KR102130023B1 - Multiple column chromatography and method for isolating exosomes - Google Patents

Abstract

The present invention relates to a multi-column for separating exosomes from a biological sample in which impurities such as lipid protein, water-soluble protein and exosomes are mixed, and a method for separating exosomes.

Description

Multi-column and exosome separation method for separating exosomes {MULTIPLE COLUMN CHROMATOGRAPHY AND METHOD FOR ISOLATING EXOSOMES}

The present invention relates to a method for separating multiple columns and exosomes to separate exosomes with high purity from a biological sample in which various biomolecules and exosomes are mixed.

Extracellular vesicles (extracellular vesicle) is a concept that includes exosomes (exosome), ectosome (ectosome), microvesicles (microvesicle) and apoptosis bodies (apoptotic body) is released or secreted from the cell, of which exosomes are 20-150 It is a nano-particle that has a size of nm and is produced from multi-vesicular bodies (MVB) in cells.

These extracellular vesicles can be relatively easily separated from various types of biofluids such as blood, lymphatic fluid, cerebrospinal fluid, urine, amniotic fluid, breast milk, saliva, semen, and, depending on the cell from which they are derived, dexosome (dendritic) They are called cell-derived), oncosomes (from cancer cells), prostasomes (from prostate cells), cardiosomes (from cardiomyocytes), and the like. Cell vesicles contain various nucleotides or labeled proteins depending on the cell or organelle within which they are derived. For example, oncosomes, which are extracellular vesicles derived from cancer cells, contain mRNA of a gene that induces growth of cancer cells, and extracellular vesicles derived from antigen-presenting cells include a major histocompatibility complex. As such, since extracellular vesicles are contained in a high concentration of biomaterials such as cell-specific proteins or nucleotides, they are present in about 0.01% of the total proteomics in normal biological fluids, so proteins or nucleotides that are difficult to detect using conventional analytical methods are also extracellular. Parcels have the advantage of being relatively easy to detect. In addition, although the types of proteins or nucleotides present in the extracellular vesicles are only a small part of the whole, exosome analysis can be very useful for the diagnosis of a specific disease because the substances of the extracellular vesicles may exhibit the unique characteristics of the cell from which they originate. It is useful, and research on this has been actively conducted recently.

It is important to obtain high purity exosomes in diagnosis or treatment methods using exosomes. Various methods have been studied, including Korean Patent Publication No. 10-2016-0115988, in relation to separation of exosomes. Examples include ultracentrifuge, density centrifuge, column use, PEG precipitation (including PEG precipitation; using ExoQuickTM, Total Exosome IsolationTM, etc.), chromatography, and immuno-magnetic Separation (immuno-magnetic separation, IMS) and acoustic purification (acoustic separation). In the case of column chromatography, a lot of attention has been recently paid in that a relatively high purity exosome can be obtained in a short time.

However, when exosomes are separated using a conventional column chromatography, there is a problem in that exosomes are eluted together with exosomes, or exosomes are lost or take a long time while performing two or more separation steps. have. Existing column chromatographic studies focused only on the exosome elution section and little in-depth analysis of lipid protein elution was done. However, since a lipid protein having a size and density similar to that of an exosome is always present in the cell culture fluid or blood, lipid protein separation is essential in exosome studies.

Under this background, the present inventors tried to develop a method for separating various impurities such as lipid proteins and water-soluble proteins separated from exosomes in a biological sample, and analyzing particles in each section eluted using a column in cell culture fluid and blood. The present invention was completed by optimizing the conditions for separating exosomes and impurities and confirming a method for obtaining pure exosomes using the conditions.

Republic of Korea Patent Publication No. 10-2016-0115988

Joanne Louise Welton, Jason Paul Webber, Laur-Alexandru Botos, Michael Jones & Aled Clayton (2015) Ready-made chromatography columns for extracellular vesicle isolation from plasma, Journal of Extracellular Vesicles, 4:1, DOI: 10.3402/jev.v4. 27269

Under this background, the present inventors completed the present invention by discovering that the exosome separation efficiency is remarkably excellent when two types of beads are stacked in a specific order and ratio.

