Preparation method of phospholipid affinity monolithic column and application of monolithic column

Abstract

The application provides a preparation method of a phospholipid affinity monolithic column, which is obtained by hydrophilic modification of a matrix, activation of the matrix by using maleimide or other reagents, and coupling of the activated matrix with a ligand with a spacer and a linker; and the application also provides application of the monolithic column. Compared with the prior art, the application has the following advantages: the application connects the matrix with a transmembrane polypeptide capable of binding to a cell membrane, and specifically adsorbs phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidic acid (PA) with a phospholipid bilayer surface, so that the purified target molecules are eluted out by EDTA or NaCl concentration, and the purpose of purification is achieved.

Description

Technical Field

[0001] This application relates to the field of biochemical purification technology, specifically to a method for preparing a phospholipid affinity monolithic column and the application of the monolithic column. Background Technology

[0002] Extracellular vesicles (EVs) can be classified into three groups based on their biogenesis: exosomes, microvesicles (MVs), and apoptotic exosomes. Exosomes are a special type of EV, surrounded by a phospholipid bilayer (approximately 50-100 nm in size) formed within endosome compartments. In contrast, MVs, also surrounded by a phospholipid bilayer and typically 100-1000 nm in size, are formed through plasma membrane budding. EVs refer only to exosomes and MVs smaller than 0.2 μm. Current research in this field mainly focuses on exosomes containing proteins and microRNAs (miRNAs). Because the proteins and miRNAs encapsulated in these exosomes are protected in the bloodstream, exosomes can provide crucial information about various diseases and medical conditions. In this regard, several exosomal proteins and miRNAs have been identified as potential diagnostic, prognostic, or therapeutic biomarkers. Furthermore, numerous studies have shown that exosomes have a wide range of biological functions, such as intercellular communication and signal transduction, and therefore can mediate therapeutic effects by transmitting genetic and non-genetic information between cells. For example, extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) can encapsulate the therapeutic and regenerative effects of MSCs on damaged tissues / organs in models of myocardial ischemia, acute renal tubular injury, stroke, acute lung injury / ischemia, and skin wounds. Therefore, extracellular vesicles hold immense potential for a wide range of clinical applications, from diagnosis to treatment.

[0003] Several techniques for separating EVs have been developed to date, including differential ultracentrifugation, size exclusion chromatography, immunoaffinity capture, and microfluidics-based techniques, as well as EV precipitation techniques. Among these, differential ultracentrifugation is considered the gold standard and most commonly used technique for EV isolation. However, EVs isolated using these techniques often contain external proteins and lipoproteins, resulting in low purity. Furthermore, this method is time-consuming and labor-intensive, making it unsuitable for high-throughput diagnostics. Collapse and damage to EV membranes or EV aggregates after separation by differential ultracentrifugation have also been reported. In particular, separating structurally and biologically intact EVs is essential for their therapeutic applications. Size exclusion chromatography has been noted to potentially produce highly purified EVs because it typically uses gravity flow, thus preserving vesicle structure and integrity to a large extent, and retaining the EV's biological activity. However, this method is limited by a lack of scalability, a feature required for therapeutic applications. Although a number of commercial isolation kits have been developed to improve yield, the purity of the EVs extracted by these kits is compromised. Significant efforts remain to develop better methods for EV separation and purification.

[0004] Compared to other separation methods, affinity chromatography (where the surface is modified with affinity ligands) offers several advantages. It is typically very mild for the target molecules, allowing them to elute readily from the column under mild conditions.

[0005] High-affinity antibody-antigen complexes are often difficult to dissociate, typically requiring extreme pH or denaturing conditions. Therefore, antibodies targeting EV surface markers (e.g., anti-CD9 and anti-CD63 antibodies) are not suitable affinity ligands for isolating intact EVs. Recently, the Tim4 phosphatidylserine binding 2+-dependent mechanism in Ca2+-bound EV membranes (which dissociates in the presence of EDTA) has been used as an affinity ligand for EV purification. Peptides with sufficient binding affinity to capture target molecules without excessive loss, while still allowing elution of bound molecules under mild conditions, have also been identified.

[0006] Due to their synthetic properties and small size, peptides exhibit excellent chemical stability, making peptide-based affinity matrices more robust than protein-based matrices and capable of withstanding extreme pH conditions during regeneration steps. Furthermore, peptides can be produced in batches at low cost under favorable manufacturing specifications; additionally, site-directed immobilization with high ligand density can be readily achieved using peptides. Cell-penetrating peptides are typically rich in arginine and lysine, enabling them to bind to cell membranes. Initial membrane binding is mediated by the interaction of arginine and lysine side chains with negatively charged groups on the cell surface. Molecular dynamics simulations indicate that both polylysine and polyarginine can bind to lipid bilayer surfaces. However, whether these peptides can serve as affinity matrices for EVs remains unclear.

