Method for purifying lipid membrane-encapsulated composite aggregates

Abstract

A chromatographic method for separating lipid membrane-encapsulated compound aggregates (LMCA) from chromatin heteropolymers (containing degradation residues of LMCA in a sample) comprises: contacting the sample with a chromatographic material having cationic metal affinity ligands on its surface, the chromatographic material carrying trivalent iron, manganese, calcium, or magnesium ions, and equilibrating it with an equilibration buffer; applying a gradient of a first elution buffer containing nonmetallic chelating salts to elute the LMCA.

Description

[0001] This invention relates to a chromatographic method for separating lipid-membrane-enclosed compound assemblages (LMCAs) from chromatin heteroaggregates, wherein the chromatin heteroaggregates contain degradation residues of LMCAs in the sample. Technical Background

[0002] In biology, the term "complex assemblage" refers to a structurally organized entity containing more than one type of biomolecule, and often more than one class of biomolecules, such as proteins, carbohydrates, and nucleic acids. Some complex assemblages are surrounded by lipid membranes, such as cellular vesicles, extracellular vesicles, and lipid-enveloped viruses. Most range in size from about 30 nm to about 300 nm, with some reaching 10 times or more.

[0003] Recent experiments have shown that unpurified formulations of lipid-membrane-enclosed compound assemblages (LMCAs) are accompanied by a specific class of contaminants containing their degradation residues [1,2]. These are detected by immunostaining with specific protein markers for particular LMCAs. Size exclusion chromatography has shown that most of these residues range in size from about 2 nm to about 50 nm.

[0004] Chromatin represents a known class of contaminants in cell culture harvests. It consists of an array of degraded nucleosomes containing host cell DNA and histones [3]. It typically binds to RNA and other proteins, resulting in heteroaggregates, where “heteroaggregates” is understood as unorganized complexes of more than one class of biochemical substances. Chromatin heteroaggregates are known to be present in cell culture harvests in the range of 2 nm to above 5 micrometers. Filtering with a 0.45-micrometer membrane can reduce the upper limit of this range to approximately 400 nm.

[0005] Recent experimental data suggest that a specific subset of chromatin hybrids contains degradation residues of LMCA[4]. This extended category of chromatin hybrids is defined by characteristic LMCA membrane protein markers of the aggregates of a given class of compounds they contain, along with excess surface-accessible nucleic acids in their chromatin components. The surface accessibility of excess nucleic acids is a key defining characteristic of these hybrids. Many types of LMCA contain nucleic acids, but as long as the LMCA remains intact, the nucleic acids are encapsulated within a lipid membrane. Chromatin hybrids containing LMCA residues range in size from less than 100 nm to greater than 1000 nm[4].

[0006] Chromatin hybrids containing LMCA residues pose particular problems for the production of therapeutic LMCA. These hybrids can lead to an overestimation of the apparent content of intact LMCA in immunological assays. This also presents a problem for affinity chromatography, which identifies LMCA via LMCA membrane proteins, as chromatin hybrids containing these proteins are also captured. The size overlap between LMCA and chromatin hybrids containing LMCA residues also makes them unseparable by size-based purification methods.

[0007] Anion exchange chromatography has the potential to separate LMCA from chromatin hybrids containing LMCA residues, but its application is limited to hybrids with a high degree of dominance of nucleic acid residues (1-3) on the outside. Such hybrids are generally not eluted in sodium chloride, but intact LMCA is eluted. Chromatin hybrids containing LMCA residues with a small amount of nucleic acid on the surface are co-eluted with intact LMCA [4]( Figure 1 This is an important issue for LCMA therapeutic applications, as regulatory agencies worldwide have consistently demanded that contaminated DNA be reduced to extremely low levels.

[0008] Sometimes LMCA presents other purification challenges, namely separating LMCA containing excess phosphatidylserine in the outer leaflet from LMCA lacking phosphatidylserine in the outer leaflet. Excess phosphatidylserine in the outer leaflet is an indicator of inflammation, infection, lesion, dying, or death of a certain LMCA-derived cell[5]. Currently, there is no known chromatographic method to separate LCMA lacking phosphatidylserine on the outer membrane surface from LCMA with phosphatidylserine on the outer membrane surface. Invention Overview

[0010] This invention relates to a chromatographic method for separating lipid membrane-encapsulated compound aggregates (LMCA) from chromatin heteropolymers (containing degradation residues of LMCA in a sample), the method comprising the following steps:

[0011] - The sample is contacted with a chromatographic material having cationic metal affinity ligands on its surface, the material carrying trivalent iron, manganese, calcium, or magnesium ions, and equilibrated with a buffer solution.

