HK1181663A

HK1181663A - Method for producing a tetranectin-apolipoprotein a-i lipid particle, the lipid particle itself and its use

Info

Publication number: HK1181663A
Application number: HK13109025.5A
Authority: HK
Inventors: 马丁．巴德尔; 莫妮卡．贝纳; 阿德尔伯特．格罗斯曼; 西尔克．莫尔
Original assignee: 霍夫曼-拉罗奇有限公司
Priority date: 2010-08-30
Filing date: 2011-08-25
Publication date: 2013-11-15

Abstract

Herein is reported a method for producing a lipid particle comprising the following steps i) providing a first solution comprising denatured apolipoprotein, ii) adding the first solution to a second solution comprising at least two lipids and a detergent but no apolipoprotein, and iii) removing the detergent from the solution obtained in step ii) and thereby producing a lipid particle.

Description

Method for producing tetranectin-apolipoprotein A-I lipid particles, the lipid particles themselves and their use

The present invention relates to the field of lipoprotein and lipid particles. Herein is reported a method of producing a lipid particle comprising an apolipoprotein, phosphatidylcholine and a lipid, and tetranectin-apolipoprotein a-I.

Background

Plasma lipoproteins are soluble protein-lipid complexes that carry out lipid transport and metabolism in the blood. Several major classes of lipoproteins are distinguished based on their density, size, chemical composition and function. Among them, High Density Lipoprotein (HDL) particles, also referred to as high density lipid particles, are composed of several subclasses varying in average molecular weight from 180kD to 360 kDa. Their average lipid and protein contents were 50 wt%, respectively. Phosphatidylcholine (PC) comprises 38% of the total lipid, followed by cholesterol esters and small amounts of other polar and non-polar lipids, including free cholesterol. The major protein component is apolipoprotein A-I (Apo A-I), representing about 60% of the total protein weight in human HDL.

Cholesterol in the human body, particularly in circulating body fluids such as blood, does not exist in the form of isolated molecules, but exists in the form of complexes with certain proteins (lipoproteins). The major fraction of cholesterol is complexed with Low Density Lipoproteins (LDL) or with High Density Lipoproteins (HDL). LDL particles contain apolipoprotein B as the major proteinaceous compound, while HDL particles contain apolipoprotein a-I as the major proteinaceous compound.

Cholesterol absorbed by HDL particles is esterified by the enzyme Lecithin Cholesterol Acyltransferase (LCAT). Cholesteryl esters have increased hydrophobicity and diffuse toward the core of HDL particles. HDL-cholesterol-ester particles can be delivered to the liver and removed from the circulation.

HDL particles and their major polypeptide apolipoprotein a-I are involved in Reverse Cholesterol Transport (RCT). Wherein apolipoprotein a-I increases cholesterol efflux from cells (e.g. from cells in the walls of blood vessels), lipid binding and activation of lecithin cholesterol acetyltransferase, and thereby clearance of cholesterol by the liver via plasma flow. This is an active transport process involving the cell membrane protein ATP-binding-cassette-transporter-a-I (ABCA-I).

Apolipoprotein a-I and apolipoprotein based therapeutics, such as reconstituted HDL particles, have been identified in the late 70 and early 80 s of the last century. Clinical evidence (implying significant plaque reduction in arteriosclerotic patients) can be shown for apolipoprotein a-I-Milano, which contains lipid particles. Apolipoprotein A-I-Milano is a dimeric form of wild-type apolipoprotein A-I designed based on naturally occurring mutants of the apolipoprotein A-I molecule. Dimers can be formed by changing amino acid residue 173 (arginine) to cysteine, thereby allowing disulfide bond formation.

In WO2009/131704, nanostructures comprising a core comprising an inorganic material suitable for sequestering cholesterol and other molecules are reported. In WO2009/097587 a method for producing a nano-scale interfacial bilayer is reported, comprising eliminating the detergent from the intermediate mixture within about 1 hour of obtaining the mixture. In WO2006/125304, pharmaceutical compositions for the treatment or prevention of coronary artery disease are reported. Compositions encoding apolipoproteins involved in lipid metabolism and cardiovascular diseases are reported in US 2002/10142953. In WO2005/084642, apoprotein-cochelate compositions are reported. In WO2007/137400, methods and compounds for treating valvular stenosis are reported. Pharmaceutical formulations, methods and dosing regimens for the treatment and prevention of acute coronary syndromes are reported in WO 2005/041866.

In US6,287,590, peptide/lipid complexes formed by co-lyophilization are reported. Apolipoprotein a-I agonists and their use for the treatment of dyslipidemia disorders (discodermers) are reported in US6,037,323.

In WO2009/097587, nanoscale interfacial bilayers, applications and production methods are reported. Hydrophobin formulations in immunogenic compositions with improved tolerability are reported in WO 2005/065708. In WO2006/069371, a method of plasma lipidation to prevent, inhibit and/or reverse atherosclerosis is reported. The formation of proteoliposomes (proteoliposomes) comprising membrane proteins is reported in FR 2915490.

Summary of The Invention

Herein is reported a method for producing a lipid particle comprising a protein. It has been found that lipid particles can be formed from a solution comprising denatured proteins by rapid dilution into a solution comprising at least one lipid and a detergent. In this step, the concentration of the detergent is reduced below the CMC. Using this method, the previous step of generating native properties (naturation) can be omitted and thus, using the method as reported herein, lipid particles can be generated faster.

In one embodiment, the dilution is from about 1: 3 (v: v) to about 1: 20 (v: v).

In one embodiment, the dilution is from about 1: 5 (v: v) to about 1: 10 (v: v).

In one embodiment, the dilution is about 1: 5 (v: v).

In one embodiment, the detergent is diluted at least about 3-fold. In one embodiment, the detergent is diluted at least about 5-fold.

One aspect as reported herein is a method for producing lipid particles, said method comprising the steps of:

i) providing a first solution comprising a denatured protein,

ii) adding the first solution to a second solution comprising at least one lipid and a detergent, but no said protein, and

iii) removing the detergent from the solution obtained in step ii) and thereby producing lipid particles.

In one embodiment, the first solution does not comprise lipids.

In one embodiment, the protein is a recombinantly produced protein.

In one embodiment, the protein is an apolipoprotein. In another embodiment, the apolipoprotein is a purified apolipoprotein.

In one embodiment, the apolipoprotein has an amino acid sequence selected from SEQ ID NO: 01, 02 and 04-52 and 66-67 or an amino acid sequence comprising at least one amino acid sequence comprising SEQ ID NO: 01, 02 and 04-52 and 66 and 67, and at least 80% of the contiguous stretch of amino acid sequence.

In one embodiment, the apolipoprotein has an amino acid sequence or is selected from the group consisting of SEQ ID NO: 01, 02 and 04-52 and 66 and 67, and at least 80% of the contiguous stretch of amino acid sequence.

In one embodiment, the apolipoprotein is apolipoprotein a-I. In one embodiment, the apolipoprotein A-I is human apolipoprotein A-I. In another embodiment, the apolipoprotein is tetranectin-apolipoprotein a-I having the amino acid sequence SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 66 or SEQ ID NO: 67.

in one embodiment, the apolipoprotein has a sequence with a mutation selected from R151C and R197C SEQ ID NO: 06.

In one embodiment, the second solution has a volume that is at least twice the volume of the first solution.

In one embodiment, the second solution has a volume of about 3 times to about 20 times the volume of the first solution. In one embodiment, the second solution has a volume of about 5 times to about 10 times the volume of the first solution.

In one embodiment, the at least one lipid is selected from the group consisting of phospholipids, fatty acids, and steroid lipids.

In one embodiment, the at least one lipid is at least two lipids, optionally independently from each other, selected from the group consisting of phospholipids, fatty acids and steroid lipids. In another embodiment, the at least one lipid is from 1-4 lipids, i.e. selected from the group comprising one lipid, two lipids, three lipids and four lipids.

In one embodiment, the second solution comprises phospholipids, lipids, and detergents.

In one embodiment, the second solution consists of phospholipids, lipids, detergents and buffer salts.

In one embodiment, the lipid is two different phospholipids. In another embodiment, the lipid is two different phosphatidylcholines. In another embodiment, the first phosphatidylcholine and the second phosphatidylcholine differ by one or two fatty acid residues or fatty acid residue derivatives, which are esterified to the glycerol backbone of the phosphatidylcholine. In one embodiment, the first phosphatidylcholine is POPC and the second phosphatidylcholine is DPPC.

In one embodiment, the detergent is selected from a sugar-based detergent, a polyoxyethylene-based detergent, a bile salt-based detergent, a synthetic detergent, or a combination thereof. In another embodiment, the detergent is selected from the group consisting of cholic acid, amphoteric detergent (Zwittergent) or salts thereof.

In one embodiment of the method as reported herein, the first solution is substantially free of lipid particles.

In one embodiment, the method comprises a step iia) after step ii) and before step iii): incubating the solution obtained in step ii). In one embodiment, the incubation and/or removal is performed at a temperature from 4 ℃ to 45 ℃.

In one embodiment, the polypeptide is incubated with the detergent for about 0.5 hours to about 60 hours. In one embodiment, the polypeptide is incubated with the detergent for about 0.5 hours to about 20 hours. In one embodiment, the polypeptide is incubated with the detergent for about 2 hours to about 60 hours. In one embodiment, the polypeptide is incubated with the detergent for about 12 hours to about 20 hours. In one embodiment, the polypeptide is incubated with the detergent for about 16 hours.

In one embodiment, the detergent is a detergent with a high CMC. In another embodiment, the detergent is a detergent with a CMC of at least 5 mM. In another embodiment, the detergent is a detergent with a CMC of at least 10 mM.

In one embodiment, the concentration of detergent in the second solution is at least 0.5x CMC.

In one embodiment, the removal is performed by diafiltration or dialysis or adsorption. In one embodiment, the adsorption is selected from affinity chromatography or hydrophobic chromatography. In one embodiment, the removal is performed by dialysis.

In one embodiment, the first solution has a first volume, the second solution has a second volume, the protein in the first solution has a defined concentration, and the lipid and the detergent in the second solution each have a defined concentration, and in step ii) the concentration of apolipoprotein, the concentration of lipid and the concentration of detergent are varied/reduced allowing the formation of lipid particles.

In one embodiment, the method comprises the steps of:

iv) purifying the lipid particle and thereby producing the lipid particle.

One aspect as reported herein is a lipid particle obtainable by a method as reported herein.

One aspect as reported herein is a pharmaceutical composition comprising the apolipoprotein-containing lipid particle obtained with the method as reported herein and the use of the lipid particle as reported herein for the preparation of a medicament for the treatment of atherosclerosis.

Detailed Description

Definition of

The term "apolipoprotein" refers to a protein contained in a lipid or lipoprotein particle, respectively.

The term "apolipoprotein a-I" refers to an amphipathic helical polypeptide having the properties of protein-lipid and protein-protein interactions. Apolipoprotein a-I is synthesized by the liver and small intestine as a prepro-apolipoprotein (prepro-apolipoprotein) of 267 amino acid residues, which is secreted as a prepro-apolipoprotein (pro-apolipoprotein), which is cleaved into a mature polypeptide of 243 amino acid residues. Apolipoprotein a-I consists of 6-8 distinct amino acid repeats, each consisting of 22 amino acid residues, separated by a linker moiety, typically proline, and in some cases, a fragment consisting of several residues. Exemplary human apolipoprotein A-I amino acid sequences are identified at GenPept database accession No. NM-000039 or database accession No. X00566; GenBank NP-000030.1(gi 4557321). Human apolipoprotein A-I (SEQ ID NO: 06) naturally occurring variants exist, such as P27H, P27R, P28R, R34L, G50R, L84R, D113E, A-A119D, D127N, deletion K131, K131M, W132R, E133K, R151C (amino acid residue 151 changed from Arg to Cys, apolipoprotein A-I-Paris), E160K, E163G, P167R, L168R, E171V, P189R, R197C (amino acid residue 173 changed from Arg to Cys, apolipoprotein A-I-ano) and E222 Mil 222K. Also included are variants with conservative amino acid modifications.

In one embodiment, tetranectin-apolipoprotein a-I comprises a fragment of a cleavage site for an immunoglobulin a protease (IgA protease). The recognition site known from IgA protease contains the following sequence, where "↓" indicates the position of the cleavage bond:

Pro-Ala-Pro↓Ser-Pro (SEQ ID NO：61)

Pro-ProSer-Pro (SEQ ID NO：62)

Pro-Pro↓Ala-Pro (SEQ ID NO：63)

Pro-Pro↓Thr-Pro (SEQ ID NO：64)

Pro-Pro↓Gly-Pro (SEQ ID NO：65)，

the first three are selected and cut more frequently.

The term "apolipoprotein mimetic" refers to a synthetic polypeptide that mimics the function of an individual apolipoprotein. For example, an "apolipoprotein A-I mimetic" is a synthetic polypeptide that exhibits biological function relative to the removal of cholesterol (i.e., reverse cholesterol efflux) comparable to native apolipoprotein A-I. In one embodiment, the apolipoprotein a-I mimetic comprises at least one amphipathic α -helix having positively charged amino acid residues clustered at the hydrophobic-hydrophilic interface and negatively charged amino acid residues clustered at the center of the hydrophilic face. To mimic the function of apolipoprotein A-I, apolipoprotein mimetics comprise a repeating polypeptide of from 15-29 amino acid residues, and in one embodiment 22 amino acid residues (PVLDEFREKLNEELEALKQKLK (SEQ ID NO: 04); PVLDLFRELLNELLEAL KQKLK (SEQ ID NO: 05)).

The term "at least one" refers to one, two, three, four, five, six, seven, eight, nine, or more. The term "at least two" refers to two, three, four, five, six, seven, eight, nine, ten or more.

The term "cardiovascular disease" generally refers to a disease or condition related to the heart or blood vessels, such as atherosclerosis, coronary heart disease, cerebrovascular disease, aortic disease (aortoiliac disease), ischemic heart disease, or peripheral vascular disease. These diseases may not be detected until the occurrence of a malignant event as a result of the disease, such as myocardial infarction, stroke, angina pectoris, transient ischemic attack, congestive heart failure, aortic aneurysm, which in most cases leads to death of the subject.

The term "cholate" refers to 3 α, 7 α, 12 α -trihydroxy-5 β -chola-24-oic acid or its salt, especially its sodium salt. Lipid particles may be formed by incubating the apolipoproteins with detergent-solubilized lipids at their respective transition temperatures.

The term "critical micelle concentration" and its abbreviation "CMC", used interchangeably, refer to the concentration of surfactant or detergent above which individual detergent molecules (monomers) spontaneously agglomerate into micelles (micelles, rods, lamellar structures, etc.).

The term "conservative amino acid modification" refers to an amino acid sequence modification that does not affect or alter the properties of the lipid particle or apolipoprotein according to the invention. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid modifications include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a "variant" protein, as used herein, refers to a molecule whose amino acid sequence differs from that of the "parent" protein by up to 10 additions, deletions, and/or substitutions, and in one embodiment, from about 2 to about 5 additions, deletions, and/or substitutions. Amino acid sequence modifications can be made by mutagenesis based on molecular modeling as described by Riechmann, L., et al, Nature (Nature) 332(1988) 323-100327, and Queen, C., et al, Proc. Natl. Acad. Sci. USA86(1989)10029-10033.