[0007] Lentivirals (LVVs), as the most important vectors in cell and gene therapy, have unique advantages compared to other retroviruses: they have a wider host range, capable of infecting both dividing and non-dividing cells. For some difficult-to-transfect cells, such as primary cells, stem cells, and undifferentiated cells, they can greatly improve the transduction efficiency of target genes, significantly increasing the probability of the target gene integrating into the host cell genome. Stable expression is also achieved. Lentivirals can effectively integrate foreign genes into the cell chromosome, and the target gene has a certain resistance to transcriptional silencing, resulting in continuous, efficient, and stable expression in target cells. Constructed lentiviral vectors can carry target genes of approximately 5kb or even longer. Therefore, in addition to small molecules such as exogenous short-hairpin RNAs (shRNAs), many cDNAs can also be cloned into lentiviral vectors. Of course, as the length of the target gene increases, the viral titer decreases. Based on the advantages of lentiviruses, routine experimental operations for transfecting target genes or RNAi genes can be performed using lentiviral vector systems. Furthermore, lentiviruses play an important role in gene editing, gene therapy, transgenic animals, and drug research.

[0008] Currently, lentiviruses are purified using a combination of tangential flow filtration (TFF) and chromatography. However, the overall purification yield is only 10-20%, the purification flow rate is slow (0.5 CV / min), the virus is easily inactivated (the number of effective virus-like particles decreases by 50% after 8 hours at 37°C), the shear force is high, and the purification impurity content is high (approximately 15%-10% of contaminating proteins), resulting in less than 50% virus-like activity. Phospholipid affinity purification, using a high-flow-rate matrix, can achieve flow rates of 2-8 CV / min, resulting in faster purification speeds and a single-step yield of 70%. Combined with VSVG pseudotype removal, the purity can reach over 95%, and the overall yield can reach 30-45%.

[0009] Currently, in the fields of cell and gene therapy and medical aesthetics, there are various enveloped viruses with phospholipid bilayers (viral vectors LVV, influenza virus), exosomes, extracellular vesicles, virus-like particles, and functional phospholipids (LNPs). These present challenges such as difficulty in purification, unstable purification processes, long purification times, low purification purity, and low yield. Summary of the Invention

[0010] To address the aforementioned technical limitations, this application proposes a method for preparing a phospholipid affinity monolithic column and its application; this overcomes the deficiencies and defects mentioned in the background art.

[0011] To achieve the above objectives, this application adopts the following technical solution:

[0012] The inventive point of this application is to provide a method for preparing a phospholipid affinity monolithic column, comprising the following steps:

[0013] S1. Prepare the matrix, modify the matrix to be hydrophilic, and then activate it with maleamide preparations;

[0014] S2. After activation, it can be coupled with a ligand containing a spacer arm and a linker.

[0015] Optionally, in the above preparation method, the matrix includes one or more of natural macromolecular sugars, polymers, inorganic materials, and composite materials; the natural macromolecular sugars are preferably agarose, dextran, or cellulose; the polymers are preferably methacrylic acid polymers, styrene-divinylbenzene polymers, epoxy resin polymers, or polyethersulfone polymers; the inorganic materials are preferably silica or alumina; and the composite materials are preferably magnetic beads.

[0016] Matrix and matrix preparation: Matrix includes, but is not limited to, the matrix currently available on the market.

[0017] Matrix materials: natural macromolecular sugars: agarose, dextran, cellulose, etc.; synthetic polymers: methacrylic acid polymers, styrene-divinylbenzene polymers, epoxy resin polymers, polyethersulfone polymers, etc.; inorganic materials: silica, alumina, etc.; composite materials: magnetic beads, etc.

[0018] Matrix shapes: membranes, microspheres, rods, monolithic columns, etc.

[0019] Optionally, in the above preparation method, the hydrophilic modification is performed on the matrix surface. The hydrophilic modification materials include, but are not limited to, one or more of the following: dextran, agarose, cellulose, cellulose acetate, starch, hydrophilic peptides, hydrophilic oligopeptides, hydrophilic proteins, hydrophilic nucleic acid chains, polyvinyl alcohol, polyethylene glycol, polyacrylic acid, polyacrylamide, polyurethane, and polypeptide amine.