[0012] - Apply a gradient of first elution buffer containing non-metallic chelating salts, thereby

[0013] -Eliminate LMCA.

[0014] In embodiments of the method of the present invention, LMCA may be selected from: cellular vesicles, extracellular vesicles, noncellular vesicles, organelles, exosomes, small extracellular vesicles, microvesicles, medium extracellular vesicles, ribosomes, mitochondria, lipid-coated viruses or lipid-coated virus-like particles, influenza viruses, coronaviruses, lentiviruses, cytomegaloviruses, liposomes, and combinations thereof.

[0015] In another embodiment of the method of the present invention, the nonmetallic chelate salt is a monovalent halide salt, particularly sodium chloride.

[0016] In another embodiment of the method of the present invention, the sample may be a cell culture harvest, a filtered cell culture harvest, a concentrated filtered cell culture harvest, or a partially purified formulation containing LMCA and chromatin hybrids, wherein the chromatin hybrids contain LMCA residues.

[0017] In another embodiment of the method of the present invention, chromatin heteropolymers containing LMCA degradation residues are gradient eluted in a second elution buffer containing chelating salts.

[0018] In another embodiment of the method of the present invention, the chelating salt of the second elution buffer is selected from: metal phosphates, or metal citrates, or metal salts of ethylenediaminetetraacetic acid (EDTA), or metal salts of ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, also known as etanic acid (EGTA), and combinations thereof.

[0019] In another embodiment of the method of the present invention, the method is combined with at least one other method for reducing chromatin heteropolymer content.

[0020] In another embodiment of the method of the present invention, the at least one other method may be treatment with nucleases and / or solid-phase treatment with anionic chromatographic ligands.

[0021] In another embodiment of the method of the present invention, the solid phase containing the anionic chromatographic ligand contains iron or manganese ions.

[0022] In another embodiment of the method of the present invention, the sample is contacted with the chromatographic material and separated from the chromatographic material after an incubation period.

[0023] In another embodiment of the method of the present invention, the sample, particularly the cell culture harvest containing exosomes, may be percolated by tangential flow filtration.

[0024] In another embodiment of the method of the present invention, the cationic metal affinity ligand may be tris(2-aminoethyl)amine (TREN).

[0025] In another embodiment of the method of the present invention, the pH range of the equilibration buffer is between pH 5 and pH 9, or pH 6 and pH 8, or preferably between pH 6.5 and pH 7.5, and its conductivity value is between 1 mS / cm and 250 mS / cm, or 2 mS / cm and 100 mS / cm, or 3 mS / cm and 50 mS / cm, or 4 mS / cm and 20 mS / cm, or preferably 5 mS / cm and 10 mS / cm.

[0026] This invention relates to a chromatographic method for isolating intact lipid membrane-encapsulated compound aggregates (LMCAs) from chromatin heteropolymers containing degradation residues of LMCAs. Surprisingly, cationic metal affinity ligands, preferably carrying ferric or manganese ions, can be used to elute intact LMCAs with a gradient of nonmetallic chelating salts (e.g., sodium chloride). Chromatin heteropolymers containing LMCA degradation residues are more robustly retained. Some can be eluted with a gradient of chelating salts (e.g., sodium phosphate). The remainder can be eluted with sodium hydroxide. Extended modifications of this invention combine it with one or more other methods for reducing chromatin heteropolymer content, such as treatment with nucleases and / or solid-phase treatment with anionic chromatographic ligands preferably carrying ferric or manganese ions.

[0027] The elements of the present invention are illustrated in the following non-limiting embodiments:

[0028] In one embodiment illustrating the core method, a cationic metal affinity column is loaded with ferric ions and then equilibrated to approximate physiological conditions. Filtered cell culture harvest containing exosomes (LMCAs) is applied to the equilibrated cationic metal affinity column, which is then washed with equilibration buffer. Intact exosomes are eluted with a sodium chloride gradient. In an alternative extension of the method, chromatin heteropolymers containing LMCA residues can be eluted with a sodium phosphate gradient. The column is cleaned with NaOH.