The term "detergent" refers to a surface active chemical. "detergents" are generally amphiphilic molecules having a non-polar hydrophobic portion and a polar hydrophilic portion. The term "zwitterionic detergent (zwitterionicodietergent)" refers to a surface active chemical compound having an overall charge of zero and comprising both at least one positively charged moiety and at least one negatively charged moiety. In one embodiment, the detergent is selected from a sugar-based detergent, a polyoxyalkylene-based detergent, a bile salt-based detergent, a synthetic detergent, or a combination thereof. The term "sugar-based detergent" refers to a detergent selected from the group consisting of n-octyl- β -D-glucopyranoside, n-nonyl- β -D-glucopyranoside, n-dodecyl- β -D-maltopyranoside or 5-cyclohexylpentyl- β -D-maltopyranoside and derivatives thereof. The term "bile salt based detergent" refers to a detergent selected from the group consisting of sodium cholate, potassium cholate, lithium cholate, 3- [ (3-chloroamidopropyl) dimethylammonio ] -yl-propanesulfonate (CHAPS), 3- [ (3 chloroamidopropyl) dimethylammonio ] -2-hydroxypropanesulfonate (CHAPSO), and derivatives thereof. The term "polyoxyalkylene based detergent" refers to a detergent selected from Tween20, Triton X-100, Pluronic F68 and derivatives thereof. The term "synthetic detergent" refers to a detergent selected from the group consisting of amphoteric detergents 3-6, amphoteric detergents 3-8, amphoteric detergents 3-10, amphoteric detergents 3-12, and derivatives thereof.

The term "high density lipoprotein particle" or its abbreviation "HDL particle", used interchangeably, refers to a lipid-protein complex comprising apolipoprotein a-I as the major protein compound.

The term "immunoassay" refers to a standard solid phase immunoassay with a monoclonal antibody, comprising the formation of a complex between an antibody adsorbed/immobilized on a solid phase (capture antibody), an antigen and an antibody directed against another epitope of the antigen conjugated to an enzyme (tracer antibody). Thus, a multilayer structure is formed: solid phase-capture antibody-antigen-tracer antibody. In reactions catalyzed by the multilayer structure, the activity of the antibody-conjugated enzyme is proportional to the antigen concentration in the incubation medium. The standard sandwich method is also referred to as a double antigen bridging immunoassay because the capture antibody and the tracer antibody bind to different epitopes of the antigen. Other types of assays are radioimmunoassays, fluorescent immunoassays and enzyme-linked immunoassays. The methods of performing the assays, as well as the practical applications and procedures, are known to those skilled in the art. The immunoassay may be performed as a homogeneous immunoassay or a heterogeneous immunoassay.

The term "increase lipid efflux" and grammatical equivalents thereof, refers to an increased level and/or rate of lipid efflux from a cell or plaque, promotes lipid efflux, enhances lipid efflux, aids lipid efflux, upregulates lipid efflux, enhances lipid efflux, and/or enhances lipid efflux. In one embodiment, lipid efflux comprises efflux of phospholipids, triglycerides, cholesterol and/or cholesterol esters.

The term "DMPC" refers to the phospholipid dimyristoylphosphatidylcholine.

The term "DPPC" refers to the phospholipid 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, also known as 1, 2-dipalmitoyl-phosphatidylcholine.

The term "multimer" refers to a complex composed of two or more monomers. Multimers are formed between monomers by non-covalent interactions. Each monomer comprises a multimerizing domain. In one embodiment, the multimer comprises 2 or 3 monomers. In another embodiment, the multimerization domains interact by non-covalent interactions between the individual multimerization domains contained in each monomer. The term "multimerization domain" refers to an amino acid sequence capable of covalently or non-covalently associating two or more monomer molecules. The multimerization domains are capable of interacting with multimerization domains of different, similar, or identical amino acid sequences. In one embodiment, the multimerization domain is a tetranectin trimerization structural element or a derivative thereof having a sequence identical to SEQ ID NO: 53 has an amino acid sequence that is at least 68% identical to the consensus amino acid sequence. In one embodiment, in SEQ ID NO: 53 by a different amino acid residue, and in another embodiment by a serine or threonine or methionine residue. A polypeptide comprising a multimerization domain may be associated with one or more other polypeptides that also comprise a multimerization domain. Multimer formation can be initiated simply by mixing the polypeptides under suitable conditions. In another embodiment, the multimerization domain has the amino acid sequence of SEQ ID NO: 53, wherein 1 to 10 residues are deleted or added at the N-terminus or C-terminus of the amino acid sequence. In another embodiment, the multimerization domain has seq id NO: 53, wherein 6 or 9 amino acid residues are deleted at the N-terminus of the amino acid sequence. In yet another embodiment, the multimerization domain has the amino acid sequence of SEQ ID NO: 53, wherein the N-terminal amino acid residue L or the N-terminal amino acid residues C and L are deleted. In one embodiment, the multimerization domain is a tetranectin trimerization structural element and has the amino acid sequence of SEQ id no: 54, or a pharmaceutically acceptable salt thereof. In one embodiment, the multimer is a homopolymer.

Multimers may be homomultimers or heteromers, as different apolipoproteins comprising a multimerization domain may be combined into the multimers. In one embodiment, the multimer is a trimeric homopolymer.

According to one embodiment, the multimerization domain is obtained from tetranectin. In one embodiment, the multimerization domain comprises a polypeptide having the sequence of SEQ ID NO: 54, or a tetranectin trimerising structural element of the amino acid sequence of seq id No. 54. The tetranectin trimerising structural element trimerisation is caused by a coiled-coil structure which interacts with the coiled-coil structures of two other tetranectin trimerising structural elements to form a trimer. The tetranectin trimerising structural element may be obtained from human tetranectin, from rabbit tetranectin, from murine tetranectin, or from a C-type lectin of shark cartilage. In one embodiment, the tetranectin trimerising structural element comprises a sequence having at least 68%, or at least 75%, or at least 81%, or at least 87%, or at least 92% identity to the consensus sequence of SEQ ID NO 53.

The term "non-covalent interaction" refers to a non-covalent binding force such as an ionic interaction force (e.g., a salt bridge), a non-ionic interaction force (e.g., a hydrogen bond), or a hydrophobic interaction force (e.g., van der waals or pi-stacking interactions).

The term "phase transition temperature" refers to the temperature required to induce a change in the physical state of a lipid from an ordered gel phase (in which the hydrocarbon chains are well-extended and tightly packed) to a disordered liquid crystal phase (in which the hydrocarbon chains are randomly oriented and mobile). The formation of the lipid particles may be carried out at or above the phase transition temperature of the phospholipid/phospholipid mixture used. The phase transition temperatures of several phosphatidylcholines and mixtures thereof are listed in table 1 below.

Table 1: phase transition temperature of pure phosphatidylcholine and phosphatidylcholine mixtures

The term "phosphatidylcholine" refers to a molecule consisting of one glycerol moiety, two carboxylic acid moieties and one phosphocholine moiety, wherein the glycerol moiety is covalently bound to the other moiety through ester linkages, i.e., two carboxylic ester linkages and one phosphoric ester linkage, respectively, wherein the phosphoric ester linkage is bound to the 1-hydroxyl or 3-hydroxyl group of the glycerol moiety. The term "carboxylic acid moiety" refers to an organic moiety comprising at least one acyl group (R-C (O). The phosphatidylcholine may be of any kind or origin. In one embodiment, the phosphatidylcholine is selected from the group consisting of egg phosphatidylcholine, soy phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine, 1-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, 1-oleoyl-2-palmitoylphosphatidylcholine, and analogs and derivatives thereof.

All phospholipids used herein may be derived from any source, i.e. if appropriate from soy, milk, egg or even from internal organs of animals other than humans, they may be derived from natural sources, or semi-synthetic sources or even fully synthetic sources.

A "polypeptide" is a polymer composed of amino acids joined by peptide bonds, whether naturally occurring or synthetically produced. Polypeptides of less than about 20 amino acid residues may be referred to as "peptides" and molecules consisting of more than two polypeptides or comprising more than one 100 amino acid residues may be referred to as "proteins". The polypeptide may also comprise non-amino acid components, such as carbohydrate groups, metal ions, or carboxylic acid esters. The non-amino acid components may be added by the cell expressing the polypeptide and may vary with the cell type. Polypeptides are defined herein by their amino acid backbone structure or the nucleic acids that encode them. Addition of e.g. carbohydrates is not usually specified, but may be present.

The term "POPC" refers to the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine, also known as 1-palmitoyl-2-oleoyl-phosphatidylcholine.

The term "rapid" refers to a process that is completed in up to 10 hours. Fast dilution is a process in which the first solution is added to the second solution in up to 10 hours. In one embodiment, the process is completed in up to 5 hours, and in another embodiment, in up to 2 hours.

The term "substantially free" means that the solution comprising protein and one or more lipids comprises less than 5% (w/w) lipid particles, less than 2.5% lipid particles, less than 1% lipid particles or less than 0.5% lipid particles.

The term "variant" also includes variants of the apolipoproteins or apolipoprotein mimetics reported herein, wherein in a variant the amino acid sequence of the respective apolipoprotein or apolipoprotein mimetic comprises one or more amino acid substitutions, additions or deletions. The modification may increase or decrease the affinity of the apolipoprotein for the apolipoprotein receptor or the apolipoprotein converting enzyme, or may increase the stability of the apolipoprotein variant compared to the respective carrier protein, or may increase the solubility of the apolipoprotein variant in aqueous solution compared to the corresponding apolipoprotein, or may increase the recombinant production of the apolipoprotein variant in/by the host cell compared to the respective apolipoprotein.

Herein reported

It has been found that lipid particles can be formed directly starting from a solution comprising denatured proteins but no detergent and no lipids, by rapid dilution into a solution comprising detergent and at least one lipid but no proteins. The generally required steps for producing native properties can be omitted, thus providing a simpler and reliable method for producing lipid particles. In addition, more uniform lipid particles are formed.

Method for producing lipid particles

Herein is reported a method for producing a lipid particle comprising a protein, the method comprising the steps of:

i) providing a first solution comprising a denatured protein,

ii) adding the first solution to a second solution comprising lipids and detergent but no protein, i.e. no said protein, and

iii) removing the detergent from the solution obtained in step ii), and thereby producing lipid particles.

In one embodiment, a method for producing a lipid particle comprising an apolipoprotein comprises the steps of:

i) providing a first solution comprising a denatured apolipoprotein,

ii) adding the first solution to a second solution comprising lipids and detergent but no apolipoprotein, and

In one embodiment, the second solution has a volume that is about 3 times to about 20 times the volume of the first solution. In one embodiment, the second solution has a volume that is about 5 times to about 10 times the volume of the first solution.

In one embodiment, the second solution comprises at least two different lipids, which are independently from each other selected from the group consisting of phospholipids, fatty acids and steroid lipids. In another embodiment, the at least two different lipids are two different phosphatidylcholines. In one embodiment, the first phosphatidylcholine is POPC and the second phosphatidylcholine is DPPC.

In one embodiment, the detergent is selected from a cholic acid, an amphoteric detergent, or a salt thereof.

Many different methods of producing lipid particles from naturally occurring or recombinantly produced polypeptides, such as e.g. apolipoprotein a-I or delipidated apolipoprotein a-I from human HDL particles, have been reported. Wherein an aqueous mixture of phospholipids (such as palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) and detergents (such as sodium cholate) is incubated, for example, with purified apolipoprotein a-I, which is used in native, i.e. non-denatured form. After formation of the lipid particles the detergent is removed by dialysis or diafiltration.

The method as reported herein allows for complete refolding and lipidation of the denatured protein in a single step. By using the method as reported herein, lipid particles with improved product quality can be obtained, the time needed for pre-treatment of proteins can be dispensed with, and large scale processing for biopharmaceutical production is for the first time possible.

The method reported herein allows for complete refolding and lipidation of denatured apolipoprotein a-I in a single step. By using the method as reported herein, lipid particles with improved product quality can be obtained, the time consumed for pre-treatment of apolipoprotein a-I can be saved, and large-scale processing of biopharmaceutical production is made possible for the first time.

The key points that must be considered for the development of a lipid particle forming process are i) the need for biological activity, and ii) the technical requirements relating to the manufacturability of the lipid particles. For example, for the formation of lipid particles comprising apolipoproteins, these requirements point in opposite directions.

From a technical point of view, saturated phospholipids comprising carboxylic acid moieties having chains below 16 carbon atoms (e.g. dipalmitoyl-sn-glycero-3-phosphocholine, DPPC; dimyristoyl-sn-glycero-3-phosphocholine, DMPC etc.) will be selected. In contrast, unsaturated phospholipids comprising a carboxylic acid moiety having a chain of at least 16 carbon atoms (e.g., palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC; stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, SOPC) are considered to be more effective and non-toxic to the liver from biological data.

The choice of phospholipid combination determines the efficacy and liver safety of the apolipoprotein containing lipid particle. In vivo studies using DMPC-containing lipid particles in rabbits, it has been found that rabbits treated with 30mg/kg show severe side effects, but survive, whereas rabbits treated with 100mg/kg die. The results clearly show that lipidation is required for the mobilization of cholesterol and thus for the efficacy of the molecule (fig. 23).

In vitro functional tests demonstrated that lipid particles comprising monophosphoryl choline, such as DPPC or POPC, activated LCAT.

It also shows that cholesterol efflux is higher for combinations of different phospholipids.

Table 2: phospholipid combinations for in vivo rabbit study preparation, except for their lipid composition

These results are also confirmed by in vivo data showing cholesterol mobilization for all combinations. However, for lipid particles containing only monophosphoryl choline DPPC, or a combination of DPPC and Sphingomyelin (SM), an increase in liver enzymes could be determined (fig. 1).

Thus, the lipid particles obtained by the method as reported herein are also an aspect.

From a technical point of view, it is more convenient to form lipid particles with pure DPPC than with pure POPC. The risk of precipitate formation is reduced by using a combination of different phospholipids. Furthermore, the 41 ℃ phase transition temperature of pure DPPC also makes it easier to prepare lipid particles compared to pure POPC with a phase transition temperature of 4 ℃. Moreover, the obtained product is more uniform. This can also be confirmed by lipid particle analysis by SEC-MALLS, an analytical tool allowing the determination of protein-lipid compositions (protein-conjugate analysis). In fig. 2, a chromatogram of a sample resolved by size exclusion chromatography (UV280 detection) is shown. Inhomogeneity of the sample can be observed by the appearance of multiple isolated or semi-dissociated peaks.

When pure POPC is used to prepare the lipid particle, the number of POPC molecules per apolipoprotein monomer in the lipid particle is in one embodiment 40-85, in one embodiment 50-80, and in one embodiment 54-75.