Accordingly, an object of the present invention is to provide a method for separating exosomes using multiple columns and exosomes for separating exosomes contained in a biological sample from lipid proteins and water-soluble proteins.

However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description.

According to an embodiment of the present invention, as a multi-column for exosome separation, porous beads having a pore size of 20 nm to 100 nm a), porous beads a) laminated on top, and porous beads having a pore size of 20 nm or less b) And a separation membrane positioned between the porous beads a) and the porous beads b), wherein the porous beads a) separate exosomes and lipid proteins in the biological sample, and the porous beads b) exo in the biological sample. Provided is a multiple column for exosome separation, which separates a little from a soluble protein.

According to one side, the volume ratio of the porous beads a) and the porous beads b) may be 95:5 to 5:95.

According to one side, the volume ratio of the porous beads a) and the porous beads b) may be 5:5 to 1:9.

According to one side, the volume ratio of the porous beads a) and the porous beads b) may be 3:7 to 1:9.

According to one side, the surface negative charge of the porous beads a) may have a higher negative charge value than the surface of the porous beads b).

According to one side, the porous beads are composed of at least one material selected from agarose, sepharose, cellulose, silica gel, dextran, N, N'-methylene bisacrylamide, methacryl, polyacrylamide and polystyrene. May be

According to one side, the biological sample may be one or more selected from the group consisting of blood, lymph fluid, cerebrospinal fluid, urine, amniotic fluid, breast milk, saliva, semen, and cell culture fluid.

According to another embodiment of the present invention, the pore size is 20nm to 100nm of the porous beads a), the porous beads a) laminated on top, the pore size of 20nm or less porous beads b) and the porous beads a) and A method for separating exosomes using multiple columns including a separation membrane located between the porous beads b), the method comprising: separating exosomes and water-soluble proteins by passing a biological sample containing exosomes through the porous beads b); And separating the exosome and the lipid protein by passing the biological sample that has passed through the porous beads b) through the porous beads a). A method of isolating exosomes is provided.

The multiple columns for exosome separation of the present invention and the exosome separation method using the same can greatly increase the purity of the separated exosomes by significantly reducing the amount of impurities such as lipid proteins and water-soluble proteins contained in the biological sample.

In particular, when performing size exclusion chromatography using the multiple columns of the present invention, since the separation efficiency of exosomes compared to lipid proteins is excellent, it can be usefully used to separate only exosomes in a sample.

The effects obtainable through the described embodiments are not limited to the above-described effects, and should be understood to include all effects that can be deduced from the specific contents for carrying out the invention described below or the configuration of the invention described in the claims. .

1 is a schematic diagram of multiple columns according to an embodiment of the present invention.
Figure 2 shows the results of separation of exosomes using a conventional single column and multiple columns of the present invention (2B: 2B single column, Dual column: multiple columns of the present invention).
A: Absorbance of fraction samples by column
B: Z-average size of particles in a column-by-column fraction sample identified through dynamic light scattering (DLS).
3 is a result of detection of exosomes and lipid proteins in the actual fraction of serum samples separated using multiple columns of the present invention (ApoB: lipid protein, CD63: exosome marker protein, 9-15: column fraction #9 -#15).
A: SDS-PAGE
B: Western blot
C: Relative detection intensity of lipid protein and exosome
Figure 4 shows the detection results of exosomes and lipid proteins for each fraction of a serum sample separated using a conventional single column and multiple columns of the present invention (serum: serum not treated separately, Marker: for molecular weight check Standard marker, ExoQuick: ExoQuick separation method, 2B column: 2B single column, Dual column: multiple columns of the invention).
A: Quantitative result of total protein in fraction
B: SDS-PAGE
C: Western blot
5 is a comparison of the relative ratio of exosomes to lipid proteins in a fraction of a serum sample separated using a conventional single column and multiple columns of the present invention (ExoQuick: ExoQuick separation method, 2B column: 2B single column, Dual column: multiple columns of the invention).
Figure 6 shows the results of detection of exosomes and lipid proteins using multiple columns with different stacking sequences of Sepharose CL-6B and Sephacryl 200-HR.
7 shows the amount of albumin, a water-soluble protein detected in a fraction of a serum sample.
Figure 8 shows the results of the detection of exosomes and lipid proteins using multiple columns prepared by different volume ratios of Sepharose CL-6B and Sephacryl 200-HR.
Figure 9 shows the amount of exosomes and lipid proteins detected in the fractions 11 to 12 of the serum sample according to the volume ratio of Sepharose CL-6B and Sephacryl 200-HR.
10 shows the CD63 density (%)/ApoB density (%) detected in the fractions 11 to 12 of the serum sample according to the volume ratio of Sepharose CL-6B and Sephacryl 200-HR.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. Unless otherwise stated, the same reference numerals in each drawing denote the same members.

