range in size from 50 to 1000 nm in diameter. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York ork. LUV is typically performed using one of three methods: detergent dilution, reverse phase evaporation, and injection. It is prepared as follows.

[0246] In detergent dilution technology, cholate, deoxycholate, octyl glucoside, heptyl glucoside, Lipid preparation using detergent solutions such as butyl glucoside and Triton X-100 Micelles are formed from the detergent. The solution is then dialyzed to remove the detergent. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York.

[0247] The reverse phase evaporation technique solubilizes lipids in an aqueous non-polar solution, resulting in the formation of reverse micelles. Upon evaporation of the solvent, the micelles aggregate to form LUVs. This method is generally used for large amounts of lipid It requires quality.

[0248] The injection method involves injecting lipids solubilized in a non-polar solution into the aqueous solution to be encapsulated. As the non-polar solution evaporates, the lipids collect at the gas / aqueous interface. When bubbled through the liquid, the lipid sheets form LUVs and oligolamellar particles. The particles are partitioned by filtration. Deamer et al., 1983, in Liposomes (Ostro, Ed. ), Marcel Dekker, Inc. New York (Deamer et al., 1976, Biochim. Biophys. Acta 443:629 and Schieren et al., 1978, Biochim. Biophys. Acta 542: 137).

[0249] An aliquot of the resulting lipid preparation is characterized to determine whether the lipid particles conform to the thermal decomposition properties disclosed herein. It is therefore desirable to determine whether the lipid component of the present invention is suitable for use as a lipid component in a cycling method. Characterization of lipid preparations can be performed using techniques including, but not limited to, size exclusion filtration, gel filtration, and the like. techniques such as denaturing gel electrophoresis, column filtration, gel permeation chromatography, and non-denaturing gel electrophoresis. This can be done using any method known in the art.

[0250] Protein Components The protein components of the lipoprotein complexes play a key role in the success of the thermal cycling method of the present invention. The present invention is not critical to the present invention. Virtually any lipid binding protein, e.g., therapeutic and / or and / or derivatives or analogs thereof providing a therapeutic or prophylactic benefit In addition, any α-helical peptide or is a peptide analogue or apolipoprotein (e.g., ApoA-I) is LCAT It is said that it can form discoid particles when activated or bound to lipids. Any other type of molecule that "mimics" the activity of apolipoproteins in that it acts like a lipoprotein. The lipid-binding protein may be included in a lipid complex and is therefore included within the definition of "lipid-binding protein." The lipid-binding proteins that can be used in the thermal cycling method include the above-mentioned Lipid-binding proteins include those described in Section 6.1.2 above. The lipid binding protein can be produced recombinantly as described above in Section 6.1. by any of the methods described herein, such as those described in Section 3 or Section 6.1.4. It can be purified by

[0251] Protein components may be derived from animal (and in particular human) origin, as is well known in the art. The enzyme can be purified from cultures containing serotype 1, serotype 2, serotype 3, or serotype 4, or can be chemically synthesized or recombinantly produced, e.g., as described in Chung et al. Cheung et al., 1987, J. Lipid Res. 28(8):913-29 See also U.S. Patent Nos. 5,059,528, 5,128,318, and 6, ,617,134; U.S. Patent Application Publication No. 20002 / 0156007, No. 2004 / Nos. 0067873, 2004 / 0077541, and 2004 / 0266660 No.; and PCT Publication Nos. WO / 2008 / 104890 and WO / 2007 / 0 See also issue 23476.

[0252] The protein component is less than the protein / peptide to lipid ratio in the desired complex. at least 5-fold higher (e.g., at least 5-fold, at least 10-fold, or at least 20-fold higher a) The ratio of protein / peptide to lipid may include lipid. For example, To prepare lipoprotein complexes with a lipid to lipid ratio of 1:200 on a molar basis, The proteins in the protein component typically account for only a small fraction of the lipids in the final complex. It can be combined with lipids in a ratio of, for example, 1:10 to 1:20. Although not intended to imply any specific mechanism, this "pre-complexation" of the protein with small amounts of lipid This is useful when the desired complex has two or more lipid species and can be prepared by thermal cycling. present in small amounts in the lipoprotein complexes produced (e.g., the desired lipoprotein complexes) 10% or less by weight, 5% or less by weight, 3% or less by weight, 2% or less by weight, or 1% or less by weight of total lipids in the body % or less by weight), allowing for a more uniform distribution of lipids.

[0253] 6.5.4. Formation of Lipoprotein Complexes by Thermal Cycling The method generally comprises: mixing a suspension containing lipid particles and a lipid-binding protein with a lipoprotein; Thermal cycling between "high" and "low" temperature ranges until a matrix complex is formed. is required.

[0254] The suspension to be thermally cycled is preferably a suspension of lipid components combined at a temperature in the high temperature range. and a protein component to form a "starting" suspension, which is then subjected to thermal cycling. do.

[0255] The optimal lipid to protein ratio in the starting suspension determines the final lipoprotein complex produced. As will be appreciated by those skilled in the art, the lipid fraction is determined by the desired stoichiometry of the components in the combination. The molar ratio of the protein fraction to the protein component determines, among other factors, the protein content of the protein component. The identity of the lipids and / or peptides, the identity and amount of lipids in the lipid fraction, and the The preferred lipids in the lipoprotein complex depend on the desired size of the lipoprotein complex. The ratio of the protein may be determined by, but is not limited to, gel electrophoretic mobility assays, size Exclusion chromatography, interaction with HDL receptor, ATP-binding cassette transporter (A Any number of factors can be considered, such as recognition by the BCA1, uptake by the liver, and pharmacokinetics / pharmacodynamics. This can be determined using functional assays known in the art, for example, gel electrophoresis. Using a dynamic mobility assay to determine the optimal ratio of lipid and protein components in the complex The complexes produced by the methods of the present disclosure have a phospholipid content in the lipid component. As a result, when charged, it resembles natural pre-β-HDL or α-HDL particles Conjugates can be designed to exhibit electrophoretic mobility. In terms of morphology, natural pre-β-HDL or α-HDL particles were analyzed to determine the mobility of the complex. It can be used as a standard for determining

[0256] In a preferred embodiment, the final complex comprises at least one apolipoprotein. or apolipoprotein mimics (most preferably mature human ApoA- I or ApoA-I peptide), at least one neutral lipid, and at least one negatively charged lipids, e.g., those described in PCT WO2006 / 100567 (the contents of which are incorporated by reference) (herein incorporated by reference).

[0257] The biological activity of apolipoproteins such as ApoA-I is determined by the parent molecules that contain apolipoproteins. Since this is thought to be mediated by the intermediate helix, an equivalent amount of ApoA-I protein was used. It is convenient to express the apolipoprotein fraction of the lipid:apolipoprotein molar ratio as ApoA-I consists of 6–10 amphipathic helices, depending on the method used to calculate the helices. It is generally accepted that the apolipoproteins contain a cyclic helix. They are expressed in units of ApoA-I equivalents based on the number of amphipathic helices they contain. For example, ApoA-I M Each molecule of is twice as large as the molecule of ApoA-I. Contains amphipathic helices and therefore typically exists as a disulfide-bridged dimer ApoA-I M can be expressed as 2 ApoA-I equivalents. Conversely, each molecule is Since poA-I contains 1 / 10 to 1 / 6 of the amphipathic helices in the molecule, it is a single amphipathic Peptide apolipoproteins containing intermediate helices are 1 / 10 to 1 / 6 ApoA- It can be expressed as I equivalent.

[0258] Generally, the lipid:ApoA-I equivalent molar ratio of the lipoprotein complex (referred to herein as "Ri ") is in the range of about 2:1 to 200:1. Ri is about 50:1 to 150:1, 75:1 to 125:1, 10:1 to 175 The weight ratio is 1:1. For phospholipids, a MW of about 650-800 is used. can be done.

[0259] In certain embodiments, the molar ratio of the components is from 2 to 6 (negatively charged lipids, e.g., D PPG): 90-120 (neutral lipid, e.g., SM): 1 (ApoA-I equivalent). In a particular embodiment described in Example 1, the complex has a lipid to tandem ratio of about 108:1. The lipid molar ratios included DPPG, SM, and ApoA-I, and DPPG accounted for 3% of the total lipids by weight. % (+ / - 1%) and SM is 97% (+ / - 5%) of the lipids by weight.

[0260] The concentrations of lipid and protein components in the starting suspension before the start of thermal cycling are 1-3 0 mg / ml concentration of ApoA-I equivalent and lipid concentrations ranging from 1 to 100 mg / ml. In a particular embodiment, the concentration of the protein component may be 1 to 30 mg / ml. 2~20mg / ml, 5~20mg / ml, 2~10mg / ml, 5~15mg / ml, The concentration of the lipid component is selected from 5 to 20 mg / ml and 10 to 20 mg / ml. 0~100mg / ml, 10~75mg / ml, 25~50mg / ml, 10~75mg / ml, 25-100mg / ml, 25-75mg / ml, and 1-75mg / ml are independently selected.