When pure DPPC is used to prepare the lipid particle, the number of DPPC molecules per apolipoprotein monomer in the lipid particle is, in one embodiment, from 50 to 150, in one embodiment, from 65 to 135, in one embodiment, from 76 to 123, and in one embodiment, from 86 to 102.

When a mixture of POPC and DPPC in a 1: 3 molar ratio is used to prepare the lipid particle, the number of phospholipid molecules per apolipoprotein monomer in the lipid particle is, in one embodiment, from about 50 to about 120, in one embodiment, from about 65 to about 105, and in one embodiment, from about 72 to about 96.

When a mixture of POPC and DPPC in a 1: 1 molar ratio is used to prepare the lipid particle, the number of lipid molecules per apolipoprotein monomer in the lipid particle is in one embodiment 50-120, in one embodiment 60-100, in one embodiment 71 to 92, and in one embodiment 71-85.

When a mixture of POPC and DPPC is used in a molar ratio of 3: 1 to prepare the lipid particle, the number of lipid molecules per apolipoprotein monomer in the lipid particle is, in one embodiment, 50 to 105.

When a mixture of POPC and DPPC is used in a molar ratio of 3: 1 to prepare the lipid particle, the number of lipid molecules per apolipoprotein monomer in the lipid particle is, in one embodiment, 60-95.

When a mixture of POPC and DPPC is used in a molar ratio of 3: 1 to prepare the lipid particle, the number of lipid molecules per apolipoprotein monomer in the lipid particle is, in one embodiment, 60 to 90.

When a mixture of POPC and DPPC is used in a molar ratio of 3: 1 to prepare the lipid particle, the number of lipid molecules per apolipoprotein monomer in the lipid particle is, in one embodiment, 60 to 88.

When a mixture of POPC and DPPC is used in a molar ratio of 3: 1 to prepare the lipid particle, the number of lipid molecules per apolipoprotein monomer in the lipid particle is, in one embodiment, 62 to 80.

When a mixture of POPC and DPPC is used in a molar ratio of 3: 1 to prepare the lipid particle, the number of lipid molecules per apolipoprotein monomer in the lipid particle is, in one embodiment, 66-86.

When a mixture of POPC and DPPC is used in a molar ratio of 3: 1 to prepare the lipid particle, the number of lipid molecules per apolipoprotein monomer in the lipid particle is, in one embodiment, 64 to 70.

When a mixture of POPC and DPPC in a 3: 1 molar ratio is used to prepare the lipid particle, the number of lipid molecules per apolipoprotein monomer in the lipid particle is, in one embodiment, about 66.

For the preparation of lipid particles comprising apolipoprotein and POPC, in one embodiment a molar ratio of apolipoprotein to POPC of 1: 40 to 1: 100, in one embodiment a molar ratio of 1: 40 to 1: 80, and in one embodiment a molar ratio of about 1: 60 is applied.

For the preparation of lipid particles comprising apolipoprotein and DPPC, in one embodiment a molar ratio of apolipoprotein to DPPC of 1: 70 to 1: 100, in one embodiment a molar ratio of 1: 80 to 1: 90, in one embodiment a molar ratio of about 1: 80 is applied.

For the preparation of lipid particles comprising apolipoprotein, POPC and DPPC, in one embodiment a molar ratio of apolipoprotein to (POPC and DPPC) (molar ratio of POPC and DPPC 1: 3) is applied in the range of 1: 60 to 1: 100, in one embodiment a molar ratio in the range of 1: 70 to 1: 90, and in one embodiment a molar ratio of about 1: 80 is applied.

For the preparation of lipid particles comprising apolipoprotein, DPPC and POPC, a molar ratio of apolipoprotein to (POPC and DPPC) (molar ratio of POPC and DPPC 1: 1) of 1: 60 to 1: 100 is applied in one embodiment, a molar ratio of 1: 60 to 1: 80 is applied in one embodiment, and a molar ratio of about 1: 70 is applied in one embodiment.

For the preparation of lipid particles comprising apolipoprotein, DPPC and POPC, a molar ratio of apolipoprotein to (POPC and DPPC) (molar ratio of POPC and DPPC 3: 1) of 1: 60 to 1: 100 is applied in one embodiment, a molar ratio of 1: 50 to 1: 70 is applied in one embodiment, and a molar ratio of about 1: 60 is applied in one embodiment.

In one embodiment, if a mixture of lipids is used to prepare the lipid particles, the mixture has a phase transition temperature of 4 ℃ to 45 ℃, in one embodiment 10 ℃ to 38 ℃, and in one embodiment 15 ℃ to 35 ℃.

For the formation of lipid particles comprising apolipoproteins, different methods are known, such as lyophilization, freeze-thawing, detergent solubilization followed by dialysis, microfluidization, sonication and homogenization.

The lipid particle may comprise, in one embodiment, an average of 1-10 apolipoprotein molecules, in one embodiment, an average of 1-8 apolipoprotein molecules per lipid particle, and in one embodiment, an average of 1-4 apolipoprotein molecules per lipid particle.

In one embodiment, the lipid particle may comprise an average number of at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 apolipoprotein molecules per lipid particle. In one embodiment, the average is 1.

In one embodiment, the lipid particle comprises one or more additional polypeptides in addition to the apolipoprotein.

Without limitation, the lipid particle may act as a carrier for enzymatic cofactors and/or lipids (especially cholesterol) used to absorb lipids.

One or more detergents may also be present in the lipid particle as reported herein. The detergent may be any pharmaceutically acceptable detergent, such as a non-ionic or ionic detergent. Nonionic detergents may be alkylene oxide derivatives of organic compounds containing one or more hydroxyl groups. In one embodiment, the nonionic detergent is selected from ethoxylated and/or propoxylated alcohol or ester compounds or mixtures thereof. In another embodiment, the ester is selected from esters of sorbitol and fatty acids,such as sorbitan monooleate or, sorbitan monopalmitate, oily sucrose esters, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene glycol ethers, polyoxyethylene-polyoxypropylene alkyl ethers, block polymers and cethyl ethers (cethyletheres), polyoxyethylene castor oil or hydrogenated castor oil derivatives and polyglycine fatty acid esters. In one embodiment, the nonionic detergent is selected fromOr

The ionic detergent may be a bile duct agent. In one embodiment, the ionic detergent is selected from cholic or deoxycholic acid, or salts and derivatives thereof, or from free fatty acids, such as oleic acid, linoleic acid and others.

In one embodiment, the ionic detergent is selected from cationic lipids such as C₁₀-C₂₄An alkylamine or alkanolamine and a cationic cholesterol ester. In one embodiment, the detergent is a detergent with a high CMC. In yet another embodiment, the detergent is a detergent with at least 5mM CMC.

In one embodiment, the lipid particle comprises less than 0.75 wt% detergent.

In one embodiment, the lipid particle comprises less than 0.30 wt% detergent.

In one embodiment, the lipid particle comprises less than 0.1 wt% detergent.

In one embodiment, the lipid particle comprises less than 0.05 wt% detergent.

In one embodiment, the detergent is selected from a sugar-based detergent, a polyoxyethylene-based detergent, a bile salt-based detergent, a synthetic detergent, or a combination thereof. In another embodiment, the detergent is a cholic acid or amphoteric detergent.

In one embodiment, in the method according to the invention, the first solution is substantially free of lipid particles.

In one embodiment, the method comprises after step ii) and before step iii) the step iia) of incubating the solution obtained in step ii) as described below. In one embodiment, the polypeptide is incubated with the detergent for about 0.5 hours to about 60 hours. In one embodiment, the polypeptide is incubated with the detergent for about 0.5 hours to about 20 hours. In one embodiment, the polypeptide is incubated with the detergent for about 2 hours to about 60 hours. In one embodiment, the polypeptide is incubated with the detergent for about 12 hours to about 20 hours. In one embodiment, the polypeptide is incubated with the detergent for about 16 hours.

In one embodiment, the incubation and/or removal is performed at a temperature of 4 ℃ to 45 ℃.

In one embodiment, the removal is performed by diafiltration or dialysis.

In one embodiment, the first solution has a first volume and the second solution has a second volume, the protein, such as an apolipoprotein, in the first solution having a defined concentration and the lipid and the detergent in the second solution having a defined concentration, respectively, wherein in step ii) the concentration of the apolipoprotein, the concentration of the lipid and the concentration of the detergent are varied/reduced allowing the formation of lipid particles. By diluting the apolipoprotein solution and adding lipids and detergents, the appropriate ratio of apolipoprotein to lipid on the one hand and lipid to detergent on the other hand is adjusted, allowing the formation of lipid particles.

In one embodiment, the method comprises the steps of:

iv) purifying the lipid particle and thereby producing the lipid particle.

For example, to produce lipid particles comprising apolipoproteins, saturated phospholipids comprising carboxylic acid moieties with chains of 16 atoms and shorter can be selected from a technical standpoint (e.g., dipalmitoyl-sn-glycero-3-phosphocholine, DPPC; dimyristoyl-sn-glycero-3-phosphocholine, DMPC, etc.). From a biological point of view, in contrast to this, it can be assumed that unsaturated phospholipids comprising a carboxylic acid moiety with a chain of at least 16C atoms (e.g. palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC; stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, SOPC) are more potent and non-hepatotoxic.

Phosphatidylcholine DPPC and POPC and mixtures thereof may be used for forming the lipid particle comprising the apolipoprotein. These exemplary phosphatidylcholines differ in one carboxylic acid moiety and have one and the same carboxylic acid moiety esterified to the phosphoglycerol backbone. When DPPC is used, it is easier to prepare lipid particles. In contrast, POPC is more effective in vitro functional assays, particularly as a substrate for activating Lecithin Cholesterol Acetyltransferase (LCAT), which is necessary for converting mobilized cholesterol to cholesterol esters. It has been found that lipid particles comprising a mixture of two phosphatidylcholines, e.g. POPC and DPPC, in different molar ratios are advantageous compared to lipid particles comprising only one phosphatidylcholine (see, e.g. fig. 4).

For example, the lipid particle may comprise POPC only. When apolipoprotein in a molar ratio of 1: 40 up to 1: 80: when lipids are used to produce lipid particles, the number of POPC molecules per apolipoprotein monomer may vary between 54-75. In one embodiment, the molar ratio of apolipoprotein to POPC is 1: 40 to 1: 80, in one embodiment 1: 50 to 1: 70, and in one embodiment about 1: 60.

Thus, for the production of lipid particles comprising apolipoprotein and POPC, the molar ratio of apolipoprotein to POPC is from 1: 40 to 1: 100, in one embodiment 1: 40 to 1: 80, and in one embodiment about 1: 60.

For example, the lipid particle may comprise DPPC only. When apolipoprotein: lipids are used in a molar ratio of 1: 40 up to 1: 80 to produce lipid particles, the number of DPPC molecules per apolipoprotein monomer may vary between 76 and 123. In one embodiment the molar ratio of apolipoprotein to DPPC is 1: 70 to 1: 100, in one embodiment 1: 75 to 1: 90, and in one embodiment about 1: 80.

For example, lipid particles can be prepared starting from a mixture of POPC and DPPC in a molar ratio of 1: 3. When apolipoprotein: lipids are used in a molar ratio of 1: 60 up to 1: 100 to produce lipid particles, the number of phospholipid molecules per apolipoprotein monomer may vary between 72-112. In one embodiment, the molar ratio of apolipoprotein to (POPC and DPPC) is 1: 70 to 1: 90, in one embodiment 1: 75 to 1: 85, and in one embodiment about 1: 80.

Thus, for the production of lipid particles comprising apolipoprotein, POPC and DPPC, the molar ratio of apolipoprotein to (POPC and DPPC) (POPC and DPPC in a molar ratio of 1: 3) is in one embodiment 1: 60 to 1: 100, in one embodiment 1: 70 to 1: 90, and in one embodiment about 1: 80.

For example, lipid particles can be prepared starting from a mixture of POPC and DPPC in a 1: 1 molar ratio. When apolipoprotein: lipids are used in a molar ratio of 1: 60 up to 1: 100 for the preparation of lipid particles, the number of phospholipid molecules per apolipoprotein monomer may vary between 71 and 111. In one embodiment, the molar ratio of apolipoprotein to (POPC and DPPC) is 1: 60 to 1: 80, in one embodiment 1: 65 to 1: 75, and in one embodiment about 1: 70.

Thus, for the production of lipid particles comprising apolipoprotein, DPPC and POPC, the molar ratio of apolipoprotein to (POPC and DPPC) (POPC and DPPC in a 1: 1 molar ratio) is in one embodiment 1: 60 to 1: 100, in one embodiment 1: 60 to 1: 80, and in one embodiment about 1: 70.

For example, lipid particles can be prepared starting from a mixture of POPC and DPPC in a molar ratio of 3: 1. When apolipoprotein: lipids are used in a molar ratio of 1: 60 up to 1: 100 for the preparation of lipid particles, the number of phospholipid molecules per apolipoprotein monomer may vary between 46-93. In one embodiment, the molar ratio of apolipoprotein to (POPC and DPPC) is from 1: 50 to 1: 70, in one embodiment from 1: 55 to 1: 65, and in one embodiment about 1: 60.

Thus, for the production of lipid particles comprising apolipoprotein, DPPC and POPC, the molar ratio of apolipoprotein to (POPC and DPPC) (wherein the molar ratio of POPC and DPPC is 3: 1) is in one embodiment 1: 60 to 1: 100, in one embodiment 1: 50 to 1: 70, and in one embodiment about 1: 60.

In one embodiment, the apolipoprotein is provided as an aqueous solution of the apolipoprotein and may be obtained from downstream processing after recombinant production or production of the apolipoprotein from any other source, and may comprise the apolipoprotein in different concentrations and purities.

Basically, the formation of lipid particles is achieved by incubating the polypeptides with detergent-solubilized lipids at their respective transition temperatures. Removal of the detergent by dialysis results in the formation of lipid particles consisting of lipid bilayers.

Basically, lipid particle formation can be achieved by incubating tetranectin-apolipoprotein a-I, or multimers thereof, with detergent-solubilized lipids at their respective transition temperatures. Removal of the detergent by dialysis results in the formation of lipid particles consisting of a lipid bilayer surrounded by alpha-apolipoprotein.

The lipid particles may be carried out by a combination of precipitation and/or chromatography stepsAnd (5) purifying. For example, excess detergent, i.e. detergent that is not part of the lipid particles, may be removed in a hydrophobic adsorption chromatography step. In one embodiment, the step of the method of purifying lipid particles comprises a hydrophobic adsorption chromatography step. In another embodiment, the chromatographic material used for the hydrophobic adsorption step is selected from the group consisting of ExtractiGel D (obtained from Pierce Biotechnology, Rockford IL, USA), CALBIOSORB^TM(available from Calbiochem, San Diego, Calif., USA), SDR30HyperD^TMSolvent-detergent removal chromatography resin (available from PALL Corporation, Ann Arbor, MI, USA). The lipid particles are recovered from the hydrophobic adsorbent material using a detergent-free solution.

In one embodiment, dialysis is used to remove detergents with high CMC.

Pharmaceutical and diagnostic compositions:

the lipid particles obtained by the method as reported herein may be used for the treatment and/or diagnosis of a disease or disorder.