[0029] In an embodiment illustrating a further extended variation of the method, ferric ions are loaded onto an anionic metal affinity solid phase and then equilibrated to approximate physiological conditions. Cell culture harvest containing exosomes is contacted with the solid phase and incubated for 1 hour, then separated from the solid phase. The major subset of chromatin, including chromatin heteropolymers associated with LMCA degradation residues, remains bound to the ferric anionic metal affinity solid and is thus removed. The treated harvest is then processed using the core method of the invention.

[0030] In an embodiment illustrating a further extended variation of the method, the cell culture harvest containing exosomes is percolated by tangential flow filtration to place it under conditions optimal for nucleases. The membrane pore size is the maximum pore size that can retain the desired LMCA so that smaller contaminants can be removed through the pores. The percolated harvest is then treated with nucleases. Tangential flow filtration is restarted to remove nucleotides released by nuclease treatment and histones released by DNA cleavage. If necessary, the exosome-containing percolate can be concentrated. The treated harvest is then processed using the core method of the invention.

[0031] In an embodiment illustrating the compound extension modification of the method, ferric ions are loaded onto an anionic metal affinity solid phase and then equilibrated to approximate physiological conditions. Cell culture harvest containing exosomes is contacted with the solid phase, incubated for 1 hour, and then separated from the solid phase. The major subset of chromatin, including chromatin heteropolymers associated with LMCA degradation residues, remains bound to the ferric anionic metal affinity solid and is thus removed. Their removal contributes to the filterability of the treated harvest. The treated harvest is concentrated and percolated through tangential flow filtration to conditions optimal for nucleases. The membrane pore size is the maximum pore size that can retain the desired LMCA so that smaller contaminants can be removed through the pores. The percolated harvest is then treated with nucleases. Tangential flow filtration is restarted to remove nucleotides released by nuclease treatment and histones released by DNA cleavage. If desired, the exosome-containing percolate can be concentrated. The treated harvest is then treated using the core method of the invention.

[0032] Brief description of the attached figures

[0033] Figure 1 The elution of exosomes and exosome fragment-chromatin mixtures from anion exchange chromatography column was depicted.

[0034] Figure 2 The elution of exosomes and exosome fragment-chromatin contaminants from a cationic metal affinity column carrying ferric ions was described, wherein exosomes were eluted in a gradient in sodium chloride, while exosome fragment-chromatin contaminants were eluted in a gradient in phosphate or subsequently in sodium hydroxide.

[0035] Detailed description of the invention

[0036] In one embodiment, the LMCA to be processed by the method of the present invention comprises cellular vesicles, or extracellular vesicles, or noncellular vesicles or organelles. In one such embodiment, the LMCA to be processed by the method of the present invention comprises exosomes, or small extracellular vesicles, or microvesicles, or medium extracellular vesicles, or ribosomes, or mitochondria. In another embodiment, the LMCA to be processed by the method of the present invention comprises lipid-coated viruses or lipid-coated virus-like particles. In one such embodiment, the LMCA to be processed by the method of the present invention comprises influenza virus, or coronavirus, or lentivirus, or cytomegalovirus, or other lipid-coated viruses.

[0037] In one embodiment, the LMCA-containing sample to be processed by the method of the present invention is a filtered cell culture harvest, or a concentrated filtered cell culture harvest, or a partially purified formulation containing the desired LMCA and chromatin hybrids, the chromatin hybrids containing LMCA residues. In one embodiment, the sample is a cell culture harvest treated to reduce nucleic acid content. In embodiments where the sample is treated to reduce nucleic acid content, the method for reducing nucleic acid content may be treatment with a nuclease. In embodiments where the sample is treated to reduce nucleic acid content, the method for reducing nucleic acid content may be treatment with an anionic metal affinity solid phase carrying a metal ion. In one such embodiment, the anionic metal affinity ligand is iminodiacetic acid (IDA). In another such embodiment, the anionic metal affinity ligand is nitric acid triacetic acid (NTA). In one such embodiment, the metal ion used to load the anionic metal affinity ligand is preferably ferric or manganese ions. In one such embodiment, the solid phase covalently linked to the anionic metal affinity ligand is a particle, or fiber, or membrane, or monolith, or other configuration, or a mixture thereof, any of which can be packed into a chromatographic column, including a chromatographic apparatus.

[0038] In one embodiment, the cationic metal affinity ligand covalently linked to the solid phase, column, or column packing is tris(2-aminoethyl)amine (TREN).

[0039] In one embodiment, the cationic metal affinity ligand carries a divalent or trivalent metal ion, such as calcium, magnesium, manganese, or preferably trivalent iron.