Various changes can be made to the embodiment described below. The embodiments described below are not intended to limit the scope of the invention to the described embodiments, and it should be understood that the scope intended to be claimed as rights through the present application includes all changes, equivalents, or substitutes thereof.

The terms used in the examples are only used to describe specific examples, and are not intended to limit the examples. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, the terms "include" or "have" are intended to indicate the presence of features, numbers, steps, actions, components, parts or combinations thereof described in the specification, one or more other features. It should be understood that it does not exclude the possibility or presence of fields or numbers, steps, actions, components, parts or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person skilled in the art to which the embodiment belongs. Terms, such as those defined in a commonly used dictionary, should be interpreted as having meanings consistent with meanings in the context of related technologies, and should not be interpreted as ideal or excessively formal meanings unless explicitly defined in the present application. Does not.

In addition, in the description with reference to the accompanying drawings, the same reference numerals are assigned to the same components regardless of reference numerals, and redundant descriptions thereof will be omitted. In describing the embodiments, when it is determined that detailed descriptions of related well-known technologies may unnecessarily obscure the subject matter of the embodiments, detailed descriptions thereof will be omitted.

As used herein, the term “exosome” refers to a cell-derived vesicle having a diameter of 20 nm to 150 nm secreted from many types of cells, including cell cultures, and refers to membrane components, proteins, and RNA. It is known to play a variety of roles such as delivery.

According to an embodiment of the present invention, as a multi-column for exosome separation, porous beads having a pore size of 20 nm to 100 nm a), porous beads a) laminated on top, and porous beads having a pore size of 20 nm or less b) And a separation membrane positioned between the porous beads a) and the porous beads b), wherein the porous beads a) separate exosomes and lipid proteins in the biological sample, and the porous beads b) exo in the biological sample. Provided is a multiple column for exosome separation, which separates a little from a soluble protein.

The porous beads a) are for separating exosomes and lipid proteins in a biological sample and can separate molecules having a size of 10 MDa or less, and the porous beads b) are for separating exosomes and water-soluble proteins in a biological sample 500 kDa It is possible to separate molecules of the following size, and each bead preferably has a negative charge. In addition, in order to allow lipid proteins that reach the bottom of the column to elute more rapidly under the influence of charge, it is preferable that the porous beads a) stacked at the bottom have a lower average surface charge than the porous beads b) stacked at the top. (See Example 5).

The separator is positioned between the porous beads a) and b) so that different types of beads do not mix when different types of beads are continuously filled in the column container. The separator may be made of a resin such as polyethylene or polypropylene, but is not limited thereto.

Porous beads filled in multiple columns of the present invention are at least one selected from agarose, sepharose, cellulose, silica gel, dextran, N, N'-methylene bisacrylamide, methacryl, polyacrylamide and polystyrene. It may be composed of a material, but is not limited thereto.

Specific examples of the porous beads include Sepharose 2B, 4B, 6B, CL-2B, CL-4B and CL-6B; Sepacryl S-200 HR, S-300 HR, S-400 HR and S-500 HR; And Toyopearl HW-55, HW-65 and HW-75 and Superdex75, preferably Sepharose CL-6B and Sephacryl 200-HR.

On the other hand, the porous beads a) and the porous beads b) are filled with a volume ratio of 95:5 to 5:95, preferably a volume ratio of 5:5 to 1:9, more preferably a volume ratio of 3:7 to 1:9 It may have been done.