The tetranectin-apolipoprotein a-I as reported herein or the lipid particle as reported herein may be used for the treatment and/or diagnosis of a disease or disorder characterized by abnormal lipid levels or deposition of lipids in a body part, such as a plaque in a blood vessel.

To determine the ability of the resulting protein-lipid complexes to support LCAT-catalyzed cholesterol esterification, cholesterol was incorporated into lipid particles as reported herein by rapid addition of a cholesterol ethanolic solution. Lipid particles comprising pure POPC are better LCAT substrates than complexes comprising DPPC independent of their apolipoprotein content (e.g. wild-type apolipoprotein a-I or tetranectin-apolipoprotein a-I).

The initial rate of cholesterol esterification in lipid particles comprising different mixtures of POPC and DPPC showed that the mixture was a better LCAT substrate than either pure phosphatidylcholine, as can be observed from the initial rate of cholesterol esterification (see table 3 and figure 4).

Table 3: initial rates of cholesterol esterification in lipid particles comprising different mixtures of phospholipids

Macrophages, such as human THP1 cells, obtained by exposing THP-1 monocytic leukemia cells to phorbol-12-myristate-13-acetate (phorbol myristate acetate) and loaded with a radiolabeled cholesterol tracer, are exposed to a cholesterol receptor test compound.

The efflux velocity induced by the receptor test compound can be calculated as the ratio of cholesterol radioactivity in the supernatant to the sum of radioactivity in the cells plus the radioactivity of their supernatant and compared to cells exposed to a medium that does not contain the receptor and analyzed by linear fit. Parallel experiments can be performed using cells exposed and not exposed to RXR-LXR agonists that are known to primarily upregulate ABCA-1 and bias efflux towards ABCA-1 mediated transport.

In cells not pretreated with RXR-LXR lipid particles, a higher increase in cholesterol efflux can be observed compared to the efflux obtained with non-lipidated tetranectin-apolipoprotein a-I. Only a small effect of the lipid mixture on efflux could be observed in the test series (figure 5). In cells pretreated with RXR-LXR, a comparable increase in cholesterol efflux in lipid particles containing non-lipidated tetranectin-apolipoprotein a-I can be observed. The overall increase was higher than that observed with cells that had not been pretreated. Only minor effects of the lipid mixture on efflux were observed in the test series (figure 6).

Different lipid particles were tested in vivo in rabbits. The lipid particles were administered as intravenous infusion and a serial blood sampling was performed within 96 hours after administration. The values of liver enzymes, cholesterol and cholesterol esters were determined. Plasma concentrations were comparable for all tested lipid particles containing the initial distribution phase, followed by a log-linear decrease in plasma concentration (fig. 7). As can be observed from table 4, the pharmacokinetic parameters were similar for all tested compounds. The half-life observed was close to 1.5 days.

Table 4: measured pharmacokinetic parameters

As can be observed from fig. 8, cholesterol was mobilized and esterified in plasma. Plasma cholesterol ester levels do continue to increase even after tetranectin-apolipoprotein a-I concentrations have begun to decrease. When plasma tetranectin-apolipoprotein A-I levels have been reduced to about 0.5mg/ml (about 50% of normal wild-type apolipoprotein A-I), increased cholesterol ester levels may still be detected.

Lipid particles comprising tetranectin-apolipoprotein a-I do not induce liver enzymes in rabbits and mice as can be observed from fig. 1 and 9. Furthermore, no hemolysis could be determined in plasma samples obtained two hours after intravenous administration (fig. 10).

Thus, aspects of the present invention are pharmaceutical and diagnostic compositions comprising apolipoprotein-containing lipid particles as reported herein or comprising tetranectin-apolipoprotein a-I as reported herein.

As shown in table 5 below, the lipid particles as reported herein have improved in vivo properties compared to non-lipidated apolipoproteins and other lipid particles.

Table 5: in vivo properties of different apolipoproteins and lipid particles

The efficiency of cholesterol mobilization into the blood can be determined by comparing the corresponding excretion of total cholesterol (respecitveexclusion) to the carrier protein concentration after in vivo administration of apolipoprotein. For quantitative evaluation, the quotient of the baseline corrected Area (AUC) under the concentration-time curve for total cholesterol and the area under the concentration-time curve for apolipoprotein was calculated.

The lipid particle as reported herein, in particular a lipid particle comprising SEQ ID NO: 01 and lipid particles of POPC and DPPC in a 3: 1 molar ratio, show increased cholesterol mobilization in vivo.

Tetranectin-apolipoprotein A-I

In addition to the lipid particles summarized above, tetranectin-apolipoprotein a-I is also reported herein.

Tetranectin-apolipoprotein A-I is a fusion protein of a human tetranectin trimerising structural element and a wild type human apolipoprotein A-I. The amino acid sequence of the human tetranectin moiety may be shortened by the first 9 amino acids, starting with an isoleucine residue at position 10 (the naturally occurring truncation site). As a consequence of this truncation, the O-glycosylation site of the threonine residue at position 4 is deleted. Between the tetranectin trimerization structural element and human apolipoprotein A-I, five amino acid residues "SLKGS" (SEQ ID NO: 03) were removed.

For improved expression and purification, constructs are produced comprising an N-terminal purification tag, e.g. a hexa-histidine-tag, and comprising an IgA protease cleavage site. As a result of the specific cleavage, the two amino acids alanine and proline-remain at the N-terminus of the tetranectin-apolipoprotein a-I according to the invention after purification and said tetranectin-apolipoprotein a-I has the amino acid sequence of SEQ ID NO: 01, or a variant thereof.

The tetranectin trimerization structural element provides a domain that allows the formation of trimerized tetranectin-apolipoprotein a-I multimers, which are composed of non-covalent interactions between each individual tetranectin-apolipoprotein a-I monomer.

By using different production methods, the purification-tag and IgA protease cleavage sites can be omitted, resulting in a polypeptide with an amino acid sequence of SEQ ID NO: 02 tetranectin-apolipoprotein A-I.

In one embodiment, the apolipoprotein can be a variant comprising conservative amino acid substitutions or an apolipoprotein a-I mimetic.

Apolipoprotein A-I can be determined enzymatically, by NMR spectroscopy, or by using monoclonal or polyclonal anti-apolipoprotein-A-I antibodies. Thus, further aspects as reported herein are polyclonal and monoclonal antibodies, which specifically bind to tetranectin-apolipoprotein a-I as reported herein. The antibodies can be obtained by methods known to those skilled in the art. In addition, the labeling of antibodies used in immunoassays can be performed by methods known to those skilled in the art.

In one embodiment, the apolipoprotein can be a variant comprising conservative amino acid substitutions, or an apolipoprotein a-I mimetic. In one embodiment, tetranectin-apolipoprotein a-I has the amino acid sequence of SEQ ID NO: 02 or SEQ ID NO: 66 or SEQ ID NO: 67, wherein X is selected from the group consisting of SEQ ID NO: 68-SEQ ID NO: 105.

thus, in one embodiment, tetranectin-apolipoprotein A-I has the amino acid sequence IVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVDEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ (SEQ ID NO: 02) below.

In one embodiment, tetranectin-apolipoprotein A-I has the following amino acid sequence (A, G, S, T) PIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVDEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ (SEQ ID NO: 66).

In one embodiment, tetranectin-apolipoprotein A-I has the following amino acid sequence (M) HHHHHHXIVNAKKDVVNTKMFEELKSRLDTLAQEVLLKEQALQTVDEPPQSPWDKDLATVYVDVLKDSGRDYVSQVSQEGSALGKQLNLKLDNWDDNTSTFSKLREQLR QEPQEDLEKWETEGLRQEVQEVKDLEVKQPYLDFQKKWQQQQQEDYLEMRQVEPLRAELQELQKLHELQKLSPLGEEMREEMREDRAHVDALRTHLAPYELRQRLAARLYLENGGGARLAYHAKEHLSLSLSSEKPALKPVLQVLESFKVSFLLEYTLNTQ (SEQ ID NO: 67), wherein X may be any of the following amino acid sequences: a, G, S, P, AP, GP, SP, PP, GSAP (SEQ ID NO: 68), GSGP (SEQ ID NO: 69), GSSP (SEQ ID NO: 70), GSPP (SEQ ID NO: 71), GGGS (SEQ ID NO: 72), GGGGS (SEQ ID NO: 73), GGGSGGGS (SEQ ID NO: 74), GGGGSGGGGS (SEQ ID NO: 75), GGGSGGGSGGGS (SEQ ID NO: 76), GGGGSGGGGSGGGGGS (SEQ ID NO: 77), GGGSAP (SEQ ID NO: 78), GGGSGP (SEQ ID NO: 79), GGGSSP (SEQ ID NO: 80), GGGSPP (SEQ ID NO: 81), GGGGSAP (SEQ ID NO: 82), GGGGGGSGP (SEQ ID NO: 83), GGGGGGGGGGGGSPP (SEQ ID NO: 84), GGSPP (SEQ ID NO: 85), GGGSGGGSAP (SEQ ID NO: 3686), SEQ ID NO: 42 (SEQ ID NO: 3988), SEQ ID NO: 36, SEQ ID NO: 3689), SEQ ID NO: 3690, GGGSGGGSGGGSGP (SEQ ID NO: 91), GGGSGGGSGGGSSP (SEQ ID NO: 92), GGGSGGGSGGGSPP (SEQ ID NO: 93), GGGGSAP (SEQ ID NO: 94), GGGGSGP (SEQ ID NO: 95), GGGGSSP (SEQ ID NO: 96), GGGGSPP (SEQ ID NO: 97), GGGGSGGGGSAP (SEQ ID NO: 98), GGGGSGGGGSGP (SEQ ID NO: 99), GGGGSGGGGSSP (SEQ ID NO: 100), GGGGSGGGGSPP (SEQ ID NO: 101), GGGGSGGGGSGGGGSAP (SEQ ID NO: 102), GGGGSGGGGSGGGGSGP (SEQ ID NO: 103), GGGGSGGGGSGGGGSSP (SEQ ID NO: 104), and GGGGSGGGGSGGGGSPP (SEQ ID NO: 105).

If a heterologous polypeptide is produced in an E.coli strain, the amino-terminal methionine residue is generally not cleaved efficiently by a protease, whereby the amino-terminal methionine residue is partially present in the produced polypeptide.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made to the process described without departing from the spirit of the invention.

Sequence listing description

SEQ ID NO: 01 tetranectin-apolipoprotein A-I (1).

SEQ ID NO: 02 tetranectin-apolipoprotein A-I (2).

SEQ ID NO: 03 peptide.

SEQ ID NO: 04 apolipoprotein a-I mimetic (1).

SEQ ID NO: 05 Apolipoprotein A-I mimetic (2).

SEQ ID NO: 06 human apolipoprotein A-I.

SEQ ID NO: 07 human apolipoprotein A-II.

SEQ ID NO: 08 human apolipoprotein A-IV.

SEQ ID NO: 09 human apolipoprotein A-V.

SEQ ID NO: 10 human apolipoprotein C-I.

SEQ ID NO: 11 human apolipoprotein C-II.

SEQ ID NO: 12 human apolipoprotein C-III.

SEQ ID NO: 13 human apolipoprotein C-IV.

SEQ ID NO: 14 human apolipoprotein D.

SEQ ID NO: 15 human apolipoprotein E.

SEQ ID NO: 16 human apolipoprotein F.

SEQ ID NO: 17 human apolipoprotein H.

SEQ ID NO: 18 human apolipoprotein L-I.

SEQ ID NO: 19 human apolipoprotein L-II.

SEQ ID NO: 20 human apolipoprotein L-III.

SEQ ID NO: 21 human apolipoprotein L-IV.

SEQ ID NO: 22 human apolipoprotein L-V.

SEQ ID NO: 23 human apolipoprotein L-VI.

SEQ ID NO: 24 human apolipoprotein M.

SEQ ID NO: 25 human apolipoprotein O.

SEQ ID NO: 26 human apolipoprotein OL.

SEQ ID NO: 27 human apolipoprotein clus.

SEQ ID NO: 28 apolipoprotein.

SEQ ID NO: 29 apolipoprotein.

SEQ ID NO: 30 apolipoprotein.

SEQ ID NO: 31 apolipoprotein.

SEQ ID NO: 32 apolipoprotein (Apolipoprotein).

SEQ ID NO: 33 apolipoprotein.

SEQ ID NO: 34 apolipoprotein.

SEQ ID NO: 35 apolipoprotein.

SEQ ID NO: 36 apolipoprotein.

SEQ ID NO: 37 apolipoprotein.

SEQ ID NO: 38 apolipoprotein (apo-lipoprotein).

SEQ ID NO: 39 apolipoprotein.

SEQ ID NO: 40 apolipoprotein.

SEQ ID NO: 41 apolipoprotein.

SEQ ID NO: 42 apolipoprotein.

SEQ ID NO: 43 apolipoprotein.

SEQ ID NO: 44 apolipoprotein.

SEQ ID NO: 45 apolipoprotein.

SEQ ID NO: 46 apolipoprotein.

SEQ ID NO: and (3) 47 apolipoprotein.

SEQ ID NO: 48 apolipoprotein.

SEQ ID NO: 49 apolipoprotein.

SEQ ID NO: 50 apolipoprotein.

SEQ ID NO: 51 apolipoprotein.

SEQ ID NO: 52 apolipoprotein.

SEQ ID NO: a 53 human tetranectin trimerization domain.

SEQ ID NO: 54 shortened human tetranectin trimerization domains

SEQ ID NO: 55 human interferon fragment.

SEQ ID NO: a 56 hexa-histidine tag.

SEQ ID NO: 57 fusion proteins.

SEQ ID NO: primer 58, N1.

SEQ ID NO: 59 primer N2.

SEQ ID NO: 60 IgA protease cleavage site.

SEQ ID NO: 61 IgA protease cleavage site.

SEQ ID NO: 62 IgA protease cleavage site.

SEQ ID NO: 63 IgA protease cleavage site.

SEQ ID NO: 64 IgA protease cleavage site.

SEQ ID NO: 65 IgA protease cleavage site.

SEQ ID NO: 66 tetranectin-apolipoprotein A-I.

SEQ ID NO: 67 tetranectin-apolipoprotein A-I with his-marker.

SEQ ID NO: 68-105 joints.

Description of the drawings

FIG. 1 results of an in vivo rabbit study with five lipid particles differing in their lipid composition. Upper part: cholesterol mobilized and therefore may show efficacy for all prepared batches. Bottom: an increase in liver enzymes was noted for lipid particles produced by using DPPC as monophosphoryl lipid.

FIG. 2 SEC-MALLS analysis of lipid particles of POPC and apolipoprotein according to the invention; the molar ratio is 1: 20-1: 160.

FIG. 3 Effect of DPPC and POPC on LCAT activity.

Figure 4 start rate of cholesterol esterification in lipid particles comprising POPC and/or DPPC.

FIG. 5 cholesterol efflux to THP-1 derived foam cells in cells stimulated with RXR-LXR agonists.