[0261] The high and low temperature ranges of the thermal cycling process are the lipid and Based on the phase transition temperature of the protein component. Alternatively, phospholipids with unsaturated fatty acid chains or The lipid components are preferably in defined or distinct phases, as may occur when a mixture of phospholipids is used. If no transition is observed, the high and low temperature ranges of the thermal cycling should be at least about 20° C. For example, in some embodiments, the low and The high temperature range is 20℃~30℃, 20℃~40℃, 20℃~50℃, 30℃~40℃, 3 Different temperatures: 0℃~50℃, 25℃~45℃, 35℃~55℃.

[0262] For lipids, the phase transition is from a tightly packed, ordered structure known as the gel state to This involves a change to a looser, less ordered structure known as the liquid state. Protein complexes are typically referred to in the art as being based on the specific lipid or mixture of lipids used. By incubating lipid particles and apolipoproteins at temperatures close to the transition temperature, The phase transition temperature of the lipid component (which can be determined by calorimetric methods) + / - 5°C to 10°C is the "low" temperature range for the methods of the present disclosure.

[0263] For proteins, the phase transition temperature is the temperature at which the protein changes from a folded 3D structure to a 2D structure. For lipids, the phase transition is from a tightly packed, ordered structure known as the gel state. This involves a change from a rigid, solid structure to a loose, less ordered structure known as the liquid state. Protein complexes are typically referred to in the art as being composed of the particular lipid or lipid mixtures used. Incubating the lipid particles and apolipoprotein at a temperature close to the transition temperature of the compound It is formed by:

[0264] Phase transition temperature of the lipid component (which can be determined by calorimetry) + / - 12°C, Preferably, + / - 10°C is the "low" temperature range in the methods of the present disclosure. In the present case, the low temperature range is + / - 3°C, + / - 5°C, or + / - the phase transition temperature of the lipid component. In one particular embodiment, the low temperature range is between the phase transition temperature of the lipid component and the temperature of the lipid component. , from above 5°C or above 10°C, to above 5°C.

[0265] For proteins, the phase transition temperature involves a change from tertiary to secondary structure. The phase transition temperature of the carbonaceous component is preferably within + / - 12°C, more preferably + / - 10°C, in the method of the present disclosure. In certain embodiments, the high temperature range is the "high" temperature range for the relative + / - 3°C, + / - 5°C, or + / - 8°C of the transition temperature. In this case, the low temperature range is from 10°C below the phase transition temperature of the protein component to 5°C above it. , below 10°C, or below 15°C.

[0266] The starting suspension is preferably started at an elevated temperature to remove at least some of the proteins in the starting suspension. 70%, at least 80%, at least 90%, at least 95%, at least 96%, At least 97%, at least 98%, or at least 99% in lipoprotein complexes The material is then subjected to thermal cycling between high and low temperatures until it is incorporated into the desired stoichiometry. The lipid and protein components were then assayed after several cycles using a small amount of lipid and protein components. Qualitatively complete conjugation can be achieved. The number of cycles depends on the protein and lipid components. minutes, depending on the duration of the cycle, but typically 5 or more cycles, 10 or more cycles Cycles, or 15 or more cycles (at both high and low temperatures) to produce a substantially intact complex. Cycles typically range from 2 minutes to 60 minutes. In the experiment, the cycle was 5-30 minutes, 10-20 minutes, 5-20 minutes, and These range from 2 to 45 minutes or 5 to 45 minutes.

[0267] The complexes produced by the above method typically have a protein component that is a mixture of phospholipids and protein. They physically bind to phospholipids with a specific stoichiometry between the proteins and have a uniform size distribution. These are supramolecular assemblies shaped as micelles, vesicles, spherical or discoidal particles. The method of the invention advantageously involves the substantial depletion of lipids and / or proteins in the starting suspension. When observed by separation methods such as chromatography, A composition that is substantially free of lipids and / or proteins is obtained. The process can be carried out in the absence of a purification step.

[0268] The methods of the present disclosure are advantageously uniform in their size distribution, eliminating the need for size fractionation. To generate a complex that avoids

[0269] In some embodiments of the present disclosure, the lipoprotein complexes are present in relatively small amounts (e.g. For example, less than 10%, less than 5%, less than 3% or less than 1% of the lipid content Contains two or more lipids, including lipids used in small amounts to optimize dispersion. The lipid component is premixed with the protein component rather than being incorporated into the lipid particles. can be done.

[0270] An aliquot of the resulting lipoprotein complex is characterized to determine whether the complex has desired characteristics, e.g. For example, substantially complete (e.g., >90%, >95%, It can be confirmed that the complex has >97% or >98% incorporation. The evaluation may be performed by, but is not limited to, size exclusion filtration, gel filtration, column filtration, gel permeation, etc. Any method known in the art, such as chromatography and non-denaturing gel electrophoresis, may be used. It can be carried out using

[0271] The homogeneity and / or stability of the lipoprotein complexes or compositions described herein may be improved by chromatographic techniques, such as, but not limited to, gel filtration chromatography The amount of the protein can be measured by any method known in the art, such as, for example, In an embodiment, a single peak or a limited number of peaks are associated with a stable complex. The stability of the complex can be monitored by monitoring the appearance of new peaks over time. The appearance of new peaks can be determined by the complex nucleation process, which is caused by particle instability. It is a sign of reorganization between the coalitions.

[0272] Preferably, thermal cycling is performed to minimize oxidation of protein and lipid components. The reaction is carried out under a blanket of an inert gas (eg, nitrogen, argon or helium).

[0273] 6.5.5. Other Methods for Producing Lipoprotein Complexes Lipoprotein complexes as described herein, such as negatively charged lipoprotein complexes, Non-limiting examples include vesicles, liposomes, proteoliposomes, micelles, and In addition to the above thermal cycling method, the granules can be prepared in various forms such as discoid particles. Thus, lipoprotein complexes can be prepared using a variety of methods known to those skilled in the art. A variety of techniques can be used to prepare liposomes or proteoliposomes. For example, apolipoproteins can be mixed with suitable phospholipids (using an ultrasonic bath or ultrasonic process). Alternatively, apo-sonication can be performed using a single aliquot of apo-sonicated IgG. Lipoproteins, e.g., ApoA-I, are combined with preformed lipid vesicles to form lipoproteins. This can result in the simultaneous formation of lipoprotein complexes. , by detergent dialysis; for example, apolipoproteins, charged phospholipids and SMs and Dialysis of a mixture of detergents such as cholate to remove the detergent and reconstitute the negatively charged ligand. to form polyprotein complexes (see, e.g., Jonas et al., 1986, Methods in Enzymol. 128:5 53-82), or by using an extrusion device, or by homogenization. It is also possible to do so.

[0274] In some embodiments, high pressure (e.g., about 40, about 50, or about 60 minutes) is used. The composite is prepared by homogenization at a pressure of 32,000 psi. In this case, a complex containing ApoA-I, SN and DPPG is prepared as follows. ApoA-I was dissolved in phosphate buffer and mixed with a high-shear mixer. The mixture is then incubated at 50° C. with a dispersion of DPPG in a buffer. The I / DPPG mixture was combined with a dispersion of SM and analyzed by dynamic light scattering or gel permeation chromatography. The reaction was incubated at 30-50°C until complex formation was essentially complete, as monitored by FTIR. Pressure equalization is achieved at over 30,000 psi.

[0275] In some embodiments, the disclosure of which is incorporated herein by reference. According to the cholate dispersion method described in Example 1 of U.S. Patent Application Publication No. 2004 / 0067873, Lipoprotein complexes can be prepared. Briefly, dry lipids are dissolved in NaHCO 3. After hydration in buffer, vortex and sonicate until all lipids are dispersed. The cholate solution is added and the mixture is stirred until lipid cholate micelles are formed. Incubate the ink for 30 min, vortexing and sonicating periodically, until clear and clear. ProApoA-I in NaHCO3 buffer is added and the solution is incubated at approximately 37°C. Incubate at ~50°C for 1 hour.

[0276] Cholate can be removed by methods known in the art. The acid salts are separated by dialysis, ultrafiltration, or by adsorption onto affinity beads or resins. In one embodiment, the affinity beads can be removed by removal of the cholate molecules. For example, BIO-BEADS (registered trademark) (Bio-Rad Laboratories, es) was added to a preparation of negatively charged lipoprotein complexes and cholate to In another embodiment, the preparation, e.g., lipoprotein complex, is adsorbed. A micellar preparation of the antibody and cholate is passed over a column packed with affinity beads.