[0020] Matrix hydrophilicity modification: When using polymer materials as the matrix, hydrophilicity modification is required. This modification involves chemically bonding the polymer to the matrix surface or pore surface to achieve hydrophilicity and avoid specific adsorption by the coating (phospholipid bilayer). Examples of materials that can be modified are listed below:

[0021] 1. Natural macromolecules:

[0022] Polysaccharides:

[0023] Glucan: The average molecular weight of glucan is 1-200,000, with preferred molecular weights being 500, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 10000, 20000, 40000, 50000, 70000, 100000, 150000, and 200000.

[0024] Agarose, cellulose, cellulose acetate, starch, etc.;

[0025] Polypeptides and proteins: Hydrophilic monopeptides, oligopeptides, polypeptides, proteins, etc., are modified to be hydrophilic, with sequences consisting of basic side chains (e.g., lysine (K), arginine (R), histidine (H)); acidic side chains (e.g., aspartic acid (D), glutamic acid (E)); uncharged polar side chains (e.g., glycine; asparagine, glutamine, serine (S), threonine (T), tyrosine (Y), cysteine ​​(C); and nonpolar side chains (e.g., alanine (A)). Valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), menthol (M), tryptophan (W), β-branched side chains (e.g., threonine (T), amino acid (V), isoleucine (I)); and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)), and other synthetic amino acids such as methionine, ornithine, citrulline, 6-aminocaproic acid, and GABA.

[0026] Nucleic acids: oligonucleotides, nucleotide-like molecules, nucleotides, and other hydrophilic nucleotide chains.

[0027] 2. Polymers:

[0028] Polyvinyl alcohol (PVA): It appears as a white, flaky, flocculent, or powdery solid. The degree of polymerization of PVA is classified into ultra-high degree of polymerization (molecular weight 250,000–300,000), high degree of polymerization (molecular weight 170,000–220,000), medium degree of polymerization (molecular weight 120,000–150,000), and low degree of polymerization (25,000–35,000). The degree of alcoholysis is generally 78%, 88%, or 98%. Partial alcoholysis typically has a degree of alcoholysis of 87%–89%, while complete alcoholysis has a degree of alcoholysis of 98%–100%, such as PVA1399, PVA1788, and PVA1792.

[0029] Polyethylene glycol: molecular weight 200-2000, preferably small molecule polyethylene glycol with hydrophilic modification.

[0030] Polyacrylic acid: molecular weight 200-5000, preferably small molecule polyacrylic acid with hydrophilic modification.

[0031] Polyacrylamide, polyurethane, polyamide.

[0032] Optionally, in the above preparation method, the ligand is a group capable of specifically adsorbing phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidic acid (PA) on the surface of the target molecule, or a group that strongly binds to phosphatidylserine (PS) on the surface of the exosome membrane in the presence of calcium ions; preferably, it is a functional transmembrane polypeptide.

[0033] The ligands can specifically adsorb onto the surface of target molecules such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidic acid (PA), or strongly bind to phosphatidylserine (PS) on the exosome membrane surface in the presence of calcium ions. This invention refers to functional transmembrane peptides that rely on hydrogen bonds and ions on the peptide surface or chelation for specific adsorption. These include transmembrane peptides containing abundant natural or synthetic basic amino acids such as arginine, lysine, citrulline, and ornithine, mixed with other amino acids, and cadherin peptides, which are a class of type 1 transmembrane proteins. They play an important role in cell adhesion, thereby ensuring that cells within tissues are bound together, and are dependent on calcium ions (Ca). 2+ It plays a role.

[0034] Optionally, the functional transmembrane peptides prepared in the above-described method include, but are not limited to, one or more of the following: polylysine 3-30, polyarginine 3-30, polyornithine 3-30, 3-30 peptides linked by lysine and arginine, 3-30 peptides linked by lysine and arginine mixed with other natural amino acids, and cadherin peptides.

[0035] Examples of peptides: Lysine-rich peptides, containing 3-30 polylysines, preferably 4-16, linked by peptide bonds between carboxyl groups and α-amino groups, including but not limited to: trilysine (KKK), tetralysine (KKKK), pentalysine (KKKKK), hexalysine (KKKKKK), heptalysine (KKKKKK), heptalysine (KKKKKK), octalysine (KKKKKKK), nonalysine (KKKKKKK). K), decalycine (KKKKKKKKK), undecyllycine (KKKKKKKKKK), dodecalycine (KKKKKKKKKK), decalycine (KKKKKKKKKK), decalycine (KKKKKKKKKK), decalycine (KKKKKKKKKK), decalycine (KKKKKKKKKKK), decalycine (KKKKKKKKKKKK), decalycine (KKKKKKKKKKKK), hexalycine (KKKKKKKKKKKKK).