As described above, when size exclusion chromatography is performed using multiple columns prepared by filling two types of beads having different pore sizes and surface negative charge values in a specific volume ratio, the elution rate of each of lipid protein, exosome, and water-soluble protein Depending on the difference, exosomes can be separated with high purity.

Exosome-containing biological sample may be one or more selected from the group consisting of blood, lymph fluid, cerebrospinal fluid, urine, amniotic fluid, breast milk, saliva, semen, and cell culture fluid, but is not limited thereto.

Exosomes may be included in the 20% to 80% section of the total eluate after the chromatography, but are not limited thereto, and according to the exosome separation method of the present invention, exosomes compared to lipid proteins in the 29% to 57% section The proportion of was found to be high.

According to another embodiment of the present invention, the pore size is 20nm to 100nm of the porous beads a), the porous beads a) laminated on top, the pore size of 20nm or less porous beads b) and the porous beads a) and A method for separating exosomes using multiple columns including a separation membrane positioned between the porous beads b), the method comprising: passing a sample containing exosomes through the porous beads b) to separate the exosomes from the water-soluble proteins; And separating the exosome and the lipid protein by passing the sample that has passed through the porous beads b) through the porous beads a). A method of isolating exosomes is provided.

The description of the multiple columns used in the exosome separation method is as described above.

In a specific embodiment of the present invention, among the multi-column fractions shown in FIG. 1, exosomes appeared in 9 to 15 sections, and in particular, in 10 to 12 sections, exosome content was relatively high compared to lipid protein and water-soluble protein. (Fig. 3). When using the multi-column, the fractionation method using PEG precipitation and the protein yield comparable to that of the conventional single column can be used to separate the exosomes because the exosome fraction of remarkably high purity can be obtained. .

Hereinafter, the present invention will be described in detail by examples. However, the following examples and experimental examples are only for illustrating the present invention, and the present invention is not limited by the following examples and experimental examples.

Example 1: multiple Column making

Sepharose® CL-6B was filled with 70% (v/v) at the bottom of the chromatography column (diameter: 10 mm, height: 50 mm) and then 30% (v/v) of Sephacryl 200-HR was charged at the top. Multiple columns were constructed so that the height ratio of the lower part to the upper part was 7:3. Here, the average surface charge values of Sepharose CL-6B and Sephacryl 200-HR are -30 and -7, respectively.

Example 2: Existing column In condition Exosome Check separation ability

A column filled with a chromatography column (diameter: 10 mm, height: 50 mm) was used, and as a sample, blood was centrifuged at 10000 rcf, 4° C. for 30 min, and the obtained supernatant serum was used. Size exclusion chromatography (SEC) was performed on the sample to confirm the effect of separating materials by a single column and multiple columns.

Specifically, beads packed in a column used Sepharose CL-2B as a single column (2B), and the one prepared in Example 1 (Dual column) was used as a multiple column. Prior to loading the sample, the beads filled with 300 ml of PBS were washed, and the prepared sample was loaded immediately when the mobile phase buffer was exhausted in addition to the filled column beads. The volume of the sample loaded on the column was not to exceed 5% of the total volume of the filled beads, and 0.3 to 0.5 ml of the sample was loaded onto the column. The pressure of the mobile phase was flowed at 2-4 Pa. The section of the solution that received 0.5 ml of eluate flowing from the time the sample was loaded was defined as one section, and the collected eluate was checked for absorbance at 280 nm, and Z-average was measured using dynamic light scattering (DLS). The size (Z-Average size) was measured and is shown in FIG. 2. The samples were all eluted within 25-30 sections.

As a result, the absorbance (FIG. 2A) and DLS analysis (FIG. 2B) showed that the fractionation of exosomes, lipid proteins, and water-soluble proteins in fractions was not easy.