[0277] In certain embodiments, the preparation is placed on BIO-BEADS® in a syringe. Loading removes cholate from the preparation of lipoprotein complexes. Seal the syringe with barrier film and incubate overnight at 4°C with shaking. Before use, the solution is injected through the BIO-BEADS® where it is absorbed by the beads. Cholate is removed by adsorption onto the cellulose membrane.

[0278] Lipoprotein complexes, such as the negatively charged lipoprotein complexes described herein, include: The complex resembles HDL, particularly in the pre-β-1 or pre-β-2 HDL populations. If the protein has a size and density that is consistent with the above, it is expected that its half-life in circulation will be increased. Stable preparations with a shelf life can be produced by lyophilization. In an embodiment, ApoA-I is co-lyophilized using co-lyophilization techniques commonly known in the art. Briefly, the co-lyophilization step is carried out using an organic solvent or A mixture of ApoA-I and lipids is solubilized in a solvent mixture, or ApoA-I and The method involves solubilizing the cellulose and lipid separately and mixing them together. Desirable characteristics of the solvent or solvent mixture include: (i) hydrophobic lipids and amphipathic proteins; (ii) the relative polarity of the medium in which the protein can be dissolved; and (iii) the potential toxicity associated with residual organic solvents. The FDA solvent guidelines for avoiding solvent contamination (Federal Register, volume 62, No. 247) are followed. (iii) a low boiling point to ensure ease of solvent removal during lyophilization; (iv) faster freezing, higher condensation temperatures and less wear on the freeze dryer; In some embodiments, glacial acetic acid is used. Combinations of methanol, glacial acetic acid, xylene, or cyclohexane can also be used. .

[0279] The ApoA-I-lipid solution is then lyophilized to obtain a homogenous powder. Optimized to produce a solution with minimal residual solvent in the lyophilized apolipoprotein-lipid powder. The selection of freeze-drying conditions depends on the nature of the solvent, the type of container, and Depending on the size, holding solution, filling volume, and characteristics of the freeze-dryer used, the skilled artisan can Therefore, it can be determined.

[0280] Using lyophilized lipoprotein complexes to prepare bulk supplies for pharmaceutical reformulation or reconstituted to obtain a solution or suspension of lipoprotein complexes. For reconstitution, individual aliquots or dosage units can be prepared. The lyophilized powder is then dispensed into a suitable volume (e.g., 5 mg of polypeptide / ml, which is administered intravenously). In some embodiments, the granules are rehydrated with an aqueous solution until the granules are ready for injection. In this method, the lyophilized powder is rehydrated using phosphate buffered saline or a physiological salt solution. The mixture can be stirred or vortexed to facilitate rehydration. The reconstitution step is carried out at a temperature equal to or higher than the phase transition temperature of the lipid component of the complex. It can be carried out at any time.

[0281] The ApoA-I-lipid complexes are lyophilized using an aqueous medium of suitable pH and osmolarity. These may form spontaneously upon hydration of the apolipoprotein-lipid powder. In the present invention, the hydration medium includes, but is not limited to, sucrose, trehalose, and glucose. In some embodiments, the complex comprises a stabilizer selected from the group consisting of glycerin and glycerin. To synthesize the lipids, the solution is heated several times above the lipid transition temperature. The ratio of the lipid to the lipid may be 1:1 to 200:1 (mol / mol), and preferably 3:1 to 2:1. lipid:protein (w / w), more preferably 2.7:1 to 2.1:1 lipid:protein The lipid:protein ratio (w / w) is, for example, 2.7:1 lipid:protein (w / w). to obtain a final complex concentration of 5-30 mg / ml, expressed in protein equivalents.

[0282] In various embodiments, the polypeptide solution is lyophilized in an aqueous NH4HCO3 solution. Then, the lipid powder and the ApoA-I powder are mixed. ApoA-I and lipids (e.g., sphingomyelin) were isolated by dissolving the AI ​​powder in glacial acetic acid. This solution is then freeze-dried and the resulting mixture is concentrated in an aqueous medium. Hydration of the resulting powder at 20° C. forms HDL-like apolipoprotein-lipid complexes. .

[0283] In some embodiments, co-freezing of phospholipids and protein solutions or suspensions is performed. Drying allows the formation of the ApoA-I-lipid complex. In an organic solvent or mixture of organic solvents A homogenous solution of ApoA-I and lipids (e.g., phospholipids) was lyophilized and then added to an aqueous buffer. Hydration of the lyophilized powder in a buffer resulted in the spontaneous formation of ApoA-I-lipid complexes. Examples of organic solvents and solvent mixtures for use in the method of the present invention include, but are not limited to, Although not used, acetic acid, acetic acid / xylene mixture, acetic acid / cyclohexane mixture, and and methanol / xylene mixtures.

[0284] An aliquot of the resulting reconstituted preparation is characterized to determine whether the complexes have the desired size distribution. For example, it is possible to characterize the size of HDL. An exemplary method for this purpose is gel filtration chromatography. A series of proteins with different diameters, as well as human HDL, were used as standards to calibrate the columns. It can be used as:

[0285] In another embodiment, ApoA-I and U.S. Patent Application Publication No. 2006 / 02173 12 and International Patent Application Publication No. WO2006 / 100567 (PCT / IB2006 / 000635) (the disclosures of which are incorporated herein by reference). The recombinant ApoA-I-lipid complex is generated by complexing the lipid with the lipid.

[0286] U.S. Patent Nos. 6,004,925, 6,037,323, and 6,046,166 and 6,287,590, which are incorporated herein by reference in their entireties. A simple method for preparing negatively charged lipoprotein complexes with similar characteristics to DL A method is disclosed, comprising: Co-lyophilization of lyophilized cellulose and formation of negatively charged lipoprotein complexes during hydration of the lyophilized powder This method has the following advantages: (1) the method requires very few steps; (2) the method uses inexpensive solvents; (3) most or all of the components involved are The molecules are used to form the designed complexes, thus being general to other methods. (4) avoiding waste of starting material that may be lost during storage; (5) providing lyophilized complexes that are highly stable during storage; (5) the resulting complex can be reconstituted immediately prior to use; (6) The complexes do not usually require further purification after formation and before use; (7) toxic compounds, including detergents, are avoided; and (7) the manufacturing process is easily scaled up. This is favorable for GMP manufacturing (i.e., in an endotoxin-free environment). It is appropriate.

[0287] Other suitable methods are described in U.S. Patent Application Publication No. 2006 / 0217312 and International Patent Application Publication No. Described in published application WO2006 / 100567 (PCT / IB2006 / 000635) No. 6,393,636, the disclosures of each of which are incorporated herein by reference.

[0288] Preferably, one of the complexes is prepared in order to minimize oxidation of the protein and lipid components. Perform two or more or all steps under an inert gas (e.g., nitrogen, argon, or helium). Run under a blanket.

[0289] The protein and lipid concentrations of the apolipoprotein-lipid particles in solution are limited. Although not a part of the current literature, protein and phospholipid assays, chromatographic methods, e.g. Equipped with a variety of detectors such as PLC, gel filtration, mass spectrometry, UV or diode array It can be measured by any method known in the art, such as GC, fluorescence, elastic light scattering, etc. Additionally, lipid and protein integrity was assessed by the same chromatographic techniques. Peptide mapping, SDS-page gel electrophoresis, N- and C- It can also be determined by terminal sequencing and standard assays for determining lipid oxidation. Cut.

[0290] Pharmaceutical Compositions The pharmaceutical compositions contemplated by the present disclosure are suitable for administration and delivery in vivo. The negatively charged lipoprotein complex is contained as an active ingredient in a suitable pharma- ceutically acceptable carrier. Peptides may contain acidic and / or basic ends and / or side chains, so that The peptidomimetic apolipoprotein may be administered in the form of a free acid or base, or in a pharma- ceutical acceptable salt. The compound may be contained in the composition in the form of an acceptable salt. Modified proteins, such as PEGylated or pegylated proteins, may also be used. The pharmaceutical compositions may comprise one or more of the following compounds: The lipoprotein complexes may include those loaded with hydrophobic, lipophilic, or non-polar active agents. stomach.

[0291] Injectable compositions may be prepared as sterile suspensions, solutions or emulsions of the active ingredient in an aqueous or oily vehicle. The composition may also contain formulatory agents, such as suspending, stabilizing and / or dispersing agents. In some embodiments, the injectable composition is phosphate buffered saline (10 Negatively charged liposomes in 10 mM sodium phosphate, 80 mg / mL sucrose, pH 8.2 The compositions for injection are delivered in unit dosage form, for example in ampoules or in multiple doses. It can be provided in multi-dose containers and may contain added preservatives. A negatively charged polymer such as ethylene vinyl acetate or any other compatible material known in the art. and providing the composition in an infusion bag made of a material compatible with the lipoprotein complexes. can be done.