[0040] In one embodiment, a cationic metal affinity solid phase carrying metal ions is equilibrated to a pH range of pH 5 to pH 9, or pH 6 to pH 8, or preferably pH 6.5 to pH 7.5, and a conductivity value of 1 mS / cm to 250 mS / cm, or 2 mS / cm to 100 mS / cm, or 3 mS / cm to 50 mS / cm, or 4 mS / cm to 20 mS / cm, or preferably 5 mS / cm to 10 mS / cm. It will be apparent to those skilled in the art that the preference for near-physiological conditions is, in part, to maintain the desired stability of LMCA. The chemoselectivity of the method of the present invention, independent of LMCA stability issues, is maintained over a wider range of pH values ​​and conductivity.

[0041] In one embodiment, an elution buffer containing a nonmetallic chelating salt is passed through a column to elute LMCA, wherein the salt is a monovalent metal halide, such as sodium chloride, or potassium chloride, or other monovalent metal halides, or sodium acetate or other monovalent metal acetates, or other non-chelating salts, preferably sodium chloride. In one such embodiment, the concentration of sodium chloride is in the range of 0.5M to 2.0M, or 0.75M to 1.5M, or 1.0M to 1.25M, or 0.8M to 1.2M, or 0.9M to 1.1M. In another such embodiment, the concentration of sodium chloride is continuously or gradually increased to a final concentration range of 0.5M to 2.0M, or 0.75M to 1.5M, or 1.0M to 1.25M, or 0.8M to 1.2M, or 0.9M to 1.1M. In any of these embodiments, the working pH can be the same as that of the equilibration buffer, preferably in the range of 6.5 to 7.5.

[0042] In one embodiment, chromatin hybrids containing LMCA residues are eluted in a separate step following LMCA elution. In one such embodiment, chromatin hybrids containing LMCA residues are eluted with a salt capable of chelating metals. In one such embodiment, chromatin hybrids containing LMCA residues are eluted with a gradient of 500 mM sodium phosphate, or 1 M potassium phosphate, or other monovalent metal phosphates, or monovalent metal citrates, or monovalent metal salts of ethylenediaminetetraacetic acid (EDTA), or monovalent metal salts of ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, also known as etaneric acid (EGTA), or combinations thereof. In some such embodiments, the chelating salt may be mixed with non-chelating salts, such as monovalent metal halide salts or metal acetates.

[0043] In one embodiment, the metal-containing cationic metal affinity column of the present invention can perform one-step or gradient elution of phosphate, citrate, EDTA, EGTA or combinations thereof in a single operation without prior elution with monovalent metal halides, monovalent metal acetates, or other non-chelating salts.

[0044] In one embodiment, LMCA elution can be performed in a single step, using a sufficiently high concentration of non-chelating salt to elute most of the intact LMCA and concentrate it in a single fraction. In another embodiment, LMCA elution can be performed in a series of steps, wherein a final step contains a sufficiently high concentration of non-chelating salt to elute most of the intact LMCA, while intermediate steps contain progressively increasing salt concentrations aimed at separating the desired intact LMCA from contaminants. In yet another embodiment, LMCA elution can be performed using a so-called linear gradient method, where the concentration of non-chelating salt is progressively increased to a final concentration sufficient to elute most of the intact LMCA.

[0045] In one embodiment, elution of chromatin hybrids can be performed by increasing the concentration of the chelating salt in one step, a series of steps, or by linearly increasing the concentration to a final concentration sufficient to elute the target substance.

[0046] In one embodiment, the method of the present invention is used for the fractionation of extracellular vesicles. In one such embodiment, the method of the present invention is used for the purification of exosomes. In one such embodiment, intact exosomes are eluted from an iron-loaded cationic metal affinity column, such as a TREN column, in the presence of chloride ions. In one such embodiment, chromatin hybrids containing exosome residues and / or other extracellular vesicle material are eluted in the presence of phosphate ions. In another embodiment involving the fractionation of extracellular vesicles, the method of the present invention is used for the fractionation of extracellular vesicles (containing exosomes) deficient in phosphatidylserine in the extracellular leaflets and extracellular vesicles (containing exosomes) containing excess phosphatidylserine in the extracellular leaflets. In one such embodiment, extracellular vesicles deficient in phosphatidylserine in the extracellular leaflets are eluted in sodium chloride, while extracellular vesicles containing excess phosphatidylserine in the extracellular leaflets are eluted in sodium phosphate. In one such embodiment, the method of the present invention is used to selectively concentrate phosphatidylserine-positive exosomes to improve the sensitivity and accuracy of diagnostic tests for the presence of phosphatidylserine-positive exosomes.