[0036] Rich in arginine polypeptides, containing 3-30 polyarginines, preferably 3-16, with arginine linkages formed by peptide bonds between carboxyl groups and α-amino groups, including but not limited to: triarginine (RRR), tetraarginine (RRRR), pentaarginine (RRRRR), hexaarginine (RRRRRR), heptaarginine (RRRRRR), octaarginine (RRRRRRRR), nonaarginine (RRRRRRRRRR), decaarginine (RRRRRRRRRR), undecaarginine (RRRRRRRRRR), dodecaarginine (RRRRRRRRRRRR), tridecaarginine (RRRRRRRRRRRR), tetradecaarginine (RRRRRRRRRRRRRR), decaarginine (RRRRRRRRRRRR), and hexadecylarginine (RRRRRRRRRRRRRRRR).

[0037] A peptide rich in ornithine, containing 3-30 polyornithines, preferably 3-16, linked by peptide bonds between the carboxyl group and the α-amino group. Examples include, but are not limited to: triornithine (Orn-Orn-Orn), tetraornithine (Orn-Orn-Orn-Orn), pentaornithine (Orn-Orn-Orn-Orn-Orn), hexaornithine (Orn-Orn-Orn-Orn-Orn-Orn), heptaornithine (Orn-Orn-Orn-Orn-Orn-Orn), octaornithine (Orn-Orn-Orn-Orn-Orn-Orn-Orn), nonaornithine (Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn), and decaornithine (Orn-Orn-Orn-Orn-Orn-Orn). Orn-Orn-Orn-Orn-Orn), Undecanoic acid (Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn), Dodecanoic acid (Orn- ... Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn), pentapolyornithine (Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Or n-Orn-Orn-Orn-Orn-Orn-Orn), ornithine (Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn);

[0038] This includes 3-30 peptides linked by peptide bonds between lysine and arginine via carboxyl groups and α-amino groups, or 3-30 peptides mixed with other natural amino acids (such as proline, glycine, serine, glutamine, alanine, valine, leucine, tryptophan, histidine, etc.) or artificially synthesized amino acids (such as ornithine, citrulline, GABA, etc.), including but not limited to:

[0039] SEQ ID No.1: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg,

[0040] SEQ ID No.2: Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln,

[0041] SEQ ID No.3: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg,

[0042] SEQ ID No.4: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Gly-Gly,

[0043] SEQ ID No.5: Tyr-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly,

[0044] SEQ ID No.6: Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg,

[0045] SEQ ID No.7: Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg,

[0046] SEQ ID No.8: Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Asp,

[0047] SEQ ID No. 9:

[0048] Gly-Leu-Trp-Arg-Ala-Leu-Trp-Arg-Leu-Leu-Arg-Ser-Leu-Trp-Arg-Leu-Leu-Tr p-Arg-Ala,

[0049] SEQ ID No. 10: Tyr-Gly-Arg-Orn-Orn-Arg-Arg-Gln-Arg-Arg-Arg.

[0050] cadherin peptide: SEQ ID No. 11: LRAHAVDVNG, etc.

[0051] Optionally, in the above preparation method, the spacer arm is a short chain used to connect the ligand and the affinity matrix.

[0052] Optionally, in the above preparation method, the connector is a substance with specific functional groups that is connected to a segment of the spacer arm and used for site-specific coupling of a hydrophilic matrix.

[0053] Spacer arms: Short-chain substances used to connect ligands and affinity matrices, increasing binding capacity, making it easier to capture target substances, and preventing target substances from flowing through. Spacer arms include, but are not limited to, 3-30 oligopeptide or polypeptide chains, 3-18 alkyl carbon chains, ether oxygen chains, 3-16 nucleotides, and dextran chains with a molecular weight of around 15 atoms, etc.

[0054] Linker: A linker is a substance with specific functional groups that is attached to a segment of the spacer arm for site-directed coupling of a hydrophilic matrix. These include, but are not limited to, hydroxyl (-OH), carboxyl (-COOH), amino (-NH2), aldehyde (-CHO), mercapto (-SH), halogen atom (-X), etc., or substances with some active functional groups such as succinimide, carbamate, maleimide, etc., which have special functional groups that can directly connect with the affinity matrix. To achieve site-directed coupling, the spacer arm is coupled to the ε-amino group on the ligand. Maleimide is preferably coupled to the mercapto group. Therefore, cysteine, cystine, mercaptoethanol, mercaptopropanol, mercaptoacetic acid, mercaptopropionic acid, mercaptobutyric acid, etc. are preferred as linkers.