[0292] A preferred formulation is one that provides a final concentration of lipoprotein of about 5 mg / mL to about 15 mg / mL. In a particular embodiment, the dosage form contains about 8 mg / Apolipoprotein AI of about 8 mg / mL to about 10 mg / mL, preferably about 8 mg / mL Contains negatively charged lipoprotein complexes at final concentrations.

[0293] Preferably, the pharmaceutical composition is inactivated to minimize oxidation of the protein and lipid components. Formulated and / or prepared under a blanket of reactive gas (e.g., nitrogen, argon or helium) is filled.

[0294] 6.7. Treatment method Lipoprotein complexes as described herein, e.g., negatively charged lipoprotein complexes. and compositions that have been shown to be useful for virtually any lipoprotein complex of interest. It can be used for the body. Lipoproteins, such as negatively charged lipoprotein complexes The protein complex is a measure of the amount of apolipoprotein (A) required by currently available treatment regimens. 20mg / kg to 100mg / kg per dose, given every 2-5 days, for an average-sized human Even when administered at doses significantly lower than 1.4g to 8g per person, This is useful for moving the rule.

[0295] As a result, the conjugates and compositions of the present disclosure are useful in treating cardiovascular diseases, disorders and / or related conditions. The present invention is particularly useful for treating or preventing cardiovascular disease, disorders and / or Methods of treating or preventing a related condition generally involve administering a low (<15 mg / kg ) a dose or amount of a lipoprotein complex or pharmaceutical composition as described herein, This includes administering the compound according to a regimen effective to treat or prevent a condition.

[0296] The lipoprotein complex is administered in an amount sufficient or effective to provide a therapeutic benefit. In the context of treating vascular diseases, disorders and / or related conditions, the therapeutic benefit may include The following can be inferred when one or more of the following occurs: Increased teratogenic transport, decreased atherosclerotic plaque volume, and reduced mean plasma triglycerides Increased hepatic transaminase (or alanine aminotransferase) levels Free cholesterol compared to baseline levels without an increase above the normal range in serum cholesterol levels Increase in the high-density lipoprotein (HDL) fraction of sterols. Complete healing is desirable, but It is not necessary for the therapeutic benefit to exist.

[0297] In some embodiments, the lipoprotein complex is administered at about 2 mg / injection. The dose is about 12 mg / kg of ApoA-I equivalent. In some embodiments, the lipoprotein complex is administered at about 3 mg / kg of ApoA-I equivalent. In some embodiments, the lipoprotein complex is administered in a dose of about 6 mg. In some embodiments, the lipoprotein is administered at a dose equivalent to 100 mg / kg of ApoA-I. The protein complex is administered at a dose equivalent to approximately 12 mg / kg of ApoA-I.

[0298] The subject to be treated is an individual suffering from a cardiovascular disease, disorder and / or related condition. The lipoprotein complexes and compositions described herein can be used to treat or prevent Non-limiting examples of such cardiovascular diseases, disorders and / or related conditions include peripheral blood Vascular disease, restenosis, atherosclerosis, as well as, for example, stroke, ischemic stroke, Transient ischemic attack, myocardial infarction, acute coronary syndrome, angina pectoris, intermittent claudication, critical limb ischemia, valve There are many different clinical manifestations of atherosclerosis, including atherosclerosis, stenosis, and atrial valvular sclerosis. Subjects were randomly assigned to receive a randomized controlled trial of pulmonary circulation therapy (NCT02246632) for the treatment of myocardial infarction (with or without ST-segment elevation) or unstable angina. The subject to be treated may be an individual who has previously suffered from an acute coronary syndrome, such as The subject may be an animal, for example a mammal, particularly a human.

[0299] In one embodiment, the method changes the patient's baseline Apoa-I after administration. and administering to a subject an amount of a charged lipoprotein complex or composition described herein that does not induce The present invention also encompasses a method of treating or preventing cardiovascular disease, comprising administering to said patient a therapeutically effective amount of

[0300] In other embodiments, the method includes increasing the level of a given concentration of a given protein by 50% or more relative to the baseline (initial) level prior to administration. Approximately 5 mg / dL to 100 mg / dL approximately 2 hours after administration or approximately 5 mg / dL to 100 mg / dL approximately 24 hours after administration 20 mg / dL-20 mg / dL higher serum levels of free or complexed apolipoproteins A lipoprotein complex (e.g., a charged A method for treating or preventing cardiovascular disease, comprising administering to a subject It encompasses the law.

[0301] In another embodiment, the method comprises increasing the initial HDL-cholesterol fraction before administration. at least about 10% higher in the circulating HDL-cholesterol fraction at least one day after administration A lipoprotein complex as described herein (e.g., The present invention relates to a method for treating or preventing cardiovascular disease, comprising administering to a subject a The present invention includes a method for preventing the

[0302] In another embodiment, the method comprises administering 30 to 300 mg / d between 5 minutes and 1 day after administration. The present invention provides a method for the preparation of a composition comprising administering to a subject ... A lipoprotein complex (e.g., a charged complex) or composition according to any one of claims 1 to 5 is administered to a subject. The present invention also encompasses methods of treating or preventing cardiovascular disease, comprising administering to a subject a therapeutically effective amount of

[0303] In another embodiment, the method comprises administering 30 to 300 mg / d between 5 minutes and 1 day after administration. L. administering to a subject a lipoprotein complex (e.g., a charged complex) or composition of The present invention encompasses a method for treating or preventing cardiovascular disease, comprising:

[0304] In yet another embodiment, the method further comprises measuring the baseline (initial) level of an increase in fecal cholesterol excretion at least one day after administration of at least about 10% A lipoprotein complex (e.g., a charged complex) as described herein in an amount effective to achieve and a method for treating or protecting against cardiovascular disease comprising administering to a subject a therapeutic agent or composition comprising administering to said ... Includes.

[0305] Lipoprotein complexes, such as the negatively charged lipoprotein complexes described herein, or or compositions, alone or in combination with other agents used to treat or prevent said conditions. Such therapies include, but are not limited to, However, simultaneous or sequential administration of the drugs included in the treatment is not required. Hypercholesterolemia, such as cholesterolemia (homogeneous or heterogeneous) or atheroma In the treatment of arteriosclerosis, charged lipoprotein preparations are used in combination with any of the currently used Cholesterol-lowering therapy with one or more of the following: It can be administered with morphine, cholesterol absorption inhibitors and / or fibrates. Such combination regimens are beneficial because each agent has an important role in cholesterol synthesis and transport. These may offer particularly beneficial therapeutic effects because they act on different targets in the Resins affect cholesterol salvage, chylomicron and LDL populations; Statins primarily affect VLDL and LDL populations; statins inhibit cholesterol synthesis. , reducing LDL population (and possibly increasing LDL receptor expression); On the other hand, lipoprotein complexes, such as the negatively charged lipoprotein complexes described herein, , affecting RCT, increasing HDL, and promoting cholesterol efflux.

[0306] In another embodiment, the negatively charged lipoprotein complexes described herein are Lipoprotein complexes or compositions may be used in combination with fibrates to treat coronary heart disease ;Coronary artery disease;Cardiovascular disease, restenosis, vascular or perivascular disease;Atherosclerosis (including the treatment and prevention of atherosclerosis) Exemplary formulations and treatment regimens are described below.

[0307] Lipoprotein complexes, such as the negatively charged lipoprotein complexes described herein, or or the composition is a fraction of small HDL fractions, e.g., pre-β, pre-γ and pre-β-like HDL. The present invention is directed to administering the compound at a dose that increases the HDL fraction, αHDL fraction, HDL3 and / or HDL2 fraction. In some embodiments, the dose can be determined using, for example, magnetic resonance imaging. When measured by imaging techniques such as MRI or intravascular ultrasound (IVUS), It is effective in achieving atherosclerotic plaque reduction. Parameters to be evaluated include, but are not limited to, atheroma volume from baseline. The parameters tracked by MRI include change in atheroma rate and change in total atheroma volume. The present invention relates to, but is not limited to, IVUS and plaque lipid composition and and calcification.

[0308] Plaque regression can be measured (over time) using patients as their own controls. 0, at the end of the last infusion, or within several weeks of the last infusion, or at the start of therapy Within three months, six months, or one year after the start of the treatment.

[0309] Intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), and intraperitoneal (IP) injections Administration can best be accomplished by any parenteral route. In certain embodiments, For this purpose, administration may be by perfusor, infiltrator, or catheter. In some embodiments, lipoprotein complexes, e.g., negatively charged The charged lipoprotein complexes were administered to achieve circulating serum concentrations equivalent to those achieved by parenteral administration. by injection, by subcutaneously implantable pump, or by depot preparation in an amount that achieves The complex can also be absorbed into, for example, a stent or other device. It is also possible.