[0047] In one embodiment, the method of the present invention is used to fractionate and separate desired lipid-coated viruses from chromatin hybrids, the chromatin hybrids comprising degradation residues of lipid-coated viruses and degradation residues of extracellular vesicles.

[0048] In one embodiment, the LMCA treated by the method of the present invention can be further processed to reduce the content of any residual contaminant species. Subsequent methods for this purpose may include filtration, precipitation, and chromatography. In one embodiment, where the subsequent purification method is chromatography, the chromatography may be size exclusion chromatography, anion exchange chromatography, or other chromatographic methods.

[0049] Those skilled in the art will understand that the ferric-loaded cationic metal affinity column of the present invention exhibits a high affinity for endotoxins due to the positive charge on the column and the high affinity of ferric ions for phosphate residues. Therefore, it can be expected that LMCA eluted in non-chelating salts will be highly deficient in endotoxins relative to the endotoxin levels in the original sample. Example

[0050] Example 1

[0051] Extracellular vesicles are separated by the method of the present invention.

[0052] A ferric ion-loaded cationic metal affinity synthesizer (TREN) was used, followed by equilibration with 15 column volumes (CV) of 50 mM Hepes, 20 mM NaCl, pH 7.0. Filtered mammalian cell culture harvest containing a mixture of exosomes (small extracellular vesicles (LMCA)), chromatin, and heteropolymers (containing chromatin and degraded vesicular fractions) was loaded onto the column. Unbound sample fractions were washed off the column with equilibration buffer, followed by a one-step elution to 1.0 M sodium chloride (50 mM Hepes, pH 7.0) for 5 CV. The column was reequilibrated with the original equilibration buffer, followed by a second elution step to 500 mM sodium phosphate, pH 7.0, and continued with 5 CV of 500 mM phosphate. The column was restored to its original equilibration conditions and then cleaned with 30 CV of 1 M NaOH and 2 M NaCl. The chromatogram is shown below. Figure 2 As shown, a group of extracellular vesicles eluted in NaCl. Although the conductivity of phosphate elution was lower than that of chloride elution, a second peak eluted in phosphate. This highlights that phosphate elution selectively elutes different groups of extracellular vesicles that sodium chloride cannot remove. This elution pattern differs from anion exchange chromatography (such as anion exchange chromatography of quaternary ammonium salts). Figure 1 (As shown).

[0053] Example 2

[0054] Isolation of the desired exosome population (LCMA) from chromatin hybrids (containing degradation residues of LMCA).

[0055] A ferric ion-loaded cationic metal affinity synthesizer (TREN) was used, followed by equilibration with 15 CV of 50 mM Tris, 20 mM NaCl, pH 7.5. Filtered mammalian cell culture harvest containing a mixture of exosomes, chromatin, and heteropolymers (including chromatin and degraded vesicular fractions) was loaded onto the column. Unbound sample fractions were washed from the column with equilibration buffer, followed by a 20 CV linear gradient elution to 50 mM Tris, 1.0 M NaCl, pH 7.0, and continued elution for 5 CV under these conditions. The column was then washed with 1 M NaOH and 2 M NaCl.

[0056] Example 3

[0057] Isolation of phosphatidylserine-positive exosome populations from phosphatidylserine-negative exosomes (LCMA)

[0058] A ferric ion-loaded cationic metal affinity precursor (TREN) was used, followed by equilibration with 15 CV of 50 mM Tris, 20 mM NaCl, pH 7.5. Filtered mammalian cell culture harvest containing a mixture of phosphatidylserine-positive and phosphatidylserine-negative exosomes was applied to the column. Unbound sample components were elute from the column. Phosphatidylserine-positive exosomes were removed by elution with 50 mM Tris, 1.0 M NaCl, pH 7.0, followed by 5 CV of further elution under these conditions. Optionally, the column was reequilibrated to its original equilibration conditions. Phosphatidylserine-positive exosomes could be recovered by eluting the column with 1 M potassium phosphate. The column was then washed with 1 M NaOH and 2 M NaCl.

[0059] Example 4

[0060] Isolation of the desired lipid-coated virus (LCMA) from chromatin hybrids (containing degradation residues of LMCA).