[0055] Spacer arm Figure 2 As shown.

[0056] The second inventive point of this application is to provide a phospholipid affinity monolithic column prepared by the above preparation method.

[0057] The third inventive point of this application is to provide a method for purifying substances containing a phospholipid bilayer, wherein a phospholipid affinity monolithic column prepared by the above preparation method is used for column chromatography; the substances containing a phospholipid bilayer include, but are not limited to, one or more of enveloped viruses, exosomes, extracellular vesicles, virus-like particles, and functional phospholipids (LNPs).

[0058] Compared with the prior art, this application has the following advantages:

[0059] This invention patent connects a matrix to transmembrane polypeptides that can bind to the cell membrane, enabling specific adsorption of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidic acid (PA) on a phospholipid bilayer surface. The target molecules are then desorbed and extracted using EDTA or NaCl concentrations, achieving the purification purpose. Attached Figure Description

[0060] Figure 1 This is a schematic diagram of the overall column chromatography packing structure.

[0061] Figure 2 This is a diagram of the spacer arm structure used in one embodiment of this application.

[0062] Figure 3This is a structural diagram of the CGGGSGGGSGGGSKKKKKKKKK sequence polypeptide used in one embodiment of this application.

[0063] Figure 4 This is a schematic diagram showing the linker, spacer arm, and ligand position of the CGGGSGGGSGGGSKKKKKKKKK sequence polypeptide used in one embodiment of this application.

[0064] Figure 5 This is a schematic diagram of a lentivirus purification process in one embodiment of this application.

[0065] Figure 6 This is a flowchart of a lentivirus purification process in one embodiment of this application. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0068] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0069] Example 1

[0070] The specific process steps involved in the purification are illustrated using lentiviruses, but are not limited to lentiviruses. They can also be used for other enveloped viruses (such as influenza virus), exosomes, extracellular vesicles, virus-like particles, functional phospholipids (LNPs), etc.

[0071] Preparation of chromatography packing material: Prepare the required matrix, modify the matrix to be hydrophilic, activate it with maleamide or other reagents, and couple it with ligands with spacer arms and linkers.

[0072] Lentiviral sample preparation: Lentiviral packaging was performed using the three-plasmid transfection method. HEK-293T cells were harvested, clarified, deep filtered, and the medium was changed to prepare the chromatographic loading solution.

[0073] Lentiviral downstream purification: A dual-affinity process is used, namely, after phospholipid affinity chromatography, it enters VSVG pseudotype affinity chromatography, and after TFF medium exchange and aseptic filtration, it is aseptically filled.

[0074] Explanation of the purpose of the downstream purification process steps for lentiviruses:

[0075] Clarification and deep filtration: The main purpose is to remove cells and cell debris from the medium;

[0076] Phospholipid affinity chromatography steps:

[0077] Buffer change and equilibration: Replace with pre-loading buffer, generally using phosphoric acid, citric acid, carbonic acid, acetic acid, barbituric acid, Tris (tris(hydroxymethylaminomethane)), 3-morpholine propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES), or 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES). The buffer system should have a pH of 6-8 and a concentration of 0-300 mM. Emulsifiers such as 0-0.1% Tween 20 and 0-1% SDS can be added to better distribute the target substance in the buffer.

[0078] Washing buffer selection: Using the above-described buffer system, add salt ion concentrations for washing. The added salts include, but are not limited to, 0-500mM sodium chloride (NaCl), 0-500mM potassium chloride (KCl), 0-300mM magnesium chloride (MgCl2), 0-300mM calcium chloride (CaCl2), 0-500mM sodium sulfate (Na2SO4), 0-500mM potassium sulfate (K2SO4), 0-200mM magnesium sulfate (MgSO4), and 0-500... 0-600mM ammonium chloride (NH4Cl), 0-500mM ammonium sulfate ((NH4)2Cl), 0-600mM arginine, 0-600mM ornithine, 0-600mM citrulline, 0-600mM lysine, etc., pH=6-8.5, preferably 0-500mM sodium chloride (NaCl), 0-500mM potassium chloride (KCl), 0-300mM magnesium chloride (MgCl2), pH=6, 0-0.01% Tween 20 for linear or gradient washing of impurities.