[0310] Administration can be accomplished through a variety of different treatment regimens. For example, several intravenous Intravenous injections should be given periodically throughout the day so that the cumulative total of injections does not reach a daily toxic dose. This method can be used with lipopolysaccharide injections at intervals of 6, 7, 8, 9, 10, 11 or 12 days. In some embodiments, the method comprises administering a lipoprotein complex. The conjugates are administered at weekly intervals.

[0311] The method includes the step of: For example, in one embodiment, the method may further include administering the lipoprotein complex once. In this study, lipoprotein complexes were administered six times with one week intervals between each administration. In some embodiments, administration is as a series of injections followed by treatment for 6 months to 1 year. The injection series can then be stopped and another series of injections can then be started. It can be administered annually or every 3 to 5 years. A series of injections is given daily (specific blood perfusion to maintain plasma levels), for several days (e.g., 4 injections over 8 days), or is administered over a period of several weeks (e.g., 4 injections over 4 weeks), followed by 6 months to 1 year later. For chronic conditions, administration can be resumed continuously. Optionally, the method may be preceded by an induction phase, in which the lipoprotein complexes are administered more frequently. This can be done.

[0312] In yet another option, a dose of about 1 to 5 mg / dose (50 to 200 mg) is administered. Starting at a low dose, then administering high doses of 200 mg to 1 g per administration. Depending on the needs of the patient, administration can be by slow infusion over a period of more than one hour, This may be by rapid infusion over an hour or less, or by a single bolus injection.

[0313] The toxicity and therapeutic efficacy of various lipoprotein complexes were evaluated using the LD50 (density for 50% of the population) ED50 (the dose that is therapeutically effective in 50% of the population) Determined using standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is defined as the LD50. It can be expressed as a ratio of ED50 to ED50. Lipoprotein complexes such as lipoprotein complexes are preferred. Limiting examples include liver function transaminases (less than or equal to 2x normal baseline levels). This is because too much cholesterol is brought to the liver, which has no way of assimilating such an amount. The movement of cholesterol from red blood cells is a sign of the inability of red blood cells to move cholesterol. Monitor the effect on red blood cells as it can make them fragile or affect their shape. It is also possible.

[0314] A few days to several days before, during or after a medical procedure (e.g., preventive treatment) Patients can be treated for up to 12 weeks. n), rotoblader or organ transplant (e.g., heart, kidney, liver, etc.), etc. The treatment may be adjunctive to or simultaneous with another invasive therapy.

[0315] In certain embodiments, the negatively charged lipoprotein complex is a cholesterol complex. is administered to patients whose cholesterol synthesis is controlled by statins or cholesterol synthesis inhibitors. In another embodiment, the negatively charged lipoprotein complexes are composed of bile salts and cholesterol. To capture sterols, it increases bile acid excretion and lowers blood cholesterol levels. To achieve this, a binding resin, such as a semi-synthetic resin, such as cholestyramine, or a fiber , for example, to a patient undergoing treatment with vegetable fiber.

[0316] 6.8. Other Uses Lipoprotein complexes as described herein, e.g., negatively charged lipoprotein complexes The subject matter and compositions can be used in vitro, for example, for diagnostic purposes to measure serum HDL. ApoA-I, ApoA-II and ApoA-II can be used in assays at 100 The peptide binds to the HDL components of serum, thus freeing negatively charged lipoprotein complexes. , as a "marker" for the HDL population, as well as the pre-β1 and pre-β2 HDL populations In addition, negatively charged lipoprotein complexes have been shown to be effective in RCTs. For this purpose, negatively charged HDL can be used as a marker for the effective HDL subpopulation. The charged lipoprotein complexes are added to or mixed with the patient serum sample. After a suitable incubation period, the incorporated negatively charged ligand can be By detecting the polyprotein complex, HDL content can be assayed. This involves the use of labeled negatively charged lipoprotein complexes (e.g., radioactively or fluorescently labeled). , enzyme labels, dyes, etc.) or for negatively charged lipoprotein complexes This can be achieved by immunoassay using specific antibodies (or antibody fragments). Cut.

[0317] Alternatively, labeled negatively charged lipoprotein complexes can be used to visualize the circulatory system. To monitor RCTs or to determine whether HDL is active in cholesterol efflux In fatty streaks, atherosclerotic lesions, etc., which should be To visualize HDL accumulation, imaging procedures (e.g., CAT scan, MRI scan) are performed. It can be used in the following cases.

[0318] Examples and Data Related to the Preparation and Characterization of Specific ProApoA-I-Lipid Conjugates No. 2004 / 0067873, the disclosure of which is incorporated by reference in its entirety. (which is incorporated herein).

[0319] Data obtained in animal model systems using specific proApoA-I-lipid complexes and U.S. Patent Application Publication No. 2004 / 0067873, the disclosure of which is incorporated herein by reference in its entirety. (incorporated herein). EXAMPLES

[0320] 7. Example 1: Development of ApoA-I expression system 7.1. Cloning of human ApoA-I in Chinese hamster ovary (CHO) cells Detection and Expression 7.1.1. Preparation of ApoA-I Expression Vector The preproApoA-I gene sequence was obtained from NCBI (P02647) and cloned Flanking sequences were added to facilitate gene expression and improve expression. The preproApoA-I coding DNA was synthesized and expressed in the Bluescript KS+ vector The 5' flanking sequence was an optimized Kozak translation sequence. HindIII and BglII restriction enzymes were used to create Bluescript The preproApoA-I insert was excised from the KS+ vector. The fragment was gel purified. and the following genetic elements: Molony Murine Sarcoma virus 5 'Human cytomegalovirus promoter fused to LTR, MoMuLV / SV package Caging region, immediate early cytomegalovirus promoter, multiple cloning site The nucleotide sequence of the ... The resulting construct was ligated into a retroviral expression vector carrying the clone. The clones were sequenced throughout the gene and flanking regions. Loan #17) was selected based on its match to the predicted DNA sequence. Expression vector for icular Stomatitis Virus envelope glycoprotein For transduction using 293GP cells cotransfected with the present plasmid, A vector was prepared by cotransfecting the cells and concentrating the vector. The supernatant was used in the CHO-S transduction step described below in Section 7.1.2.

[0321] 7.1.2. Preparation of Mammalian Cell Lines for Expression of ApoA-I Chinese hamster ovary cells (CHO- S) in a three-round fashion using the retroviral vector described above in Section 7.1.1. The cells were then subjected to transduction (1X, 2X and 3X). For cryopreservation after each transduction, The pooled population was expanded and a sample of cells was submitted for gene copy analysis. The numbers are shown in Table 2 below. The 3X transduced cell lines were cultured twice in 125 mL flasks for 16 days. The productivity in the fed-batch test was analyzed, and the results are shown in Table 3.

[0322] [Table 2]

[0323] [Table 3]

[0324] 7.1.3. Stability of Cell Lines Expressing ApoA-I The master cell bank producing ApoA-I, CHO-S-ApoA-IR (3 X) Clone 17 was examined for its stability of viability, proliferation rate, protection of gene insert, and Non-GMP characterization studies were performed to assess the stability and consistency of product secretion during long-term culture. .

[0325] Cell lines were thawed from a master cell bank and cultured in PF CHO LS medium (HyClone, The cells were cultured in 125 mL shake flasks using a 100-mL shake flask (Logan UT). The cells were serially cultured by successive passage from generation 4 to generation 43. At the end of the culture, cells derived from all generations were frozen. Cell samples were thawed and used to characterize ApoA- A final culture run was performed to compare protein production from each sample. The supernatants were tested on day 12 using reverse phase HPLC analysis to determine the level of ApoA-I production. The ApoA-I concentrations ranged from 1259 mg / L to 1400 mg / L for the various samples. / L (Figure 2).

[0326] To compare the stability of gene inserts, CHO cell samples were used for DNA isolation. The number of gene inserts in each sample was determined using real-time PCR on genomic DNA. The PCR-based index of repeat count is based on the standard deviation of overlap between generations 0 and 43. The production and gene expression of ApoA-I from the master cell bank cell lines were significantly different. Copy index values ​​were found to be stable over the 43 generations examined.