[0061] A ferric ion-loaded cationic metal affinity synthetate (TREN) was used, followed by equilibration with 15 CV of 50 mM Tris, 20 mM NaCl, pH 7.5. Filtered mammalian cell culture harvest containing a mixture of coronavirus, chromatin, and heteropolymers (including chromatin and degraded LMCA residues) was loaded onto the column. Unbound sample components were washed from the column with equilibration buffer, followed by a 20 CV linear gradient elution to 50 mM Tris, 1.0 M NaCl, pH 7.0, and continued elution for 5 CV under these conditions. The column was then washed with 1 M NaOH and 2 M NaCl.

[0062] Example 5

[0063] Reduce chromatin before performing the method for purifying exosomes according to the present invention.

[0064] Ferric ion-loaded iminodiacetic acid particles were equilibrated to 20 mM Tris, 50 mM NaCl, pH 7.5. These were mixed with filtered cell culture harvest containing exosomes and contaminants at a 5% particle ratio and incubated for 2 hours. The particles were allowed to settle, and the supernatant was filtered through a 0.45 μm membrane filter. Removal of solids facilitated supernatant filtration by tangential flow filtration (TFF) with a pore size range of 300 kDa to 750 kDa, achieving a concentration 20–100 times higher. During TFF, the buffer composition was changed to 20 mM Tris, 20 mM NaCl, pH 7.5, after which the sample was processed using the method of the present invention as described in Example 2.

[0065] Example 6

[0066] Reduce chromatin before performing the method for purifying exosomes according to the present invention.

[0067] Ferric ion-loaded iminodiacetic acid particles were equilibrated to 20 mM Tris, 50 mM NaCl, pH 7.5. These were mixed with filtered cell culture harvest containing exosomes and contaminants at a 5% particle ratio and incubated for 2 hours. The particles were allowed to settle, and the supernatant was filtered through a 0.45 μm membrane filter. Removal of solids facilitated supernatant filtration by tangential flow filtration with a pore size range of 300 kDa to 750 kDa, achieving a concentration 20-100 times. During TFF, the buffer composition was changed to 20 mM Tris, 500 mM NaCl, 5 mM Magnesium Chloride, pH 7.5. The sample was treated with a salt-tolerant nuclease, and then TFF was restarted to remove released nucleotides, histones, and excess magnesium ions, while the buffer composition was changed to 50 mM Tris, 20 mM NaCl, pH 7.5. In this step, the sample could be further concentrated. The sample was then processed using the method of the present invention as described in Example 2.

[0068] Example 7

[0069] After performing the exosome purification method of this invention, chromatin is reduced.

[0070] Following the methods of Example 5 or Example 6, the sample was diluted or buffer-exchanged to conditions close to 50 mM Tris, 20 mM Sodium Chloride, and pH 7.5, according to the method of the present invention described in Example 2. A strong anion exchange column was equilibrated to these conditions and loaded with the sample. Exosomes were eluted from the column with a linear gradient of 20 CV to 1 M NaCl. The column was then cleaned with 1 M NaOH. Further treatment steps further reduced chromatin and other contaminant levels.

[0071] References

[0072] All references cited in this article are fully incorporated through citation, and the degree of incorporation does not contradict the views expressed in this article.

[0073] [1] K Vrabec, T Lojpur, P Gagnon, Emerging tools for exosome purification and in-process monitoring, Bioprocess International eBook, March 22, 2019, https: / / bioprocessintl.com / analytical / downstream-validation / emerging-tools-for-exosome-purification-and-in-process-monitoring

[0074] [2] P Gagnon, K vrabic, T Lojpur, A Strancar, Setting a cornerstone for platform purification of exosomes, Bioprocess International 18(2020)28-51, https: / / bioprocessintl.com / downstream-processing / separation-purification / setting-a-cornerstone-for-exosome-purification-platform

[0075] [3] P Gagnon, R Nian, J Lee, L Tan, SM Abdul-Latiff, CL Lim, C CHuah, X Bi, YSYan, W Zhang, HT Gan, Nonspecific interactions of chromatin with immunoglobulin G and protein A, and their impact on purification performance, J Chromatogr A 1340(2014)68-78.