[0079] Elution buffer selection: Using the above-mentioned buffer system, salt ion concentrations are added for elution. The added salts include, but are not limited to, 150-1000mM sodium chloride (NaCl), 150-1000mM potassium chloride (KCl), 100-600mM magnesium chloride (MgCl2), 150-1000mM sodium sulfate (Na2SO4), 150-1000mM potassium sulfate (K2SO4), 100-500mM magnesium sulfate (MgSO4), 200-1000mM ammonium chloride (NH4Cl), 200-1000mM ammonium sulfate ((NH4)2Cl), etc. Preferred options are 150-1000mM sodium chloride (NaCl), 150-1000mM potassium chloride (KCl), and 100-600mM magnesium chloride (MgCl2). The pH is 7-8, and 0-0.01% Tween 20 is used for linear or gradient washing of impurities.

[0080] CIP (Cleaning in Place): Cleaning is performed using 0.5M NaOH.

[0081] Chemical solvent resistance: 0.5M NaOH, 10mM HCl, 0.1M citric acid (pH3), 6M urea, 6M guanidine hydrochloride, 30% isopropanol, 20% ethanol.

[0082] Preferred:

[0083] Binding buffer, 20 mM Bis-Tris, pH 6.0, 0.055% Tween 20.

[0084] Wash buffer: 20 mM Bis-Tris, pH 6.0; 150 mM NaCl; 0.005% Tween 20; Elution buffer: 20 mM Tris-HCl, pH 7.5; 500 mM NaCl

[0085] Cleaning buffer: 0.5M NaOH

[0086] Purpose of phospholipid affinity chromatography:

[0087] It adsorbs substances containing phospholipids, such as intact LVV viruses and their envelopes, and removes substances without phospholipids, such as VSVG proteins, host proteins, and endotoxins.

[0088] Combined with lentivirus capabilities, 10^9 to 10^14 Vp / ml.

[0089] VSVG pseudotype affinity chromatography.

[0090] Example 2

[0091] This case study includes monolithic column fabrication techniques, using a 5ml monolithic column as an example:

[0092] 1) Mix glycidyl methacrylate: ethylene glycol dimethacrylate: cyclohexanol: dodecanol: sodium persulfate = 20:20:50:9:1 (weight ratio), dissolve by ultrasonic vibration, take 6ml, pour into the designated mold, place in an 80℃ water bath, react for 24h, mold, and demold.

[0093] 2) Using a special tool, the above monolithic column is trimmed into a 5ml column with a bottom diameter of 20mm and a height of 16mm. It is then placed in a specific component and the pore-forming agent is washed with alcohol.

[0094] 3) Test pressure, flow rate, and integrity.

[0095] Example 3

[0096] This case study illustrates the use of dextran hydrophilization modification on monolithic columns, using a 5ml monolithic column as an example:

[0097] The column was placed in 10 ml of 5% dextran with a molecular weight of 1000, and 5 ml of 40% NaOH and 1% sodium borohydride were added. The reaction was carried out at 60°C for 10 h.

[0098] The column was washed with water until pH=7, and the inoculation amount was measured to be 7.5 μmol / ml.

[0099] Example 4

[0100] This case study involves monolithic column coupling with ligands, using a 5ml monolithic column as an example:

[0101] 1) Take 0.1g SMCC (4-(N-maleimide methyl)cyclohexane-1-carboxylic acid succinimide ester), dissolve it in DMSO, and circulate it in the column of Example 3 at 1ml / min for 2 hours, then empty the column.

[0102] 2) Dissolve SEQ ID No. 12: CGGGSGGGSGGGSKKKKKKKKK in 1 mol sodium sulfate, 20 mM sodium hydrogen phosphate buffer solution, adjust the pH to 7, and synthesize the polypeptide sequence shown in SEQ ID No. 12: CGGGSGGGSGGGSKKKKKKKKK using a polypeptide synthesizer. Figure 3 (As shown) Continue the cycle at 1 ml / min for 2 hours, then empty the syringe.

[0103] 3) The column was cleaned with an aqueous solution to obtain column #1, and the ligand concentration was measured to be 296 nmol / ml.

[0104] The sequence of the polypeptide linker, spacer arm, and ligand positions is shown in the diagram below. Figure 4 As shown.

[0105] Example 5

[0106] This case study involves monolithic column coupling with ligands, using a 5ml monolithic column as an example:

[0107] 1) Dissolve 0.1 g of SMCC (4-(N-maleimide methyl)cyclohexane-1-carboxylic acid succinimide ester) in DMSO. Circulate the solution to the column from Example 3 at a rate of 1 ml / min for 2 hours, then empty the column.