FIG. 6 cholesterol efflux to THP-1 derived foam cells following activation of the ABCA-I pathway using RXR-LXR agonists.

FIG. 7 time-dependent plasma concentrations of different apolipoprotein compositions.

Figure 8 time and concentration progression of cholesterol mobilization and esterification in plasma.

FIG. 9 comparison of liver enzyme release of different compositions comprising the apolipoprotein of the invention in mice after a single intravenous injection of 100 mg/kg..

Figure 10 in vivo rabbit study-spontaneous haemolysis in plasma.

FIG. 11 analytical SEC of lipid particles using 250mM Tris-HCl, 140mM NaCl, pH7.5.

FIG. 12 uses 50mM K₂HPO₄Analysis SEC of lipid particles, 250mM arginine hydrochloride, 7.5% trehalose, at pH7.5.

FIG. 131: 20-1: 320 molar ratio of non-denaturing PAGE of lipid particles of POPC and tetranectin-apolipoprotein A-I (lane 1: non-denaturing marker; lane 2: molar ratio 1: 320; lane 3: molar ratio 1: 160; lane 4: molar ratio 1: 80; lane 5: molar ratio 1: 80 (f/t); lane 6: molar ratio 1: 40; lane 7: molar ratio 1: 20; lane 8: apolipoprotein (hexamer formation)).

FIG. 141: 20-1: 160 molar ratio of POPC to tetranectin-apolipoprotein A-I lipid particles by SEC-MALLS analysis.

Figure 15 superposition of SEC chromatograms (UV280 signal) of lipid particles of POPC and tetranectin-apolipoprotein a-I.

FIG. 16 SEC-MALLS analysis of lipid particles of POPC and tetranectin-apolipoprotein A-I at a 1: 40 molar ratio.

FIG. 17 non-denaturing PAGE of lipid particles of DPPC and tetranectin-apolipoprotein A-I obtained with a molar ratio of 1: 20 to 1: 100 (1: molecular weight marker; 2: lipid-free tetranectin-apolipoprotein A-I; 3: 1: 20; 4: 1: 40; 5: 1: 60; 6: 1: 80; 7: 1: 100).

FIG. 18 SEC-MALLS analysis (UV280 signal) of lipid particles of a mixture of POPC: DPPC ═ 3: 1 and tetranectin-apolipoprotein A-I obtained at a molar ratio of 1: 60 (uppermost curve) -1: 100 (lowest curve)

FIG. 19 non-denaturing PAGE SDS of lipid particles of tetranectin-apolipoprotein A-I using cholate, amphoteric detergents 3-8, 3-10 and 3-12. Lane 1 on each gel: pure apolipoprotein; lane 2 on each gel: 0.1xCMC cholate lipidated samples were used as reference.

Figure 20 SEC-MALLS protein conjugate analysis of lipid particles of tetranectin-apolipoprotein a-I using 3x CMC amphoteric detergent 3-8 and POPC (apolipoprotein: phospholipid molar ratio 1: 60).

Figure 21 SEC-MALLS protein conjugate analysis of lipid particles of tetranectin-apolipoprotein a-I using 2x CMC amphoteric detergent 3-10 and POPC (apolipoprotein: phospholipid molar ratio 1: 60).

FIG. 22 SEC-MALLS protein conjugation analysis of lipid particles of tetranectin-apolipoprotein A-I using POPC. The following steps: a lipid particle formed from native tetranectin-apolipoprotein a-I; the following: lipid particles formed from denatured tetranectin-apolipoprotein a-I.

FIG. 23 results of in vivo rabbit study with tetranectin-apolipoprotein A-I lipidated with DMPC (1: 100) (dimyristoylphosphatidylcholine) (a) and with tetranectin-apolipoprotein A-I not lipidated in PBS (b).

FIG. 24 SE-HPLC chromatograms of lipid particles comprising wild-type apolipoprotein A-I (A) and tetranectin-apolipoprotein A-I (B) as reported herein, stored at 5 ℃ and 40 ℃.

Materials and methods

Size exclusion-HPLC:

chromatography was carried out on a Tosoh Haas TSK3000SWXL column on an ASI-100HPLC system (Dionex, Idstein, Germany). The elution peak was monitored by UV diode array detector (Dionex) at 280 nm. After dissolving the concentrated sample to 1mg/ml, the column was washed with a buffer consisting of 200mM potassium dihydrogen phosphate and 250mM potassium chloride pH7.0 until a stable baseline was obtained. The analysis run was carried out in 30 minutes at room temperature under conditions of constant solvent composition (isocratic conditioning) using a flow rate of 0.5 ml/min. The chromatograms were manually integrated with Chromeleon (Dionex, Idstein, Germany). Polymerization in% was determined by comparing the area under the curve (AUC) for the high molecular weight form with the AUC for the monomer peak.

Dynamic Light Scattering (DLS):

DLS is a non-invasive technique for measuring particle size, typically in the sub-micron size range. In the present invention, a Zetasizer Nano S device (Malvern Instruments, Worcestershire, UK) with temperature controlled quartz cuvettes (25 ℃) was used to monitor the size range between 1nm and 6 μm. The intensity of the backscattered laser light was detected at an angle of 173 °. The intensity fluctuates at a rate dependent on the particle diffusion rate, which in turn is controlled by the particle size. Particle size data can thus be generated by analysing fluctuations in scattered Light intensity (Dahneke, B.E (ed.), Measurement of Suspended particles by quasi-electronized Light Scattering, Wiley Inc. (1983); Pecora, R., dynamic Light Scattering: Application of Photon Correlation Spectroscopy, Plenum Press (1985)). The size distribution of the intensity is calculated using the multiple narrow modes of the DTS software (Malvern). Experiments were performed with undiluted samples.

SEC-MALLS：

SEC-MALLS is a combination of size exclusion chromatography with three detector systems: i) UV detection, ii) refractive index detection and iii) light scattering detection. For size separation, a Superose6 column from GEHealthcare 10/300GL column was used. The process was carried out with a constant solvent composition using PBS buffer, pH7.4, using a flow rate of 0.4 ml/min. Three detector systems are connected in series. The signal of intact lipid particles (protein-lipid particles) was monitored by a refractive index detector, while the UV absorbance measured at 280nm determined the signal induced by the protein fraction. The proportion of the lipid fraction was obtained by simple subtraction of the protein UV signal from the complete signal. Applying light scattering allows the detection of the molecular weight of the respective species and thus the lipid particle can be described in full and in detail.

Detergent assay

The determination of residual detergent was performed by reverse phase chromatography (RP-ELSD) coupled with an evaporative light scattering detector. Luna C184.6x150mm, 5 μm,as a column. After centrifugation through a 10kDa membrane, 90. mu.l of the flow-through was used for HPLC separation. Elution was carried out under conditions of constant solvent composition with a 74% (v/v) methanol solution containing 0.1% (v/v) trifluoroacetic acid. The column temperature was set at 30 ℃. Detection was carried out by means of an evaporative light scattering detector, using a spray temperature of 30 ℃, an evaporation temperature of 80 ℃ and an air flow of 1.0 l/min. By using in the case of cholate salts ranging from 0.22 mug to 7.5 mug of cholate,a standard curve was established to quantify residual detergent.

Protein determination:

the protein concentration was determined by measuring the Optical Density (OD) at 280nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.

Recombinant DNA technology:

DNA is manipulated using standard methods, such as those described in Sambrook, j, et al, Molecular cloning: a laboratory manual (molecular cloning guidelines); cold Spring Harbor laboratory, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

Example 1

Preparation and description of E.coli expression plasmids

The tetranectin-apolipoprotein A-I fusion polypeptide is prepared by a recombination mode. The amino acid sequence of the expressed fusion polypeptide in the direction from N-terminal to C-terminal is as follows:

-the amino acid methionine (M),

an interferon sequence fragment having the amino acid sequence of CDLPQTHSL (SEQ ID NO: 55),

-a GS linker,

hexahistidine tag having the amino acid sequence of HHHHHHHH (SEQ ID NO: 56)

-a GS linker,

an IgA protease cleavage site having the amino acid sequence VVAPAP (SEQ ID NO: 60), and

-having the sequence of SEQ ID NO: 02 amino acid sequence of tetranectin-apolipoprotein A-I.

The tetranectin-apolipoprotein a-I fusion polypeptide as described above is a precursor polypeptide from which it is released by enzymatic cleavage in vitro using IgA protease.

The precursor polypeptide-encoding fusion gene is assembled by ligating appropriate nucleic acid fragments using known recombinant methods and techniques. The nucleic acid sequence prepared by chemical synthesis was confirmed by DNA sequencing. The preparation was used to generate the nucleic acid encoding SEQ ID NO: 31 of the fusion protein of SEQ ID NO: 01 of the tetranectin-apolipoprotein A-I expression plasmid.

Preparation of E.coli expression plasmid

Plasmid 4980(4980-pBRori-URA3-LACI-SAC) is an expression plasmid for the expression of core-streptavidin in E.coli. It was produced by ligating the 3142bp long EcoRI/CelII-vector fragment from plasmid 1966(1966-pBRori-URA 3-LACI-T-repeat; reported in EP-B1422237) with the 435bp long core-streptavidin coding EcoRI/CelII-fragment

The core-streptavidin E.coli expression plasmid comprises the following elements:

the origin of replication from the vector pBR322 for replication in E.coli (corresponding to the 2517-3160bp position described by Sutcliffe, G., et al, Quant.biol.43(1979) 77-90),

the URA3 gene of Saccharomyces cerevisiae (Rose, M.et al, Gene29(1984)113-124) encoding orotidine 5' -phosphate decarboxylase, which allows plasmid selection by complementation of an E.coli pyrF mutant strain (uracil auxotrophy),

core-streptavidin expression cassette comprising

T5 hybrid promoter (T5-PN 25/03/04 hybrid promoter as described in Bujard, H., et al, methods. enzymol. 155(1987)416-433 and Stueber, D., et al, Immunol. methods (immunological methods) IV (1990) 121-152), including the synthetic ribosome binding site as described in Stueber, D., et al (see above),

core-the streptavidin gene,

two phage-derived transcription terminators,. lambda. -T0 terminator (Schwarz, E., et al, Nature272(1978)410-414) and fd-terminators (Beck E. and Zink, B.Gene1-3(1981)35-58),

the lacI repressor gene from E.coli (Farabaugh, P.J., Nature274(1978) 765-769).

The final expression plasmid for expression of the tetranectin-apolipoprotein A-I precursor polypeptide was prepared by excision of the core-streptavidin structural gene from vector 4980 using unique flanking EcoRI and CelII restriction enzyme cleavage sites, and insertion of the precursor polypeptide-encoding nucleic acid flanking the EcoRII/CelII restriction sites into a 3142bp long EcoRI/CelII-4980 vector fragment.

Example 2

Expression of tetranectin-apolipoprotein A-I

For the expression of the fusion protein, an E.coli host/vector system was used which enables antibiotic-free plasmid selection by complementation of E.coli auxotrophs (PyrF) (EP0972838 and US6,291,245).

Escherichia coli K12 strain CSPZ-2(leuB, proC, trpE, th-1, ApyrF) was transformed by electroporation with expression plasmid p (IFN-His 6-IgA-tetranectin-apolipoprotein A-I). Transformed E.coli cells were first cultured on agar plates at 37 ℃.

Fermentation protocol 1:

for the preliminary fermentation, M9 medium according to Sambrook et al (Molecular Cloning: A laboratory Manual; Cold spring harbor laboratory Press; 2 nd edition (12 months 1989) supplemented with about 1g/l L-leucine, about 1g/l L-proline and about 1mg/l thiamine-HCl was used.

For the pre-fermentation, 2ml of the original inoculum storage ampoule was inoculated in 300ml of M9-medium in a 1000ml Erlenmeyer-flash with baffles. Incubation was carried out at 37 ℃ for 13 hours on a rotary shaker until an optical density of 1-3 (578nm) was observed.

For the fermentation, a batch medium according to Riesenberg et al (Riesenberg, D., et al, J.Biotechnol.20(1991)17-27) was used: 27.6g/l glucose H₂O，13.3g/l KH₂PO₄，4.0g/l(NH₄)₂HPO₄1.7g/l citrate, 1.2g/l MgSO₄*7H₂O, 60mg/l iron (III) citrate, 2.5mg/l CoCl₂*6H₂O，15mg/l MnCl₂*4H₂O，1.5mg/l CuCl₂*2H₂O，3mg/l H₃BO₃，2.5mg/l Na₂MoO₄*2H₂O，8mg/l Zn(CH₃COO)₂*2H₂O, 8.4mg/l Titriplex III, 1.3ml/l Synperonic 10% antifoam. Batch medium was supplemented with 5.4mg/L thiamine-HCl and 1.2g/L L-leucine and L-proline, respectively. Feed 1 solution contained 700g/l glucose supplemented with 19.7g/l MgSO₄*7H₂And O. The base solution used for pH adjustment was 12.5% (w/v) NH supplemented with 50g/l L-leucine and 50g/l L-proline, respectively₃An aqueous solution. All ingredients were dissolved in deionized water.

The fermentation was carried out in a 10l Biostat C DCU3 fermenter (Sartorius, Melsungen, Germany). Starting with 6.41 sterilized fermentation batch medium plus 300ml of inoculum from the pre-fermentation, batch fermentations were carried out at 37 ℃ at pH 6.9. + -. 0.2, 500mbar and an aeration rate of 10 l/min. After the initial glucose supplementation was completed, the temperature was adjusted to 28 ℃ and the fermentation entered fed-batch mode. At this point, the relative value of dissolved oxygen (pO2) was maintained at 50% (DO-stat, see e.g. Shay, L.K., et al, J.Indus.Microbiol.Biotechnol.2(1987)79-85) by adding feed 1 in combination with constantly increasing stirrer speed (550 rpm-1000rpm in 10 hours and 1000rpm-1400rpm in 16 hours) and aeration rate (10 l/min-16l/min in 10 hours and 16l/min-20l/min in 5 hours). When the pH reached the lower limit of adjustment (6.70) after about 8 hours of incubation, the addition of the base solution resulted in the supply of additional amino acid. Expression of the recombinant therapeutic protein was induced by the addition of 1mM IPTG at optical density 70.

At the end of the fermentation, the cytosolic and soluble expressed tetranectin-apolipoprotein a-I is transferred to insoluble protein aggregates, so-called inclusion bodies, using a heating step (in which the entire medium in the fermenter is heated to 50 ℃ for 1 or 2 hours) before collection (see, for example, EP-B1486571). Subsequently, the contents of the fermenter were centrifuged in a non-countercurrent centrifuge (13,000rpm, 13l/h) and the harvested biomass was stored at-20 ℃ until further processing. The insoluble protein aggregate form of the synthetic tetranectin-apolipoprotein a-I precursor protein, the so-called Inclusion Bodies (IBs), is found only in the insoluble fraction of cell debris.

The insoluble protein aggregates form (so-called Inclusion Bodies (IBs)) of the synthetic fusion protein are found only in the insoluble cell debris fraction.