[0327] [Table 4]

[0328] 7.2. Cell Growth and Harvesting of ApoA-I 7.2.1. Inoculum and scale-up to 200 L production bioreactor C stably transfected with the human ApoA-I gene from a master cell bank Thaw a vial of HO-S cells in a 37 °C water bath and add 35 mL of HyQ PF-CHO L A single shake flask (250 mL) containing cell culture medium (Thermo Fis The initial cell count was determined using a hemocytometer. , 2.48x10 5 The viability of the culture was 93.8%. The cells were incubated in a humidified 5% CO2 environment at 37 °C in an Orbit shaking incubator. The subculture step was performed at approximately 1.6x10 5 Inoculation of cells / mL The density was targeted. The cell count and viability on day 4 in the flask was 15.01x10 5 The number of cells / mL and viability was 93.9%. The 250 mL flask was replaced by a 1 L flask. The average viable cell count on day 3 from a 1 L flask was 13.56 x 10 5 Thin The viability was 95.3%. 0mL and 1.75x10 5 10 in initial culture volume and initial target cell density in cells / mL L disposable Wave Bag (GE Healthcare Bioscience Wave Bioprocess Corp, Somerset, NJ The cells were subcultured in the Wave Bioreactor System 20EH. rOperating settings were 37°C incubation temperature, 15.0 cpm shaking speed, 10. 5.0% C in aeration gas using a shaking angle of 9° and a gas flow rate of 0.25 L / min After 3 days of culture, the viable cell density in the Wave Bag was 10.73x10 5 cells / mL, and fresh HyQ PF-CHO LS was added to bring the culture volume to 5000. After an additional 3 days, the viable cell density was 13.25x10 5 cells / mL, Wav The volume in the eBag was transferred to a 30 L bioreactor. Temperature, pH, dissolved oxygen, pressure, The operating set points in the 30 L bioreactor for agitation rate and agitation speed were 37 °C, pH 7.0, 40% air saturation, 1 psig pressure, and 50 rpm agitation speed. .

[0329] The viable cell density in the 30L bioreactor was 10.80x10 on day 3 of culture. 5 cell / mL, which is 2.40x10 5 200 L bioreactor at initial target density of cells / mL The entire contents of the 30L were transferred, and the weight after inoculation was The amount, cell density and viability were 134.5Kg, 2.37x10, respectively. 5 cells / mL and 90.9%. The results of 20 studies on temperature, pH, dissolved oxygen, pressure, and agitation speed were The operating set points for the 0 L bioreactor were 37°C, pH 7.0, and 40% empty, respectively. The conditions were: air saturation, 1 psig pressure, and 35 rpm agitation speed. On day 3, the cell density was 1 5.71x10 5 cells / mL, which corresponds to the full volume of 60 L (v / v) in the bioreactor. All medium (AGT CD CHO 5X, Invitrogen) and 200 mM L- It was sufficient to add glutamine (final concentration 10 mM) solution. On the second day, glucose levels dropped to below 5 g / L, which was the same as the first day after 3 g / L of glucose (2 On day 9, the viable cell density was 33.20x10 5 Pi in cells / mL The viability of the cells reached 92.5%. The viability of the cells reached 78.5% on day 13. The actors were harvested. The cell counts and viabilities of the cultures throughout the culture period are shown in Figure 3A. The ApoA-I concentration in the culture medium throughout the culture period is shown in FIG. 3B.

[0330] 7.2.2. Cell harvest, cell isolation and storage The contents of the bioreactor were then transferred to two Cuno (Rutherford, NJ, USA) 60M02 Maximizer filter, followed by Millipore 0.22μ By passing it through the m Opticap (Billercia, MA, USA), The medium from the bioreactor was harvested into 200 L bags. The clarified medium was then The clarified medium was stored at 2-8°C until the homogenization step. Filter through a 2 L sterile PET bottle (Billerica, MA, USA). The mixture was dispensed into G bottles (ThermoFisher, Marietta, OH, USA). The samples were then frozen at -20°C until shipment. EXAMPLES

[0331] 8. Example 2: Development of ApoA-I purification system 8.1. Materials and Methods Expression, Primary Isolation and Conditioning. ApoA-I clarified cells obtained as described in Example 1 were Cell growth medium (approximately 1.8 L) was thawed by storage at ambient temperature. The thawed medium was Anion exchange chromatography was performed by lowering the pH to 5.3 ± 0.2 with 1 M HCl. The conditions were then adjusted for graphy.

[0332] Purification of ApoA-I. A GE Healthcare anion exchange column was loaded onto a 20 ml TAMP A buffer. The column was equilibrated with 100 mM sodium phosphate, pH 5.3. The pH of the column effluent was approximately 5. Equilibration was deemed complete when the elution volume was 5.5. The conditioned filtrate was diluted with water at 3.7 cm / ml The column was loaded with 25-35 g of ApoA-I / L of anion exchange resin at a flow rate of n. ApoA-I was washed through the column and collected in the TAMP A flow-through.

[0333] The ApoA-I containing flow-through from the anion exchange column was purified using 1M NaOH. The pH was adjusted to 8.0±0.2. The solution was then pumped at approximately 12.5 L / h / m 2 At a flow rate of 0. The mixture was filtered through a 2 μm Planova 20N filter (Asahi Kasei Medical). The viruses and virus particles were removed.

[0334] A Source 30 reversed-phase chromatography column with a bed height of 25 cm was used. Add TAMP D buffer (2 mL) until equilibrated when the eluate reaches pH 9.5. 0 mM ammonium carbonate, pH 9.5). The filtrate containing the AI ​​was loaded onto the column at a flow rate of 2.8 cm / min. ApoA-I was adsorbed onto the matrix and incubated at 35–50% acetate in TAMP D buffer. The column was eluted with a gradient of acetone.

[0335] A reversed-phase silica C18 column (300 Å 10 μm) packed to a bed height of 25 cm was used. Add TAMP E buffer (1 mL) until the eluate is equilibrated when it reaches pH 9.5. The C18 column was then washed with 4.00 mM ammonium carbonate, pH 9.5. The loading was 7 g ApoA-I / 1 L of matrix. ApoA-I was eluted in a 40-50% acetonitrile gradient in TAMP E buffer. He made him do so.

[0336] The fractions were pooled and concentrated approximately 2.5-fold, and then eluted with 15 volumes of TAMP C buffer (3 mM The concentrate was diafiltered against sodium phosphate, pH 8, to remove 100% EDTA from the C18 column. Acetonitrile was removed from the ApoA-I-containing fraction eluted from the diafilter. The pH of the AI ​​solution was lowered to approximately 6.0 using dilute phosphoric acid, and then the solution was incubated in a Mustang Q anionizer. DNA and The Mustang Q filtrate was diluted with 5 volumes of TAMP C to remove lysates and host cell proteins. Diafiltered against buffer.

[0337] The diafiltered filtrate is then separated into 28,000 daltons of ApoA-I, which is retained by the membrane. A polyethersulfone membrane (Fil) with a molecular weight cut-off of 10,000 Daltons was used as a The final ultrafiltration was performed using a chromatograph (Tron Omega series). The plate was scanned and the ratio of the intensity of the purified ApoA-I band region to the total intensity of all bands was measured. The protein solution had a concentration of 7.8–17 g / L of pure ApoE, as determined by It contained AI.

[0338] 8.2.Results Characterization of ApoA-I. The purity of the ApoA-I product was assessed by SDS-PAGE. The results showed that the ELISA kit was >99% pure and contained low levels of DNA and host cell proteins. and the amount of truncated ApoA-I was undetectable. See FIG. EXAMPLES

[0339] 9. Example 3: Optimization of Lipoprotein Complex Components 9.1. Preparation of apolipoprotein and phospholipid components ProApoA-I: The protein proApoA-I contains approximately 90 mg of protein. In individual lyophilized 100 mL flasks, the Unite de Biotechn ologie, Institut Meurice, Hte Ecole Lucia De Brouckere, 1 Avenue Emile Gryzon, B-107 0 Anderlecht, Belgium. Batch no. The protein was kept at approximately 4°C until use. The proApoA-I content was 3.225 mg / mL, and the urea content was approximately 0.011 mg / mL. Approximately 630 mg of proApoA-I was dissolved in 25.6 mL of acetic acid / water 5%. A solution of proApoA-I was prepared by the above procedure. The final concentration of the solution was 25 mg / mL. It was.

[0340] ApoA-I: ApoA-I was prepared as described in Example 1 above.

[0341] Sphingomyelin: egg-derived sphingomyelin (Coatsome® NM -10) is NOF Corporation, 1-56, Ohama-Cho, Amagasaki-S The batch number was 05. The sphingomyelin was kept at approximately -20°C until use. The purity of sphingomyelin was 99.1%. 799.4 mg of purified sphingomyelin was Spiking was prepared by dissolving in 6 mL of acetic acid / water 5% to give a final concentration of 50 mg / mL. A solution of engomyelin was prepared.

[0342] Phosphatidylglycerol: 1,2-dipalmitoyl-SN- as the sodium salt Glycero-3-phosphatidylglycerol (DPPG-Na, Coatsome Trademark) MG-6060LS) is a trademark of NOF Corporation, 1-56, Oohama-Cho, Am The product was supplied by Agasaki-Shi, 660-0095, Japan. The batch number was 0309651L. DPPG-Na was stored at approximately -20°C until use. The purity of DPPG-Na was 99.2%. DPPG by dissolving in 1 mL of acetic acid / water 5% to obtain a final concentration of 50 mg / mL A solution of -Na was prepared.