[0108] 2) Dissolve the VSVG protein antibody in 1 mol sodium sulfate and 20 mM sodium hydrogen phosphate buffer solution, adjust the pH to 7 (using antibody library screening, VSVG protein antibody, and modified with cysteine), continue to cycle at 1 ml / min for 2 hours, and then cycle empty.

[0109] 3) The column was cleaned with an aqueous solution to obtain column #2, and the ligand concentration was measured to be 78 nmol / ml.

[0110] Example 6

[0111] This case study includes sample preparation for LVV lentivirus loading:

[0112] 1) HEK293 cells were cultured in suspension and transfected using the three-plasmid transfection method. Cells were harvested and clarified. The viral supernatant was pretreated by a low-retention membrane filter with reduced pore size (from 0.8 μm to 0.45 μm) to obtain 980 ml of sample solution.

[0113] 2) After qPCR testing, the viral-like concentration was 7.2*10^7 TP / ml.

[0114] Example 7

[0115] This case study includes LVV lentivirus, phospholipid affinity, and affinity procedures such as equilibration, loading, washing, elution, and CIP.

[0116] Column Case 4: Column 1#

[0117] Loading flow rate: 4 CV / min (20 ml / min);

[0118] Equilibration buffer: 20 mM Bis-Tris, pH 6.0, 0.005% Tween 20;

[0119] Equilibrium volume: 20 CV (100 ml);

[0120] Sample solution: The above sample solution

[0121] Sample volume: 750ml

[0122] Sample loading rate: 4 CV / min (20 ml / min)

[0123] Washing buffer: 20 mM Bis-Tris, pH 6.0, 150 mM NaCl; 0.005% Tween 20.

[0124] Washing volume: 10 CV (50 ml)

[0125] Washing flow rate: 4 CV / min (20 ml / min)

[0126] Elution buffer: 20 mM Tris-HCl, pH 7.5; 500 mM NaCl

[0127] Elution volume: 10 CV (50 ml)

[0128] Elution flow rate: 2 CV / min (10 ml / min)

[0129] CIP: 0.5M NaOH

[0130] CIP flow rate: 2 CV / min (10 ml / min)

[0131] CIP volume: 20CV (100ml).

[0132] To better demonstrate the effects before and after purification, the p24 ELISA method (which measures the amount of p24 capsid protein in the viral supernatant and then correlates the p24 content level with the viral titer) was used to detect the viral titer, and qPCR was used to test the protein titer and the number of virus-like particles. The specific data are shown in Table 1.

[0133] Table 1

[0134] Harvest concentration 7.2*10^7 TP / ml 1.2*10^6 ng / ml 1.0*10^5ng / ml Harvest volume 980ml 980ml 980ml Sample volume 750ml 750ml 750ml Elution volume 15ml 15ml 15ml Elution concentration 3.13*10^9 TP / ml 1.6*10^3 ng / ml 1.7*10^3 ng / ml yield 87％

[0135] As can be seen from the table above, the concentrations of HCP and HCD decreased to 1.3% and 1.7% of their original levels, respectively.

[0136] Example 8

[0137] This case study includes LVV lentivirus, VSVG pseudotype affinity, and affinity procedures such as equilibration, loading, washing, elution, and CIP.

[0138] Column Case 4: Column 2#

[0139] Sample loading rate: 4 CV / min (20 ml / min)

[0140] Equilibration buffer: 50mM HEPES, 150mM NaCl, pH 7.5

[0141] Equilibrium volume: 20 CV (100 ml)

[0142] Sample solution: Example 6 eluent was diluted 1:3 with 50mM HEPES, pH 7.5, and adjusted to a volume of 200ml.

[0143] Sample volume: 200ml

[0144] Sample loading rate: 4 CV / min (20 ml / min)

[0145] Washing buffer: 50mM HEPES, 150mM NaCl, pH 7.5

[0146] Washing volume: 10 CV (50 ml)

[0147] Washing flow rate: 4 CV / min (20 ml / min)

[0148] Elution buffer: 50 mM HEPES, 150 mM NaCl, 800 mM arginine, pH 7.5; Elution volume: 10 CV (50 ml)

[0149] Elution flow rate: 2 CV / min (10 ml / min)

[0150] CIP: 50mM sodium phosphate buffer, pH 12

[0151] CIP flow rate: 2 CV / min (10 ml / min)

[0152] CIP volume: 20 CV (100 ml)

[0153] To better demonstrate the effects before and after purification, P24 and ELISA methods were used to detect viral titers, and qPCR was used to test protein titers and virus-like particle counts. Specific data are shown in Table 2.