[0076] [4] S. Staubach, P. Gagnon, K. Vrabec, T. Lojpur, S. Peljhan, B. Giebel, A. Strancar, Challenges in industrial process development of exosome-based therapies: characterizing and managing diversity, BioProcess International, 2020, https: / / bioprocessintl.com / analytical / downstream-development / classification-of-extracellular-vesicles-using-chromatography-for-exosome-therapy-production

[0077] [5]R Sharma, X Huang, R Brekken, A Schoit, Detection of phosphatidylserine-positive exosomes for diagnosis of early-stage malignancies, Br J Cancer 117(2017)545-552.

Claims

1. A chromatographic method for separating LMCA from chromatin hybrid samples containing degradation residues of LMCA, wherein LMCA is a lipid membrane-encapsulated compound aggregate. The sample is contacted with a chromatographic material having cationic metal affinity ligands on its surface, the material containing ferric, manganese, calcium, or magnesium ions, and equilibrated with a buffer solution. A gradient of first elution buffer containing nonmetallic chelating salts is applied, thereby... LMCA was washed away.

2. The chromatographic method according to claim 1, characterized in that, The LMCA is selected from: cellular vesicles, extracellular vesicles, noncellular vesicles, organelles, exosomes, microvesicles, ribosomes, mitochondria, lipid-coated viruses or lipid-coated virus-like particles, influenza viruses, coronaviruses, lentiviruses, cytomegaloviruses, liposomes, and combinations thereof.

3. The chromatographic method according to claim 2, characterized in that, The extracellular vesicles are small or medium-sized extracellular vesicles.

4. The chromatographic method according to claim 1, characterized in that, The nonmetallic chelate salt is a monovalent halide salt.

5. The chromatographic method according to claim 4, characterized in that, The monovalent halide salt is sodium chloride.

6. The chromatographic method according to claim 1, characterized in that, The sample is a cell culture harvest, a filtered cell culture harvest, a concentrated filtered cell culture harvest, or a partially purified formulation containing LMCA and chromatin hybrids, wherein the chromatin hybrids contain LMCA residues.

7. The chromatographic method according to claim 1, characterized in that, The chromatographic method involves gradient elution of chromatin heteropolymers containing LMCA degradation residues in a second elution buffer containing chelating salts.

8. The chromatographic method according to claim 7, characterized in that, The chelating salt of the second elution buffer is selected from: metal phosphates, or metal citrates, or metal salts of ethylenediaminetetraacetic acid (EDTA), or metal salts of ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and combinations thereof.

9. The chromatographic method according to any one of claims 1 to 8, characterized in that, The chromatography method is combined with at least one other method to reduce the content of chromatin heteropolymers.

10. The chromatographic method according to claim 9, characterized in that, Other methods for reducing chromatin heteropolymer content include nuclease treatment and / or solid-phase treatment with anionic chromatographic ligands.

11. The chromatographic method according to claim 10, characterized in that, The solid phase containing anionic chromatographic ligands contains iron or manganese ions.

12. The chromatographic method according to claim 10, characterized in that, The sample is in contact with the chromatographic material and then separated from the chromatographic material after an incubation period.

13. The chromatographic method according to claim 10, characterized in that, The sample contained exosomes, which were percolated by tangential flow filtration.

14. The chromatographic method according to claim 13, characterized in that, The sample was a cell culture harvest.

15. The chromatographic method according to claim 1, characterized in that, The cationic metal affinity ligand is selected from tris(2-aminoethyl)amine (TREN).

16. The chromatographic method according to claim 1, characterized in that, The pH range of the equilibration buffer is from pH 5 to pH 9.

17. The chromatographic method according to claim 16, characterized in that, The pH range of the equilibration buffer is from pH 6 to pH 8.

18. The chromatographic method according to claim 17, characterized in that, The pH range of the equilibration buffer is between pH 6.5 and pH 7.

5.

19. The chromatographic method according to claim 1, characterized in that, The conductivity of the equilibration buffer solution ranges from 1 mS / cm to 250 mS / cm.

20. The chromatographic method according to claim 19, characterized in that, The conductivity of the equilibration buffer solution is from 2 mS / cm to 100 mS / cm.

21. The chromatographic method according to claim 20, characterized in that, The conductivity of the equilibration buffer solution is from 3 mS / cm to 50 mS / cm.

22. The chromatographic method according to claim 21, characterized in that, The conductivity of the equilibration buffer solution is from 4 mS / cm to 20 mS / cm.

23. The chromatographic method according to claim 22, characterized in that, The conductivity of the equilibration buffer solution is from 5 mS / cm to 10 mS / cm.

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