[0343] Phosphatidylcholine: Di-palmitoylphosphatidylcholine (DPPC) was obtained commercially. were obtained from public sources.

[0344] 9.2. Preparation of Lipoprotein Complexes The following lipoprotein complexes were prepared: (a) Neutral lipoprotein complexes: Formulation A: Protein : The weight ratio of phospholipids is 1:2.5 Pro ApoA-I Yobi S M; b. Formulation B: Protein : The weight ratio of phospholipids is 1:2.7 Pro ApoA-I Yobi S M; c. Formulation C: Protein : The weight ratio of phospholipids is 1:3.1 Pro ApoA-I Yobi S M; d. Formulation D: with a 50:50 SM:DPPC wt ratio , Li Polyprotein wt: Total phospholipids wt ratio is 1:2.7 proApoA-I, SM and DPPC; e. Formulation E: Protein : The weight ratio of phospholipids is 1:2.7 Pro ApoA-I And SM. (b) Negatively charged lipoprotein complexes: a. Formulation F: having a SM:DPPC:DPPG wt:wt ratio of 48:48:4; Lipoprotein wt:total phospholipids wt ratio is 1:2.7 Pro-ApoA-I, SM, D PPC and DPPG; b. Formulation G: having a SM:DPPC:DPPG wt:wt ratio of 73:23:4; Lipoprotein wt:total phospholipids wt ratio is 1:2.7 Pro-ApoA-I, SM, D PPC and DPPG; c. Formulation H: Lipoprotein wt having a SM:DPPG wt:wt ratio of 97:3 t: total phospholipid weight ratio is 1:2.7 ApoA-I, SM and DPPG; d. Formulation I: Lipoprotein wt having a SM:DPPG wt:wt ratio of 97:3 t: total phospholipid weight ratio is 1:3.0 ApoA-I, SM and DPPG; e. Formulation J: Lipoprotein wt having a SM:DPPG wt:wt ratio of 97:3 t: total phospholipid weight ratio is 1:3.3 ApoA-I, SM and DPPG.

[0345] 9.3. Rate and uniformity of formation of lipoprotein complexes The samples are injected into the HPLC system and examined for size and distribution of lipoprotein complexes. The formation of lipoprotein complexes from formulations A to J was examined by simultaneous homogenization. The complexes were prepared by and sampled at the indicated times.

[0346] Figure 5 shows the results of the study, which includes formulations A through C, which contain different lipoprotein:SM wt:wt ratios. and neutral phospholipids of 50:50 SM:DPPC in a 1:2.7 ratio. Exemplary HP for neutral lipoprotein complex according to formulation D containing lipids and neutral phospholipids The LC chromatograms are shown. A lipoprotein:SM wt:wt ratio of 1:2.7 was optimal. Formulation D, which contained a mixture of SM and DPPC, showed less complex formation.

[0347] The addition of phosphatidylcholine as a second neutral phospholipid resulted in a slow decomposition rate. Complete complex formation was obtained. Figure 6 shows the results of lipolysis of formulation D at 10, 20, 30 and 60 min. In contrast, as shown in FIG. Method B rapidly formed a complex.

[0348] Addition of the negatively charged phospholipid, DPPG, to SM and DPPC resulted in 20, 40, and 60% phospholipids in Fig. 8. The HPLC chromatograms of lipoprotein complexes of formulation F at 60 and 120 min are shown. As expected, this resulted in even less complex formation.

[0349] As shown in FIG. 9, SM was used as the phospholipid at a protein:lipid weight ratio of 1:2.7. Lipoprotein complexes containing α-HDL formed more pre-B HDL complexes and Lipoproteins with the same protein:lipid weight ratio but containing DPPC and / or DPPG It does so more quickly than protein complexes. Thus, lipoproteins containing only SM as neutral lipids The protein complexes contained DPPC in addition to SM, with or without the addition of DPPG. The complex forms more homogeneous lipoprotein complexes at a faster rate than complexes without the complex.

[0350] Finally, the phospholipid fraction contained SM and DPPG in a weight ratio of 97:3, 1:2. Negatively charged lipoprotein complexes containing an apolipoprotein:phospholipid weight ratio of 7-1:3. The aggregates consisted of complexes containing an apolipoprotein:phospholipid weight ratio of 1:3.3 and uncharged lipids. Compared to the polyprotein complex, it showed optimal homogeneity and no free lipid peak. See FIG. 10, which shows the HPLC chromatograms for formulations E, H, I and J. For further testing in animals (Example 6), lipoproteins according to formulations B and H Conjugates are selected and, based on the results of the animal studies, are evaluated clinically in human patients (see Example 6). and 8), the lipoprotein complex of formulation H was selected. EXAMPLES

[0351] 10. Example 4: Formation of Lipoprotein Complexes Using a Thermal Cycling-Based Method 10.1. Procedure for preparing ApoA-I / DPPG / sphingomyelin complex Overview of Phosphate buffer with a protein concentration of 1-30mg / ml, typically 5-20mg / ml The frozen ApoA-I solution in 100% aqueous sodium chloride (pH 7-9) was thawed at 2-8°C for approximately 24-96 h. Prepare and weigh out sodium dihydrogen phosphate and sodium hydrogen phosphate in the following manner. to the oA-I solution to give a final peptide concentration of 10 mM in pH 7.4 buffer. .

[0352] The DPPG solution was dissolved in phosphate buffer (10 mM sodium phosphate, pH 8.0) at 50 Prepare by heating DPPG powder (NOF Corp.) to a temperature of at least 1. After thawing at ambient temperature for 5 h, it is weighed and added to the buffer container. DPPG is then added to the , ULTRA-TURRAX® High Performance Dispenser (IKA® Work The dispersion was carried out at a temperature of 50°C using a DPPG suspension and Heat the ApoA-I solutions to 57°C. Combine them and incubate at 57°C under nitrogen for 30 minutes. Heat. Allow the precomplex solution to cool to room temperature.

[0353] Sphingomyelin (SM) powder (NOF Corp.) was thawed at ambient temperature and then placed in a glass tube. Weigh out the phosphate buffer (10 mM sodium phosphate) thorium, pH 8.0) was heated to 50°C and mixed with SM powder to give 220 mg / ml SM Disperse SM powder into suspension using ULTRA-TURRAX® The dispersion was cooled to 4°C and then passed through a homogenizer. Dynamic Light Scattering (DLS) ) to monitor SM particles down to 55–70 nm Zeta (Z) average size (using intensity measurements). For example, input 32000+ / -3000bar and 10~18℃. Temperature and preferably output temperature of 30-40°C (and not exceeding 59°C) Nano Achieved using a DeBee homogenizer to obtain particles with a Z-average size of 58 nm It is possible.

[0354] For complexation, the ApoA-I / DPPG mixture and the SM dispersion were separately heated to 57°C. The warmed SM dispersion was added to the ApoA-I / DPPG mixture at an initial temperature set point of 57°C. After stirring to combine, the solution is cooled to 37°C and then heated in a series of Cycle (57°C to 37°C) to form the ApoA-I / DPPG / SM complex. This heating-cooling process continues with a contact time between temperatures of 5 to 30 minutes. The cooling cycle is continued until most of the protein components are incorporated into lipoprotein complexes. The size and distribution of the complexes during thermal cycling were analyzed by gel permeation chromatography. The reaction is monitored by gel permeation chromatography (GPC).

[0355] The ApoA-I / DPPG / SM complex was prepared according to the procedure described above (and illustrated in FIG. 11). The ApoA-I protein was prepared according to the sequence shown in FIG. The complex had an amino acid sequence similar to that of sphingomyelin (S M) and 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-rac -glycerol) (dipalmitoylphosphatidylglycerol or DPPG) The ratio of ApoA-I protein to total lipids was 1:2.7 weight / weight (w / w). , which is equivalent to a molar ratio of 1:108. On the left is Lauda Ecoline Sta r edition Type 26LE water bath, in which the samples are thermally cycled On the right side is a shell and tube heat exchanger with a holding capacity of 18 ml (model EF-C50-HE) and connected by a peristaltic pump, arranged as described in FIG. The combined lipid and protein were incubated at temperatures between 57°C and 37°C for 5 minutes at each temperature using a heat exchanger. The protein components were cycled.