Example 3: in serum Exosome Comparison of the tendency of lipid protein elution

To compare the elution of exosomes, lipid proteins, and water-soluble proteins for each section in serum samples, the cells were fractionated by the method described in Example 2 using multiple columns of Example 1, and SDS-PAGE and Western blot ( Western blot). As the primary antibody, the antibody against the exosome marker protein CD63 (sc-15363 manufactured by Santa Cruz) was diluted 1:500, and the lipid protein antibody ApoB-100 antibody (sc-25542 manufactured by Santa Cruz) 1:1000 After dilution with, it was confirmed whether or not it was eluted using diluted antibodies.

As a result, the albumin (~65 kDa), a water-soluble protein, was detected from section #13 onwards (FIG. 3A), and the lipid protein was detected most clearly at section #9 and up to #11. Exosomes were detected in a small amount in #9, and after appearing the darkest in the #11 and #12 sections, the detected amount gradually decreased (Fig. 3B). When comparing the amount of lipid protein and exosome in the fractions of each section in sections #9 to #15, lipid protein appeared mainly in section 9 (#9), but from section 10, exosomes were detected more than lipid protein. In the 11th section (#11), exosomes appeared most in the fraction, in the 12th section (#12), almost no lipid protein in the fraction was detected, and in the 13th section (#13), the soluble protein was detected. Therefore, it was confirmed that sections 10 to 12 (#10-#12) were more suitable for obtaining exosomes (FIG. 3C).

Example 4: separation method and On the column Follow Exosome Confirmation of the separation efficiency of lipid proteins

Each of the serum samples described in Example 2 was subjected to sedimentation (Exoquick) using centrifugation, size exclusion chromatography using a conventional single column (2B column), and size exclusion chromatography using multiple columns of the present invention. The fractions of exosomes and lipid proteins separated are shown in FIG. 4.

Specifically, the sedimentation method was performed through the addition and centrifugation of a commercialized product, ExoQuick ^TM (System Biosciences, Inc.), and the existing single column was used to be filled with Sepharose CL-2B, and multiple columns were used in Example 1 The multiple columns disclosed in were used. The total amount of protein in the sample before fractionation and in the fractionated sample was quantified, and how the detection amount of the exosome and the lipid protein and the relative amount of the exosome and the lipid protein differed were confirmed by SDS-PAGE and Western blot. As in Example 3, an antibody against the exosome marker protein CD63 and an antibody against the lipid protein marker protein ApoB were used, and the CD63 density (%)/ApoB density (%) was used by Bio-Rad ChemiDoc image software. Then, it was calculated through density measurement of the blotted sample part.

As a result, the total amount of protein contained in the fractionated sample was found to be the lowest when using the 2B column (FIG. 4A), and when using Exoquick, the lipid protein was found to contain a large amount (FIG. 4B).

However, in the Western blot result, it was confirmed that exosomes were least detected in the fraction using the 2B column, and many lipid proteins were detected. On the other hand, in the experimental group using the multiple column of Example 1, it was found that almost no lipid protein was detected (FIG. 4C). In particular, the results of confirming CD63 density (%)/ApoB density (%) indicating the separation efficiency of exosomes and lipid proteins showed the highest value when using the multiple columns of Example 1 (FIG. 5).

That is, the multi-column of the present invention has superior ability to remove the lipid protein contained in the sample and separate only exosomes compared to the existing single column.

Example 5: Bead Evaluation of separation efficiency according to the stacking order

Sephacryl 200-HR 70% at the bottom of the multi-column (A) of Example 1 and the chromatography column (diameter: 10 mm, height: 50 mm) in order to evaluate the separation efficiency according to the stacking order of beads packed in multiple columns. After stacking (v/v), a multi-column (B) in which 30% (v/v) of Sepharose® CL-6B was stacked was prepared.

Size exclusion chromatography was performed by the method described in Example 2 using the prepared two multi-columns, and the amounts of detection of lipid proteins and exosomes for each elution section were shown in FIG. 6. As a result, in the multi-column (A) of Example 1, lipid proteins were hardly detected, whereas in the multi-column (B), lipid proteins were detected in most elution sections, and the detection amount of exosomes was multi-column (A). It was confirmed to be more.