Samples recovered from the fermentor were analyzed by SDS-polyacrylamide gel electrophoresis, one before induction and the other at designated time points after induction of protein expression. From each sample, the same amount of cells (OD) was added_Target= 5) were resuspended in 5mL PBS buffer and disrupted by sonication on ice. Next, 100. mu.L of each suspension was centrifuged (15,000rpm, 5 minutes), and each supernatant was recovered and transferred to a separate vial. This is to distinguish between soluble and insoluble expressed target proteins. To each supernatant (═ soluble) fraction was added 300. mu.L and to each pellet (═ insoluble) fraction was added 400. mu.L of SDS sample buffer (Laemmli, U.K., Nature227(1970) 680-. The sample was heated at 95 ℃ for 15 minutes with shaking to dissolve and reduce all proteins in the sample. After cooling to room temperature, 5. mu.L of each sample was transferred to a 4-20% TGX Criterion Stain Free polyacrylamide gel (Bio-Rad). In addition, 5. mu.l of a molecular weight Standard (Precision Plus Protein Standard, Bio-R)ad) and 3 amounts (0.3. mu.l, 0.6. mu.l and 0.9. mu.l) of quantification standard with known product protein concentration (0.1. mu.g/. mu.l) were set on the gel.

Electrophoresis was run at 200V for 60 minutes, and then the gel was transferred to a GelDOC EZ imager (Bio-Rad) and treated with UV irradiation for 5 minutes. The gel images were analyzed using Image Lab analysis software (Bio-Rad). Using three criteria, a linear regression curve was calculated with a coefficient > 0.99 and used to calculate the concentration of the target protein in the original sample.

Fermentation scheme 2:

For pre-fermentation, 300ml of modified M9-medium in a 1000ml Erlenmeyer flask with baffles was inoculated from 1-2ml in agar plates or with a primary inoculum storage ampoule. Incubation was carried out at 37 ℃ for 13 hours on a rotary shaker until an optical density of 1-3 (578nm) was observed.

For fermentations and high yields expressing tetranectin-apolipoprotein a-I, the following batches of media and feeds were used:

8.85g/l glucose, 63.5g/l yeast extract, 2.2g/l NH₄Cl, 1.94g/l L-leucine, 2.91g/l L-proline, 0.74g/l L-methionine, 17.3g/l KH₂PO₄*H2_O，2.02g/lMgSO₄*7H₂O, 25.8mg/l thiamine-HCl, 1.0ml/l Synperonic 10% antifoam. Feed 1 solution contained 333g/L yeast extract and 333 g/L85% -glycerol supplemented with 1.67g/L L-methionine and 5g/L L-leucine and L-proline, respectively. Feed 2 was a solution of 600g/l L-proline. The alkaline solution used for pH adjustment was a 10% (w/v) KOH solution, and a 75% glucose solution was used as an acid. All ingredients were dissolved in deionized water.

The fermentation was carried out in a 10l Biostat C DCU3 fermenter (Sartorius, Melsungen, Germany). Starting with 5.15l of sterilized fermentation batch medium plus 300ml of inoculum from the pre-fermentation, batch fermentations were carried out at 25 ℃ at pH 6.7. + -. 0.2, 300mbar and an aeration rate of 10 l/min. Before the start of the glucose supplementation was consumed, the culture reached an optical density of 15 (578nm) and the fermentation entered the feed-batch mode, at which time feed 1 started at 70 g/h. Glucose concentration was monitored in the culture, increasing feed 1 to a maximum of 150g/h while avoiding glucose accumulation and keeping the pH close to the upper adjustment limit of 6.9. At an optical density of 50 (578nm), feed 2 started at a constant feed rate of 10 ml/h. Dissolved oxygen (pO) was maintained by increasing the stirrer speed (500-1500 rpm), aeration rate (10-20 l/min) and pressure (300-500 mbar) in parallel₂) The relative value of (A) is more than 50%. Expression of the recombinant therapeutic protein was induced by the addition of 1mM IPTG at an optical density of 90.

Seven samples recovered from the fermentor were analyzed by SDS-polyacrylamide gel electrophoresis, one before induction and the other at designated time points after induction of protein expression. From each sample, the same amount of cells (OD) was added_Target= 5) were resuspended in 5mL PBS buffer and disrupted by sonication on ice. Next, 100. mu.L of each suspension was centrifuged (15,000rpm, 5 minutes), and each supernatant was recovered and transferred to a separate vial. This is to distinguish between soluble and insoluble expressed target proteins. To each supernatant (═ soluble) fraction was added 300. mu.L and to each pellet (═ insoluble) fraction was added 200. mu.L of SDS sample buffer (Laemmli, U.K., Nature227(1970) 680-. The sample was heated at 95 ℃ for 15 minutes with shaking to dissolve and reduce all proteins in the sample. After cooling to room temperature, 5. mu.L of each sample was transferred to a 10% Bis-Tris polyacrylamide gel (Novagen). In addition, 5. mu.l of a molecular weight Standard (Precision Plus Protein Standard, Bio-Rad) and 3 kinds of quantitative standards (0.3. mu.l, 0.6. mu.l and 0.9. mu.l) having a known concentration of the product Protein (0.1. mu.g/. mu.l) were set on the gel.

The electrophoresis was run at 200V for 35 minutes, followed by staining the gel with Coomassie Brilliant blue R dye, destaining with heated water and transferring to a densitometer for digitization (GS710, Bio-Rad). The gel images were analyzed using Quantity One1-D analysis software (Bio-Rad). Using three criteria, a linear regression curve was calculated with a coefficient > 0.98 and used to calculate the concentration of the target protein in the original sample.

At the end of the fermentation, the cytosolic and soluble expressed tetranectin-apolipoprotein a-I is transferred to insoluble protein aggregates, so-called inclusion bodies, using a heating step (in which the entire medium in the fermenter is heated to 50 ℃ for 1 or 2 hours) before collection (see, for example, EP-B1486571). After the heating step, the IBs form of the synthetic tetranectin-apolipoprotein a-I precursor protein is found only in the insoluble fraction of cell debris.

The contents of the fermenter were cooled to 4-8 ℃, centrifuged with a non-countercurrent centrifuge (13,000rpm, 13l/h), and the harvested biomass was stored at-20 ℃ until further processing. Depending on the expressed construct, the total harvested biomass yield ranged between 39g/l and 90g/l dry matter.

Example 3

Preparation of tetranectin-apolipoprotein A-I

By adding to potassium phosphate buffer or Tris buffer (0.1M, supplemented with 1mM MgSO)₄pH6.5) were resuspended and the collected bacterial cells were subjected to inclusion body preparation. After the addition of DNAse, the cells were disrupted by homogenization at a pressure of 900 bar. A buffer solution containing 1.5M NaCl and 60mM EDTA was added to the homogenized cell suspension. After adjusting the pH to 5.0 with 25% (w/v) HCl, the final inclusion body slurry was obtained after a further centrifugation step. The slurry was stored at-20 ℃ in single-use sterilized plastic bags until further processing.

The inclusion body slurry (about 15kg) was dissolved in guanidine hydrochloride solution (150 l, 6.7M). After clarification of the solubilizate by depth filtration, the solution is added to the Zn-chelated affinity chromatography material. The fusion polypeptide was purified by Zn-chelate chromatography material and cleaved by IgA protease. Subsequently, the polypeptide is further purified by anion exchange chromatography and cation exchange chromatography steps. These steps are carried out in a solution comprising urea (7M), i.e. under denaturing conditions. These steps are used to remove polypeptide fragments, endotoxins and other impurities. Diafiltration in a solution containing 6.7M guanidine hydrochloride was performed. The final solution obtained contained denatured tetranectin-apolipoprotein a-I.

Example 4

Refolding and lipidation of tetranectin-apolipoprotein A-I

a) General procedure

Pure crystalline POPC or DPPC (Lipoid, Switzerland) was dissolved in an aqueous buffer (lipidation buffer) containing cholate in a 1: 1.35 molar ratio phospholipid cholate. The mixture was incubated under nitrogen atmosphere and protected from light at room temperature (POPC) or at 55 ℃ (DPPC) until a clear solution was obtained. The clarified lipid-bile acid salt solution was cooled to 4 ℃ (POPC) or stored at 41 ℃ (DPPC). Purified tetranectin-apolipoprotein A-I was added at 4 deg.C (POPC) or 41 Deg.C (DPPC) at defined apolipoprotein: phospholipid ratios. For lipid particle formation, the reaction mixture was incubated overnight at 4 ℃ (POPC) or 41 ℃ (DPPC) under nitrogen atmosphere and protected from light. Finally, cholate was removed by extensive dialysis (4 ℃/41 ℃) against the lipidation buffer. Finally, the sample is centrifuged to remove precipitated material.

Cholate-solubilized lipid solutions containing pure POPC or pure DPPC were prepared as above. Preparation of lipid mixtures by combining lipid solutions in the required ratios followed by the respective T_m(T_mPhase transition temperature). As described for the pure lipid solutions, but at the respective T of the selected lipid mixture_mLipid particle formation of tetranectin-apolipoprotein a-I was performed.

The following lipidation buffers have been tested:

1. 50mM potassium phosphate buffer supplemented with 250mM arginine hydrochloride, 7.5% sucrose at pH7.5

2. 50mM dipotassium phosphate buffer supplemented with 250mM arginine hydrochloride, 7.5% sucrose, 10mM methionine at pH7.5

3. 250mM TRIS-hydroxyaminomethane (TRIS) supplemented with 140mM NaCl, 10mM methionine at pH7.5

4. 50mM dipotassium phosphate buffer supplemented with 250mM arginine hydrochloride, 7% trehalose, 10mM methionine at pH7.5.

Homogeneity of lipid particles formed from tetranectin-apolipoprotein a-I samples was assessed by analytical SEC (figures 11 and 12). In summary, the choice of lipid buffer had only a minor effect compared to the choice of phospholipids. DPPC-lipid particles eluted as one major peak, and POPC-lipid particles showed two peak patterns. The choice of lipidation buffer is influenced by the purification process of the apolipoproteins and the supply of stable lipid-free apolipoproteins. It was shown that lipid particles could be formed independently of the lipidation buffer. Of the buffers tested, the most suitable lipidation buffer was identified as 250mM Tris, 140mM NaCl, 10mM methionine, pH7.5.

The lipidation mixture contains a defined amount of apolipoproteins, respectively, and the amount of phospholipids, e.g. POPC, is calculated accordingly. The molar amount of lipids was calculated based on tetranectin-apolipoprotein a-I monomer.

b) POPC and cholate

Table 6: lipid particles were formed using pure POPC with tetranectin-apolipoprotein a-I as an example. And calculating the molar ratio of the apolipoprotein to the phospholipid of the protein monomer. Comparison: apolipoprotein without addition of lipid (pure Apo) and lipid without addition of apolipoprotein (Apo-free).

Clarification after centrifugation

The molar ratio from 1: 40 to 1: 160 remained clear throughout the process. No turbidity by excess phospholipid nor protein precipitation was observed.

Lipid particle samples were analyzed by non-denaturing PAGE (see figure 13). The most uniform band pattern was found with sample 1: 80 (lane 4). In addition, 1 Xfreeze/thaw (-80 ℃) did not change the appearance of the sample (lane 5). The banding pattern for samples 1: 320 and 1: 160 showed heterogeneous products, resulting in multiple bands (lanes 2 and 3). Samples 1: 40 and also 1: 20 had additional bands below the main product band (lanes 6 and 7). The migration pattern of pure tetranectin-apolipoprotein A-I is not apparent in lane 8 of FIG. 13.

The SEC-MALLS assay is used to obtain more detailed information on the homogeneity of lipid particles and their apolipoprotein-phospholipid composition (protein-conjugate analysis). Fig. 14 shows the chromatograms of the SEC resolved samples (UV280 detection). Here, the 1: 160 sample was divided into three separate peaks. The 1: 80 sample was shown to contain at least two species of different sizes, as shown as a double peak. The peak obtained from sample 1: 20 shows the most uniform product.

In step 5, experiments were performed using tetranectin-apolipoprotein A-I (3.84 mg/ml; 10 mg/sample) and the apolipoprotein: phospholipid molar ratio was increased from 1: 40 to 1: 80. At molar ratios below 1: 40, lipid particle formation is incomplete. By experiment it was excluded that at molar ratios above 1: 80, the sample became turbid after removal of cholate by dialysis. Moreover, at higher lipid ratios, the lipid particles become more heterogeneous.

Table 7: lipid particle formation of tetranectin-apolipoprotein a-I using pure POPC. The molar ratio of apolipoprotein to phospholipid was calculated based on tetranectin-apolipoprotein a-I monomer.

Volume 2.6ml before and after dialysis

Within SD of said method

All samples remained visually clear during the-3 ℃ transition temperature incubation. After removal of cholate by dialysis, an increased turbidity of the samples 1: 40 to 1: 65 was observed. The precipitate can be removed by centrifugation and the sample subsequently remains clear.

The SEC-MALLS assay is used to obtain detailed information about the homogeneity of the formed lipid particles and their apolipoprotein-phospholipid composition (protein-conjugate analysis). All lipid particles were comparably homogeneous on analytical size exclusion chromatography (SEC; fig. 15), showing a small post peak, which is more pronounced at lower molar ratios. In addition, there is a significant shift in the peak mode from higher molar ratios to higher molecular weights. The respective retention times are provided in table 8.

Table 8: summary of size exclusion chromatography results: percent calculation by integration of area under the curve (AUC)

Protein-conjugate analysis (summarized in table 8) enables to calculate the total molecular weight of the protein (MW protein) as well as the lipid component (MW lipid) of each lipid particle eluted from the SEC column. The composition of the lipid particles (n-protein and n-POPC) can be calculated based on the molecular weight of the tetranectin-apolipoprotein a-I monomer (32.7kDa) and POPC (760 Da). The molecular weight of the apolipoprotein fraction found in the major peak of the lipid particles at all molar ratios, corresponding to the tetranectin-apolipoprotein a-I trimer of each lipid particle, is about 100 kDa. The ratio of n (POPC)/n (protein monomers) provides the number of POPC molecules per tetranectin-apolipoprotein a-I monomer in the lipid particle. The number of POPC molecules per tetranectin-apolipoprotein A-I monomer varies between 54 and 75, although molar ratios from 1: 40 up to 1: 80 are applied. The% protein value is a parameter of the degree of lipidation. The lower the percentage of protein in the lipid particle, the higher the degree of lipidation.

Table 9: as shown in fig. 16, a summary of protein conjugate analysis of POPC and tetranectin-apolipoprotein a-I lipid particles.

c) DPPC and cholate

Before lipidation, 50mM KH pH7.5 was used₂PO₄Tetranectin-apolipoprotein A-I was dialyzed against 250mM arginine hydrochloride, 7% trehalose, 10mM methionine. In step 5, tetranectin-apolipoprotein A-I (3.84mg/ml, 3 mg/sample) was lipidated using a molar ratio (lipid concentration increase) of 1: 60 to 1: 100. The lipidation buffer was 250mM Tris-HCl, 140mM NaCl, 10mM methionine, pH7.5.