[0356] Add 180 mg of DPPG to 2.82 grams of 10 mM phosphate buffer, pH 8.0. The mixture was added at 50°C for 10 minutes using the ULTRA-TURRAX® dispenser. The suspension was dispersed by adding 22 grams of SM powder to 78 grams of 10 mM phosphate buffer. The pH was adjusted to 8.0. The solution was incubated at 50°C for 20-40 minutes in ULTRA-TURRAX (registered trademark). The suspension was dispersed using a N ® dispenser set to an average particle size of 60 nm. SM was homogenized using anoBEE. DPPG, SM and proteins were incubated at 57°C. 18 mg (0.3 ml) of DPPG was dissolved in 10 mM phosphate buffer pH 7.4 was added to 214 mg of ApoA-I protein. After 30 min at 57°C, 559 1 mg of SM particles (2.54 ml) was added. The solution was then subjected to thermal cycling. The complex was formed.

[0357] ApoA- after 30, 60, 120, 180 and 210 min of thermal cycling Gel permeation chromatography showing I / DPPG / SM complex formation was performed using the same method as in FIG. 13A. 13E. As cycle time increases, as indicated by the increase in sharpness of the major GPC peaks, With increasing concentration, smaller complexes are produced. The peak corresponding to the uncomplexed protein also appears at times. It disappears with time.

[0358] 10.2. Formation of the ApoA-I / sphingomyelin complex Following the procedure above, but without precomplexing ApoA-I with DPPG, ApoA-I / The protein component was 5 ml of ApoA-SM complex at a concentration of 8.9 mg / ml. The lipid components were suspended in 10 mM phosphate buffer, pH 8.0, homogenized, and incubated for 60 min. 0.5 ml of egg sphingomyelin (220 mg / ml) was used to form lipid particles of 1 nm. The protein and lipid components were mixed at 50°C in a ratio of 1:2.5 wt:wt. The resulting suspension was subjected to thermal cycling for 18 hours (108 cycles) using the thermal cycling apparatus shown in FIG. The gel was subjected to thermal cycling for 10 min (10 min cycling time) and 20 min cycling time. This indicates that the protein / lipid complexes formed are essentially homogeneous (G in FIG. 14). See PC chromatogram).

[0359] 10.3. ApoA-I / DPPG / N-palmitoyl-4-hydroxysphinganib Formation of the choline-1-phosphocholine (phytosphingomyelin) complex ApoA-I / DPPG / phytosphingomyelin complex was prepared according to the procedure described above. Phytosphingomyelin particles were prepared with a size of approximately 183 nm (measured by DLS). and added to a 7.8 g / L protein:DPPG mixture to give a final ratio of 1:2.7. The protein:lipid (SM and DPPG) ratio achieved was xysphinganine-1-phosphocholine (phyto-sphingomyelin) and protein Quality: The suspension containing the DPPG component was incubated at 37°C for 10 min and 40°C for a total of 2 h. Thermocycling was performed for 6 cycles at 57°C for 10 min. Gel Permeation Chromatography The results show that the protein / lipid complexes formed are essentially homogeneous (Figure 15). See GPC chromatogram).

[0360] 10.4. Formation of ApoA-I / DPPG / synthetic palmitoyl sphingomyelin The ApoA-I / DPPG / synthetic palmitoyl sphingomyelin complex was prepared as follows: The synthetic palmitoyl sphingomyelinase was prepared in 10 mM phosphate buffer, pH 7.4. 8.8 mL of a solution of phosphorus (220 mg / ml) was mixed until a particle size of 3300 nm was reached. ApoA-I (945 mg at 14 mg / ml) was diluted with 0.03% by weight of DPPG (6 0 mg / ml) and heated at 50°C for 30 minutes. The phosphomicelles were mixed with the protein / DPP solution at an apolipoprotein:phospholipid weight ratio of 1:2.7. The protein and lipid suspension was incubated for a total of 240 minutes or until suitable incubation time. Heat-cool cycles of alternating 37°C and 57°C every 10 minutes were used until the particle size distribution was reached. The size and distribution of the complexes during thermal cycling were analyzed by GPC. After complexation, the concentration was adjusted to 8.0 mg ApoA-I / ml, and then the solution was isotonic. Combined with sucrose (40 mg / ml) and mannitol (20 mg / ml) for The lipoprotein complexes were assayed by GPC and were essentially homogeneous. (See the GPC chromatogram in FIG. 16).

[0361] 10.5. Formation of ApoA-I / DPPG / phytosphingomyelin The ApoA-I / DPPG / phytosphingomyelin complex was prepared as follows. Phytosphingomyelin (220 mg / m l) 2.0 mL of the solution was subjected to ULTRASOUND at 50°C for 40 min until a particle size of 990 nm was reached. ApoA-I (at a concentration of 7.8 mg / ml) was dispersed in A-TURRAX®. 15.6 mg at 57°C) was combined with 0.03% by weight of DPPG (60 mg / ml) and incubated at 57°C. The phytosphingomyelin solution was mixed with apolipoprotein A solution at a ratio of 1:2.7. The protein and DPPG mixture were then mixed in a weight ratio of 0.1:0.25 wt. The lipid suspension was stirred for 37 min every 10 min for a total of 240 min or until a suitable particle size distribution was achieved. The cells were thermocycled using alternating heating-cooling cycles of 57°C and 57°C. The population of the protein complex was determined by GPC and found to be approximately 92.6% homogeneous. (See GPC chromatogram in Figure 17).

[0362] 10.6. Complex formation with ApoA-I peptides ApoA-I peptide (H-Lys-Leu-Lys-Gln-Lys 5 -Leu-A la-Glu-Leu-Leu 10 -Glu-Asn-Leu-Leu-Glu 15 -A rg-Phe-Leu-Asp-Leu 20 -Val-Inp 22 -OH; SEQ ID NO: 4) The solution is used to mix the ApoA-I peptide, A complex of DPPG and sphingomyelin was generated. The phospholipid components were 48.5: Egg sphingomyelin (SM), 1,2-dipalmitoyl-sn, in a weight ratio of 48.5:3 -glycero-3-phosphocholine (dipalmitoylphosphatidylcholine, DPPC) and and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycero Dipalmitoylphosphatidyl-glycerol (DPPG) is a peptide. The ratio of drug to total phospholipid complex is 1:2.5 (w / w). In buffered saline (12 mM sodium phosphate, 130 mM sodium chloride, pH 8.2) The starting solution was diluted with 50 ml of CER-522 complex at a flow rate of 1 ml / min for 2 hours. Complexes were allowed to form by thermal cycling between 30°C and 37°C.

[0363] Gel permeation chromatography demonstrates that the protein / lipid complexes formed are essentially homogeneous. , indicating that the majority of the protein is incorporated into lipoprotein complexes (Figure 1 (See GPC chromatogram in Figure 18).

[0364] 10.7. Effect of lipid particle size and number of thermal cycles on complex formation The effect of lipid particle size and number of thermal cycles on particle size was examined. Four different starting lipid particles were Preparations of 100 nm diameters (with zeta averages of 85 nm, 77 nm, 66 nm and 60 nm) were 19A-19D, respectively, by passing the SM solution through the NanoBEE in a single pass. Each flow-through was analyzed for its zeta average. The lipid particle size was The lipid particles were collected until the desired size was achieved. The lipid particle size was determined by 5 or 7 cycles of 3 min at 37°C and 10 min at 57°C. , and were tested in the complex formation assay of Section 10.1.

[0365] When the lipid component is mixed with the protein component, the resulting suspension is cloudy. As the mixture is mixed, this turbidity decreases and the solution becomes clearer.

[0366] After five cycles, the suspension of complexes prepared from 60 nm lipid particles was the clearest. The GPC chromatograms (Figures 20A-20D) were characterized by the uniformity of the main peaks. As shown in Fig. 1, all samples showed complete or nearly complete complexation. When starting from particles, the resulting suspension of lipoprotein complexes is detected by GPC There was no possible uncomplexed protein, because all proteins were lipidated after five cycles. The resulting conjugate was 98% pure, suggesting that the conjugate was conjugated to a high quality substance. Afterwards, all four suspensions became clearer, indicating an even higher degree of complexation. The suspensions made with 60 nm lipid particles appeared the clearest.

[0367] In a separate study, 450 nm and 40 nm SM particles were analyzed using the method described in Section 10.1. The GPC chromatogram in Figure 21A shows the complex with ApoA-I and DPPG. As shown (peak at 9.8 min), when 450 nm SM particles were used as the lipid component, In this case, most, but not all, ApoA-I was incorporated into lipoprotein complexes. As shown in the GPC chromatogram in FIG. 21B (peak at 9.551 min), the lipid composition When 40 nm SM particles were used as the target particle, a much smaller fraction of ApoA-I was expressed as lipoprotein. was incorporated into the protein complex.

[0368] 10.8. Effect of starting temperature on complex formation The effect of initial thermal cycling temperature on complex formation ...

Claims

[Claim 1] The invention described in the present specification.

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