[0154] Table 2

[0155]

[0156]

[0157] The HCP content decreased to 5.6 ng / ml, and the HCD content decreased to 39 ng / ml.

[0158] Example 9

[0159] This case study includes an LVV lentivirus, and the overall process yield is shown in Table 3:

[0160] Table 3

[0161] Single-step yield 95％ 87％ 79％ 55％ 35.91％

[0162] The final purity can reach 90.6%.

[0163] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing a phospholipid affinity monolithic column, characterized in that, Includes the following steps: S1. Prepare the matrix, modify the matrix to be hydrophilic, and then activate it with maleamide preparations; S2. After activation, it can be coupled with a ligand containing a spacer arm and a linker; The matrix is ​​selected from one or more of natural macromolecular sugars, high molecular polymers, inorganic materials, and composite materials; The natural macromolecular sugars mentioned are agarose, dextran, or cellulose; When the matrix contains natural macromolecular sugars and the natural macromolecular sugar is selected as agarose, the hydrophilic modification material is selected from one or more of the following: dextran, cellulose, cellulose acetate, starch, hydrophilic peptides, hydrophilic oligopeptides, hydrophilic proteins, hydrophilic nucleic acid chains, polyvinyl alcohol, polyethylene glycol, polyacrylic acid, polyacrylamide, polyurethane, and polypeptide. When the matrix contains natural macromolecular sugars and the natural macromolecular sugar is selected as dextran, the hydrophilic modification material is selected from one or more of the following: agarose, cellulose, cellulose acetate, starch, hydrophilic peptides, hydrophilic oligopeptides, hydrophilic proteins, hydrophilic nucleic acid chains, polyvinyl alcohol, polyethylene glycol, polyacrylic acid, polyacrylamide, polyurethane, and polypeptide amine. When the matrix contains natural macromolecular sugars and the natural macromolecular sugar is selected as cellulose, the hydrophilic modification material is selected from one or more of the following: agarose, dextran, cellulose acetate, starch, hydrophilic peptides, hydrophilic oligopeptides, hydrophilic proteins, hydrophilic nucleic acid chains, polyvinyl alcohol, polyethylene glycol, polyacrylic acid, polyacrylamide, polyurethane, and polypeptide. When the matrix is ​​selected as one or more of polymers, inorganic materials, and composite materials, the hydrophilic modification material is selected as one or more of agarose, dextran, cellulose, cellulose acetate, starch, hydrophilic peptides, hydrophilic oligopeptides, hydrophilic proteins, hydrophilic nucleic acid chains, polyvinyl alcohol, polyethylene glycol, polyacrylic acid, polyacrylamide, polyurethane, and polypeptide. The polymer is a methacrylic acid polymer, a styrene-divinylbenzene polymer, an epoxy resin polymer, or a polyethersulfone polymer; The inorganic material is silicon dioxide or aluminum oxide; The composite material is a magnetic bead; The hydrophilic modification is achieved by chemically bonding to the matrix surface or pore surface; The maleamide formulation was selected as SMCC; The ligand is selected as a functional transmembrane polypeptide; The functional transmembrane peptide is selected from one or more of the following: polylysine 3-30, polyarginine 3-30, polyornithine 3-30, 3-30 peptides linked by lysine and arginine, 3-30 peptides linked by lysine and arginine mixed with other natural amino acids, and cadherin peptides. The spacer arm is a short chain used to connect the ligand and the affinity matrix, selected as a 3-30 oligopeptide or polypeptide chain, a 3-18 atom alkyl carbon chain, an ether oxygen chain, a 3-16 nucleotide amount, or a dextran chain with a molecular weight within 500. The connector is a substance with a specific functional group that is connected to a segment of the spacer arm and used for site-specific coupling of a hydrophilic matrix, and is selected from one or more of cysteine, cystine, mercaptoethanol, mercaptopropanol, mercaptoacetic acid, mercaptopropionic acid and mercaptobutyric acid.

2. The phospholipid affinity monolithic column prepared by the preparation method according to claim 1.

3. A method for purifying a substance containing a phospholipid bilayer, characterized in that, Column chromatography was performed using the phospholipid affinity monolithic column as described in claim 2; the substance containing the phospholipid bilayer was selected from one or more of enveloped viruses, exosomes, extracellular vesicles, virus-like particles, and functional phospholipids (LNPs).

Images