Table 10: sample review of lipid particles of apolipoprotein and DPPC

Calculated for protein monomers

During the formation of the lipid particles, no protein precipitation or clouding by excess lipid was observed. The higher the yield of tetranectin-apolipoprotein a-I in the final product, the more DPPC is available for lipidation.

Residual lipid-free apolipoprotein was found in the 1: 20 sample on native PAGE (lane 3, FIG. 17). On native PAGE, the 1: 40 and 1: 60 samples appeared most homogeneous (lanes 4 and 5), while the 1: 80 and 1: 100 samples contained additional higher molecular bands on the major lipid particle band (lanes 6 and 7).

SEC-MALLS protein conjugate analysis was used to characterize the composition of the lipid particles obtained after DPPC lipid particle formation (MW DPPC: 734 Da). Uniform SEC peaks were obtained at molar ratios of 1: 80 and below. At higher lipid ratios, pre-peaks appear (see, e.g., 1: 90 samples in Table 11).

Table 11: SEC-MALLS protein conjugate analysis of lipid particles of DPPC and tetranectin-apolipoprotein A-I

The highest degree of lipidation (lowest percentage of protein) was found with a molar ratio of 1: 80 to 1: 90. In addition, DLS revealed the most uniform particle formation at ratios of 1: 80 to 1: 90 (> 98%), with particle sizes ranging from 14 to 17 nm.

d)75％DPPC/25％POPC

Lipid particle formation was carried out accordingly as reported in the examples items a) -c), using the following parameters:

protein: tetranectin-apolipoprotein A-I is 3.84mg/ml, 3 mg/sample

Lipidation buffer: 250mM Tris-HCl, 140mM NaCl, 10mM methionine pH7.5

Lipidization: at 34 deg.C

And (3) dialysis: at 4 deg.C

Molar ratio tested: in step 5, 1: 60 to 1: 100, with increased lipid

Lipid particle formation is simple and easy and comparable to the process performed with pure lipids. All samples remained clear during the method and dialysis. The yield of lipid particles was similar for all ratios tested (-85%). SEC-MALLS analysis showed that a molar ratio of 1: 80 resulted in the most homogeneous lipid particle with 90.9% main peak, no pre-peak and 9.1% post-peak. Protein conjugate analysis showed the presence of 1 tetranectin-apolipoprotein a-I trimer/lipid particle in the main species of all samples (see figure 18 and tables 12 and 13).

Table 12: summary of SEC results; percent calculation by AUC integration

Table 13: summary of protein-conjugate analysis of 75% DPPC/25% POPC and tetranectin-apolipoprotein a-I lipid particles.

e)50％DPPC/50％POPC

protein: tetranectin-apolipoprotein A-I is 3.84mg/ml, 3 mg/sample

Lipidation buffer: 250mM Tris-HCl, 140mM NaCl, 10mM methionine pH7.5

Lipidization: at 27 deg.C

And (3) dialysis: at room temperature

Molar ratio tested: in step 5, 1: 60 to 1: 100, with increased lipid

All samples remained clear during the process and dialysis. The yield of lipid particles was similar for all ratios tested.

Table 14: summary of SEC results; the percentage was calculated by integration of AUC.

A lipid mixture of 50% DPPC and 50% POPC was used for lipid particle formation of tetranectin-apolipoprotein a-I, obtaining the most homogeneous product at a molar ratio of 1: 70 (see table 14). The product was 89.9% pure with respect to the main peak and contained one single tetranectin-apolipoprotein a-I trimer (see table 15).

Table 15: protein conjugate analysis of lipid particles with 50% DPPC/50% POPC and tetranectin-apolipoprotein a-I summary

f)25％DPPC/75％POPC

protein: tetranectin-apolipoprotein A-I is 3.84mg/ml, 3 mg/sample

Lipidation buffer: 250mM Tris-HCl, 140mM NaCl, 10mM methionine pH7.5

Lipidization: at 18 deg.C

And (3) dialysis: at room temperature

Molar ratio tested: in step 5, 1: 60 to 1: 100, with increased lipid

Lipid particle formation is simple and easy and comparable to the process using pure lipids. All samples remained clear during the method and dialysis.

Table 16: summary of SEC results; the percentage was calculated by integration of AUC.

Lipid mixtures of 25% DPPC and 75% POPC were used to form tetranectin-apolipoprotein a-I, yielding the most homogeneous product at a molar ratio of 1: 60 (see table 17). The product was 90.2% pure with respect to the main peak and contained one single tetranectin-apolipoprotein a-I trimer (see table 15).

Table 17: summary of protein conjugates of lipid particles of 25% DPPC/75% POPC and tetranectin-apolipoprotein a-I.

g) Forming lipid particles using amphoteric detergents

The formation of lipid particles was carried out accordingly (using the following parameters) as reported in the examples under a) -c), except that the cholate was replaced by a synthetic detergent amphoteric detergent:

protein: tetranectin-apolipoprotein A-I at 23.5mg/ml

Buffer solution: 50mM Tris-HCl, 7.2M guanidine hydrochloride, 10mM methionine, pH8

Lipidation buffer: 250mM Tris-HCl, 140mM NaCl, pH7.5

100% POPC, apolipoprotein: phosphatide molar ratio is 1: 60

Table 18: sample overview of various methods and observations/parameters of lipid particle formation

Lipid particles comprising tetranectin-apolipoprotein a-I were analysed on non-denaturing PAGE. Lipid-free tetranectin-apolipoprotein A-I migrates at 140kDa (lane 1 in FIG. 19), whereas the lipid particle shows a characteristic shift towards higher molecular weights between 232kDa and 440 kDa.

Lipid-free tetranectin-apolipoprotein a-I was detected in all samples prepared with only 0.1x CMC of the respective detergent, but no lipid particles were detected (fig. 19, lanes 2, 8, 13, and 19). However, 0.5x CMC concentration of detergent was sufficient to enable amphoteric detergents 3-8 and 3-10 to form tetranectin-apolipoprotein a-I into lipid particles (lanes 3, 9, and 14). With amphoteric detergent 3-12, no lipid particle formation occurred until a concentration of 2.0x CMC was reached (lane 21).

Figure 20 shows SEC-MALLS chromatograms of lipid particles comprising tetranectin-apolipoprotein a-I using 3x CMC amphoteric detergent 3-8 and POPC (apolipoprotein: phospholipid molar ratio 1: 60). The results of the protein-conjugate analysis are summarized in table 18. The lipid particle fraction consists of two different species, as shown in the SEC chromatogram in two overlapping peaks. However, these two species are very similar, differing mainly in the number of tetranectin-apolipoprotein a-I molecules per particle (4.2 for peak 1 and 3.5 for peak 2).

Table 19: summary of protein-conjugate analysis of lipid particles formed in the presence of amphoteric detergent 3-8.

Figure 21 shows a chromatogram of the SEC-MALLS analysis and a summary of the protein-conjugate analysis of lipid particles comprising tetranectin-apolipoprotein a-I using 2x CMC amphoteric detergent 3-10 and POPC (apolipoprotein: phospholipid molar ratio 1: 60). The two peaks contain lipid particles containing 3.5 and 5 tetranectin-apolipoprotein a-I molecules, respectively.

Table 20: summary of protein-conjugate analysis of lipid particles formed in the presence of amphoteric detergent 3-10.

The results of lipid particle formation comprising tetranectin-apolipoprotein a-I using amphoteric detergents 3-12 and POPC (apolipoprotein: phospholipid molar ratio 1: 60) are summarized in table 21. The lipid particle fraction consists of two different species, as shown in the SEC chromatogram in two overlapping peaks. However, these two species are very similar, differing mainly in the number of tetranectin-apolipoprotein a-I molecules per particle.

Table 21: summary of protein-conjugate analysis of lipid particles formed in the presence of amphoteric detergent 3-12.

The results of lipid particle formation comprising tetranectin-apolipoprotein a-I using cholate and POPC (apolipoprotein: phospholipid molar ratio ═ 1: 60) are summarized in table 21. The lipid particle fraction consists of two different species, as shown in the SEC chromatogram in two overlapping peaks. However, these two species are very similar, differing mainly in the number of tetranectin-apolipoprotein a-I molecules per particle.

Table 22: summary of protein-conjugate analysis of lipid particles formed in the presence of cholate

Example 5

Rapid dilution method for refolding and lipid particle formation

a) POPC and sodium cholate

Tetranectin-apolipoprotein A-I was expressed in E.coli and purified according to examples 1-3 (method 1). After purification, the buffer was changed by diafiltration to a solution containing 250mM Tris, 140mM NaCl, 6.7M guanidine hydrochloride at pH 7.4. The protein concentration was adjusted to 28 mg/ml.

Lipid stock solutions were prepared by dissolving 100 mol/l POPC in a pH7.4 buffer containing 250mM Tris-HCl, 140mM NaCl, 135mM sodium cholate at room temperature. The lipid stock solution was incubated at room temperature for 2 hours. Refolding buffer was prepared by diluting 77ml of lipid stock mixture into 1478ml of 250mM Tris-HCl, 140mM NaCl, pH 7.4. The buffer was stirred at room temperature for an additional 7 hours.

Refolding and lipid particle formation was initiated by adding 162ml tetranectin-apolipoprotein A-I in 250mM Tris, 140mM NaCl, 6.7M guanidine hydrochloride, pH7.4 to the refolding buffer. This resulted in a 1: 10 dilution of guanidine hydrochloride. The solution was incubated at room temperature for 16 hours while stirring was continued. The removal of the detergent was performed by diafiltration.

Table 23: summary of protein conjugate analysis of lipid particles obtained by rapid dilution with POPC

Tetranectin-apolipoprotein A-I was expressed in E.coli and purified according to examples 1-3 (method 2). After purification, the buffer was changed by diafiltration to a solution containing 50mM Tris, 10mM L-methionine, 6.7M guanidine hydrochloride, pH 7.4. The protein concentration was adjusted to 20.4 mg/ml.

A lipid stock solution was prepared by dissolving 100 mol/L phospholipid (POPC: DPPC ratio 3: 1) in a buffer pH7.4 containing 250mM Tris-HCl, 140mM NaCl, 10mM L-methionine, 135mM sodium cholate at room temperature. Refolding buffer was prepared by diluting 3.7ml of lipid stock solution into 35.6ml of 250mM Tris-HCl, 140mM NaCl, pH 7.4. The buffer was stirred at room temperature for an additional 2 hours.

Refolding and lipid particle formation was initiated by adding 9.8ml of tetranectin-apolipoprotein A-I in 50mM Tris, 10mM L-methionine, 6.7M guanidine hydrochloride, pH8.0 to the refolding buffer. This resulted in a 1: 5 dilution of guanidine hydrochloride. The solution was incubated at room temperature overnight while continuing to stir. The removal of the detergent was performed by diafiltration.

Table 24: protein conjugate analysis of lipid particles obtained by rapid dilution with POPC/DPPC/cholate mixtures was summarized.

b) POPC, DPPC and sodium cholate

Tetranectin-apolipoprotein A-I was expressed in E.coli and purified according to examples 1-3. After purification, the buffer was changed by diafiltration to a solution containing 250mM Tris, 140mM NaCl, 6.7M guanidine hydrochloride at pH 7.4. The protein concentration was adjusted to 30 mg/ml.

Two separate lipid stock solutions were prepared. Solution A was prepared by dissolving 100 mol/l POPC in a buffer pH7.4 containing 250mM Tris-HCl, 140mM NaCl, 135mM sodium cholate at room temperature. Solution B was prepared by dissolving 100moles/l DPPC in 250mM Tris-HCl, 140mM NaCl, 135mM sodium cholate pH7.4 at 41 ℃. Lipid stock solutions A and B were mixed in a 3: 1 ratio and incubated at room temperature for 2 hours. Refolding buffer was prepared by diluting 384ml of lipid stock solution into 6365ml of 250mM Tris-HCl, 140mM NaCl, pH 7.4. The buffer was stirred at room temperature for another 24 hours.

Refolding and lipid particle formation was initiated by adding 750ml tetranectin-apolipoprotein A-I in 250mM Tris, 140mM NaCl, 6.7M guanidine hydrochloride, pH7.4 to the refolding buffer. This resulted in a 1: 10 dilution of guanidine hydrochloride. The solution was incubated at room temperature for at least 12 hours while stirring was continued. The removal of the detergent was performed by diafiltration.

Table 25: protein conjugate analysis of lipid particles obtained by rapid dilution with POPC DPPC 1: 1

c) Different guanidine hydrochloride concentrations

The tetranectin-apolipoprotein A-I according to the invention was expressed in E.coli and purified from inclusion bodies by metal chelate affinity chromatography (see examples 1-3). After purification, the buffer was changed by diafiltration to a solution containing 250mM Tris, 140mM NaCl, 6.7M guanidine hydrochloride at pH 7.4. The protein concentration was adjusted to 28 mg/ml.

Lipid stock solutions were prepared by dissolving 100 mol/l POPC in a pH7.4 buffer containing 250mM Tris-HCl, 140mM NaCl, 135mM sodium cholate at room temperature. The lipid stock solution was incubated at room temperature for 2 hours. Refolding buffers were prepared by diluting the lipid stock solution into 250mM Tris-HCl, 140mM NaCl, pH 7.4. The buffer was stirred at room temperature for another 12 hours. Varying amounts of tetranectin-apolipoprotein a-I were diluted into refolding buffer: 1: 5, 1: 7.5, 1: 10, 1: 12.5. This resulted in different residual concentrations of guanidine hydrochloride in the refolding buffer. The solution was allowed to stir at room temperature o/n to initiate refolding and lipid particle formation. The detergent was removed by dialysis.

Table 26: summary of protein conjugate analysis of lipid particles obtained by rapid dilution with different dilution ratios.

d) POPC and sodium cholate in the presence of urea

Tetranectin-apolipoprotein A-I was expressed in E.coli and purified according to examples 1-3. After purification, the buffer was changed by diafiltration to a solution containing 250mM Tris, 140mM NaCl, 6.7M urea, pH 7.4. The protein concentration was adjusted to 28 mg/ml.

Refolding and lipid particle formation was initiated by adding 162ml tetranectin-apolipoprotein A-I in 250mM Tris, 140mM NaCl, 6.7M urea, pH7.4 to the refolding buffer. This resulted in a 1: 10 dilution of urea. The solution was incubated at room temperature for 16 hours while stirring was continued. The removal of the detergent was performed by diafiltration.

e) POPC and sodium cholate and wild type apolipoprotein A-I

In another exemplary second method, human apolipoprotein A-I (wild-type apolipoprotein A-I) in 6.7M guanidine hydrochloride, 50mM Tris, 10mM methionine, pH8.0 is diluted 1: 5(v/v) into a lipidation buffer to give a protein concentration of 0.6 mg/ml. The lipidation buffer consisted of 7mM cholate, 4mM POPC and 1.3mM DPPC, corresponding to a lipid: protein ratio of 240: 1. SEC-MALLS was used to analyze complex formation. About two apolipoprotein molecules are found in a complex consisting of about 200 lipid molecules.