Example 6: Bead In bulk Separation efficiency evaluation

Sepharose® CL-6B and Sephacryl 200-HR in order from the bottom of the column for chromatography (diameter: 10 mm, height: 50 mm) in order to evaluate the separation efficiency according to the volume ratio of beads packed in multiple columns in a volume ratio of 9: 1 , 7:3, 5:5, 3:7, 1:9 multi-columns stacked in a ratio, and a single column of Sepharose CL-6B and Sephacryl 200-HR was prepared.

Size exclusion chromatography was performed by the method described in Example 2 using the prepared column. As a result, since the water-soluble protein having a low molecular weight and passing through both types of beads elutes after 13 sections regardless of the volume ratio of the beads to be filled (Fig. 7), 11 to 12 sections are set as Region of interest (ROI). The detection amounts of lipid proteins and exosomes were analyzed and shown in FIGS.

8 and 9, CD63 density (%) and ApoB density (%) when the volume ratio of Sepharose #CL-6B and Sephacryl 200-HR in the Region of interest (ROI) of 11 to 12 is 3:7 ) Value, the difference is the largest, referring to Figure 10, CD63 density (%) / ApoB density (%) value shows a tendency of the quadratic function to increase in the interval of the volume ratio 5:5 to 1:9 You can confirm that.

As described above, although the embodiments have been described by a limited embodiment and drawings, those skilled in the art can make various modifications and variations from the above description. For example, even if the described techniques are performed in a different order than the described method, and/or the described components are combined or combined in a different form from the described method, or replaced or replaced by another component or equivalent Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (14)

As a multiple column for exosome separation,
Porous beads having a pore size of 20 nm to 100 nm a), porous beads a) are stacked on top, porous pores having a pore size of 20 nm or less b) and a separator positioned between the porous beads a) and the porous beads b) Including,
The porous bead a) is Sepharose CL-6B, the porous bead b) is Sephacryl 200-HR, and the porous bead a) separates exosomes and lipid proteins in the biological sample, and the porous beads b) Is to separate the exosomes and water-soluble proteins in the biological sample, multiple columns for exosome separation.
According to claim 1,
The volume ratio of the porous beads a) and the porous beads b) is 95:5 to 5:95, multiple columns for exosome separation.
According to claim 2,
The volume ratio of the porous beads a) and the porous beads b) is 5:5 to 1:9, multiple columns for exosome separation.
According to claim 3,
The volume ratio of the porous beads a) and the porous beads b) is 3:7 to 1:9, multiple columns for exosome separation.
According to claim 1,
The surface negative charge of the porous beads a) has a higher negative charge value than the surface negative charge of the porous beads b), multiple columns for exosome separation.
delete According to claim 1,
The biological sample is one or more selected from the group consisting of blood, lymph fluid, cerebrospinal fluid, urine, amniotic fluid, breast milk, saliva, semen and cell culture fluid, multiple columns for exosome separation.
Porous beads having a pore size of 20 nm to 100 nm a), porous beads a) are stacked on top, porous pores having a pore size of 20 nm or less b) and a separator positioned between the porous beads a) and the porous beads b) As a method for separating exosomes using multiple columns, comprising:
Separating the exosome from the water-soluble protein by passing a biosample containing exosome through the porous beads b); And
Separating the exosome and the lipid protein by passing the biological sample that has passed through the porous beads b) through the porous beads a); Including,
The porous beads a) is Sepharose CL-6B, the porous beads b) is Sephacryl 200-HR, exosome separation method.
The method of claim 8,
The volume ratio of the porous beads a) and the porous beads b) is 95:5 to 5:95, exosome separation method.
The method of claim 9,
The volume ratio of the porous beads a) and the porous beads b) is 5:5 to 1:9, exosome separation method.
The method of claim 10,
The volume ratio of the porous beads a) and the porous beads b) is 3:7 to 1:9, exosome separation method.
The method of claim 8,
The surface negative charge of the porous beads a) has a higher negative charge value than the surface negative charge of the porous beads b), exosome separation method.
delete The method of claim 8,
The biological sample is one or more selected from the group consisting of blood, lymph fluid, cerebrospinal fluid, urine, amniotic fluid, breast milk, saliva, semen and cell culture fluid, exosome separation method.

Images