Table 27: summary of protein conjugate analysis

Example 6

Lipid particle formation starting from denatured or native proteins

The method as reported in example 4 (first method) requires native apolipoprotein for lipid particle formation, whereas the method reported in example 5 (second method) starts lipid particle formation with fully denatured apolipoprotein.

In an exemplary first method, denatured tetranectin-apolipoprotein A-I in 6.7M guanidine hydrochloride, 50mM Tris, 10mM methionine, pH8.0 was extensively dialyzed against a buffer pH7.5 consisting of 250mM Tris, 140mM NaCl, 10mM methionine, at a protein concentration of 3.46 mg/ml. Next, a mixture of POPC and cholate was added to produce a final concentration of 6mM POPC and 8mM cholate in solution. This corresponds to a ratio of 60 molecules of POPC/tetranectin-apolipoprotein A-I monomer (60: 1). Subsequently, the detergent was removed by diafiltration. The protein-lipid complexes formed were analyzed by SEC-MALLS. Using this method, heterogeneous products are formed in which about 60% of the species formed include more than three tetranectin-apolipoprotein a-I monomers.

In an exemplary second method, denatured tetranectin-apolipoprotein A-I in 6.7M guanidine hydrochloride, 50mM Tris, 10mM methionine, pH8.0 was diluted 1: 10(v/v) directly into the lipidation buffer to give a protein concentration of 2.5 mg/ml. The lipidation buffer consisted of 6mM cholate and 4.5mM POPC, corresponding to a lipid to protein ratio of 60: 1. Using this method, a homogeneous product was formed, comprising more than 90% of the single forming species, in which 60 molecules of lipid were bound per tetranectin-apolipoprotein a-I molecule (see fig. 22).

Table 28: summary of protein conjugate analysis

Example 7

Lipidation of insulin-F with cholate-and amphoteric detergent-solubilized POPC/DPPC

Selection for lipidsThe particle-forming protein is commercially available insulin (II)InsulinLispro, Lilly). The molecular weight of the protein is 5808 Da. To increase the detection limit of insulin in lipid particles, the protein was treated with NHS-fluorescein (6- [ fluorescein-5 (6) -carboxyamino-amino group)]N-hydroxysuccinimide hexanoate, Sigma Aldrich #46940-5 MG-F).

Amphoteric detergent-and cholate-mediated lipidation of NHS-fluorescein-labeled insulin (insulin-F) was performed as reported in example 4 using a 1: 1 mixture of POPC and DPPC. 0.5mM lipid mixture was dissolved in 1XCMC cholate, 2 XCMC amphoteric detergent 3-8 or 5 XCMC amphoteric detergent 3-10 in PBS pH 7.4. The dissolution of the lipids was achieved in an ultrasonic bath at 45 ℃ for 1 hour. insulin-F was added to the dissolved lipids in a molar ratio of protein to lipid of 1: 2 (amphoteric detergent 3-8) or 1: 1.2 (amphoteric detergent 3-10 and cholate). The lipidation mixture was incubated for 1 hour at room temperature, followed by extensive dialysis using PBS ph7.4 to remove the detergent.

The formed lipid particles and control samples were analyzed on SE-HPLC using fluorescence detection (494nm ext., 521nm em.) and UV280 absorption. Three different samples were analyzed on SE-HPLC for each lipidation method: insulin-F dissolved in PBS, liposomes without insulin F in PBS and lipid particles comprising insulin-F. The non-lipidated insulin-F eluted from the column at an elution time of about 40min and peaks were detected by fluorescence and UV280 detection. The lipidated insulin F-sample eluted from the column as two separate peaks, which were detected by fluorescence and UV 280. Late peaks (peak maxima at about 40min) migrated with insulin-F control samples. The early peak at 15min elution time was of higher molecular weight than pure insulin-F and consisted of lipidated insulin-F. The protein without lipid particles eluted at an elution time of 15 min.

Example 8

Application of apolipoprotein

a) Effect of DPPC and POPC on LCAT Activity

Lipid particles comprising Palmitoyl Oleoyl Phosphatidylcholine (POPC) or dipalmitoyl phosphatidylcholine (DPPC) and recombinant wild-type apolipoprotein a-I or tetranectin-apolipoprotein a-I were examined for their ability to support cholesterol esterification by LCAT.

Tritiated cholesterol (4%; relative to molar-based phosphatidylcholine content) was incorporated into the lipid particles by addition of a cholesterol ethanolic solution. In the presence of 125. mu.l (10mM Tris, 150mM NaCl, 1mM EDTA, 1mM NaN)₃(ii) a pH 7.4; 2mg/ml HuFAF albumin; 4mM β -mercaptoethanol) was tested at 37 ℃ for 1 hour for the ability of the resulting protein-lipid complexes to support LCAT catalyzed cholesterol esterification. The reaction was stopped by adding chloroform: methanol (2: 1) and the lipids were extracted. The "percent" esterification was calculated after separation of cholesterol-cholesterol esters by TLC and scintillation counting. Since less than 20% of the tracer is incorporated into the formed ester, the reaction rate can be considered constant under the reaction conditions. Using XLfit software (IDBS), data were fitted to the meter-man equation (Michaelis Mentenequation). For a display of these results, see FIG. 3.

b) Effect of DPPC/POPC mixtures on LCAT Activity

Lipid particles were prepared by mixing recombinant wild-type apolipoprotein A-I with 3H cholesterol, a DPPC/POPC mixture and cholate in a molar ratio of 1: 4: 80: 113, and using the cholate as a detergent. The DPPC/POPC mixture comprises any 100% POPC; 75% POPC; 50% POPC; 25% POPC.

After removal of cholate by dialysis, the resulting protein-lipid complexes were tested for their ability to support LCAT-catalyzed cholesterol esterification. By adding ethanol solution of cholesterol³H-cholesterol (4%; relative to the molar-based phosphatidylcholine content) is incorporated in the lipid particles. Presence existenceIn 125. mu.l (10mM Tris, 150mM NaCl, 1mM EDTA, 1mM NaN)₃(ii) a pH 7.4; 2mg/ml HuFAF albumin; 4mM β -mercaptoethanol) was tested at 37 ℃ for 1 hour for the ability of the resulting protein-lipid complexes to support LCAT catalyzed cholesterol esterification. The reaction was stopped by adding chloroform: methanol (2: 1) and the lipids were extracted. The "percent" esterification was calculated after separation of cholesterol-cholesterol esters by TLC and scintillation counting. Since less than 20% of the tracer is incorporated into the ester, the reaction rate can be considered constant under the reaction conditions. Using XLfit software (IDBS), the data were fit to the mi-man equation and are shown in fig. 4.

Table 3 a: apparent kinetic parameters

c) Efflux of cholesterol into THP-1 derived foam cells

Macrophages such as human THP-1 cells are obtained by exposing THP-1 monocytic leukemia cells to phorbol-12-myristate-13-acetate (phorbol myristate acetate). Subsequently, the cells are contained by being present³Acetylated LDL of H cholesterol tracer was further cultured for loading. These model foam cells were then exposed to cholesterol receptors for 4h-8h test compounds (see below).

Cell culture supernatants were harvested and cells were cut in 5% NP 40. The efflux fraction was calculated as the ratio of cholesterol radioactivity in the supernatant to the sum of the radioactivity in the cells plus the supernatant. The efflux of cells exposed to medium containing no receptor was subtracted and the efflux velocity was calculated by linear fitting. The efflux speed (relative efflux speed) was normalized to that of the wild-type apolipoprotein A-I at 10. mu.g/ml as a reference using the efflux from the cells. The relative efflux velocities obtained in two separate experiments were plotted as a function of cholesterol receptor concentration and the data were fit to the mie-man equation.

Parallel experiments were performed using cells exposed to RXR-LXR agonists known to upregulate ABCA-1 transporters and bias cholesterol transport toward ABCA-1 mediated efflux.

Only minor effects of the lipid mixture were observed in the test series (fig. 5 and table 29).

Table 29: different samples.

RXR-LXR pre-treatment of foam cells strongly increased efflux to non-lipidated species with a 6-fold increase in maximal velocity compared to untreated cells. The effect on lipid particles was much smaller with a 2-fold increase, reflecting a smaller contribution of the ABCA-1 transporter to cholesterol efflux (figure 6).

d) In vivo studies

5 lipid particle variants were studied:

i) POPC only

ii) DPPC only

iii)POPC∶DPPC 3∶1

iV)POPC∶DPPC 1∶1

V)DPPC∶SM 9∶1

Rabbits were infused intravenously at 80mg/kg over 0.5h (n-3 rabbits/test compound) followed by serial blood sampling over 96h post-infusion.

Apolipoprotein levels were analyzed by ELISA:

-drug level

Data on plasma values of liver enzymes, cholesterol esters.

The plasma concentrations were very similar for all tested compositions, showing a very insignificant initial "distribution" phase followed by a log-linear decrease in concentration (figure 7, table 3).

Table 3: pharmacokinetic data.

The Pharmacokinetic (PK) parameters determined were similar for all compounds tested. In addition, low intra-individual variability was also found. The half-life measured was close to 1.5 days, i.e., increased compared to wild-type apolipoprotein A-I. The volume of distribution was similar to the plasma volume (in rabbits, approximately 40 ml/kg).

f) Cholesterol mobilization

Mobilize and esterify cholesterol in plasma. Indeed, plasma cholesterol ester levels continue to increase even after tetranectin-apolipoprotein a-I has begun to decrease. When plasma tetranectin-apolipoprotein A-I levels had decreased to 0.5mg/ml (about 50% of normal wild-type apolipoprotein A-I), increased cholesterol ester levels were still detectable (FIG. 8).

g) Liver enzyme release

Lipid particles comprising tetranectin-apolipoprotein a-I containing POPC did not induce liver enzyme release (figure 1). Similar to rabbits, a single intravenous injection of tetranectin-apolipoprotein a-I comprising POPC or a mixture of POPC/DPPC according to the invention is safe in mice. The apolipoprotein composition comprising DPPC POPC in a 1: 3 molar ratio was comparable to POPC alone (fig. 9).

No significant hemolysis was observed until 2 hours post infusion in any of the five preparations. Hemolysis was measured by photoelectric colorimetry as the red color in plasma samples obtained 2 hours after intravenous administration of tetranectin-apolipoprotein a-I. 100% of whole blood hemolysis (produced by 0.44% Triton X-100-final concentration) was used for calibration (FIG. 10).

h) Anti-inflammatory effect of tetranectin-apolipoprotein A-I on human umbilical vein endothelial cells

HUVECs (human umbilical vein endothelial cells) from 5-10 passages were incubated for 16 hours in each tetranectin-apolipoprotein A-I preparation and stimulated with TNF α for the last 4 hours. VCAM1 surface expression was detected by FACS using specific antibodies.

Example 9

Lipid particle stability

Wild-type apolipoprotein a-I comprising an N-terminal histidine-tag and an IgA protease cleavage site was expressed in e.coli and purified by column chromatography as reported in the above examples. Histidine-tag was removed by IgA protease cleavage. Lipid particles (HDL particles) were assembled using a 1: 150 ratio of protein to Lipoid S100 soy phospholipid mixture. The particles were stored in a buffer containing 5mM sodium phosphate and 1% sucrose, pH 7.3. SE-HPLC revealed three separate peaks after incubation after lipidation and 10 days of incubation. After incubation at 40 ℃, a major peak (47% of total protein) at a retention time of 10.8 minutes could be detected, which was not present in samples stored at 5 ℃. The peak at 10.8 minutes shows the formation of soluble large molecular weight assemblies due to protein instability.

HDL particles containing tetranectin-apolipoprotein A-I as reported herein obtained starting from a POPC: DPPC mixture (POPC to DPPC ratio 3: 1) were also incubated at 5 ℃ and 40 ℃. Incubation at elevated temperature resulted in a slight degree of front formation, but no significant shift to the high molecular weight assemblies at 10.8 minutes (< 2% increase at 11 minutes). This demonstrates increased HDL particle stability compared to particles comprising wild-type apolipoprotein a-I.

Example 10

Cholesterol mobilization

The efficiency of cholesterol mobilization into the blood can be determined by comparing the respective excretion of total cholesterol to the apolipoprotein concentration after in vivo administration of apolipoprotein. For quantitative assessment, the quotient of the baseline corrected area under the concentration-time curve (AUC) of total cholesterol and the area under the concentration-time curve of apolipoprotein was calculated.

In this experiment, the following substances were analyzed:

-expressing the wild type apolipoprotein a-I comprising an N-terminal histidine-tag and an IgA protease cleavage site in escherichia coli and purifying by column chromatography as reported in the above examples; removal of the histidine-tag by IgA protease cleavage; assembling lipid particles (HDL particles) using a 1: 150 ratio of protein to Lipoid S100 soy phospholipid mixture;

apolipoprotein a-I Milano variant: lipid particles (HDL particles) were assembled using a 1: 40 ratio of protein to POPC,

-tetranectin-apolipoprotein a-I as reported herein; lipid particles (HDL particles) were assembled using a 1: 60 ratio of protein to (POPC and DPPC) (POPC and DPPC ratio 3: 1).

Three HDL particles were applied to rats. See the values obtained for each AUC ratio shown in table 30.

Table 30: cholesterol mobilization

Claims

1. A method for producing lipid particles, the method comprising the steps of:

i) providing a first solution comprising a denatured protein,

ii) adding said first solution to a second solution comprising at least one lipid and a detergent, but no said protein, and

2. The method of claim 1, wherein the second solution has a volume of about 3 times to about 20 times the volume of the first solution.

3. The method according to any one of the preceding claims, characterized in that the first solution is free of lipids.

4. The method according to any one of the preceding claims, characterized in that the protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 01, 02, 04-52, 66 or 67, or at least one polypeptide comprising the amino acid sequence of SEQ ID NO: 01, 02, 04-52, 66 or 67, or a fragment thereof comprising at least 80% of the amino acid sequence of seq id no.

5. The method according to claim 4, characterized in that the protein is a polypeptide having the sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 66 or SEQ ID NO: 67, or a tetranectin-apolipoprotein a-I.

6. Method according to any one of the preceding claims, characterized in that said at least one lipid is two different phosphatidylcholines.

7. The method of claim 6, wherein the first phosphatidylcholine is POPC and the second phosphatidylcholine is DPPC.

8. Method according to any one of the preceding claims, characterized in that the detergent is selected from cholic acid, amphoteric detergent or their salts.

9. The method according to any of the preceding claims, characterized in that the method comprises after step ii) and before step iii) the following steps:

iia) incubating the solution obtained in step ii).

10. Method according to any one of the preceding claims, characterized in that the incubation and/or removal is carried out at a temperature of 4-45 ℃.

11. The method according to any one of claims 9 and 10, characterized in that the incubation is carried out for about 2 hours to about 60 hours.

12. Method according to any one of the preceding claims, characterized in that the detergent is a detergent with a high CMC.

13. Method according to any one of the preceding claims, characterized in that the removal is carried out by diafiltration or dialysis or adsorption.

14. Lipid particles obtained by the method of any one of claims 1-13.

15. A pharmaceutical composition comprising the lipid particle of claim 14.