阅读说明：本技术 针筒式重力柱与固相萃取仪相结合的固相萃取系统及方法 (Solid phase extraction system and method combining syringe type gravity column and solid phase extractor ) 是由 臧美翎 董翠 于 2021-06-08 设计创作，主要内容包括：本发明提出了一种针筒式重力柱,该针筒式重力柱包括：具有空腔的柱体,所述柱体的上端设置有进液口,所述柱体的下端设置有出液口,所述柱体的空腔内自下而上依次连接填充有第一过滤层、填料层、缓冲层和第二过滤层。由此,可以实现高通量自动化萃取,提高纯化效率,节省溶液使用量,目标物得率、纯度和获得量均较高,尤其适用于蛋白纯化,有助于实现规模化研发应用。(The invention provides a syringe type gravity column, which comprises: the cylinder with cavity, the upper end of cylinder is provided with the inlet, the lower extreme of cylinder is provided with the liquid outlet, it has first filter layer, packing layer, buffer layer and second filter layer to connect gradually to fill in the cavity of cylinder from bottom to top. Therefore, high-throughput automatic extraction can be realized, the purification efficiency is improved, the solution usage amount is saved, the target product yield, purity and yield are high, the method is particularly suitable for protein purification, and the method is favorable for realizing large-scale research and development.)

1. A syringe-type gravity column, comprising: a column body with a cavity, a liquid inlet is arranged at the upper end of the column body, a liquid outlet is arranged at the lower end of the column body,

the hollow cavity of the column body is sequentially filled with a first filter layer, a filler layer, a buffer layer and a second filter layer from bottom to top.

2. The syringe-type gravity column according to claim 1, wherein the buffer layer is formed of a buffer solution comprising a 10-30 vol% ethanol solution.

3. The syringe-type gravity column according to claim 1, wherein a height ratio of the filler layer to the buffer layer is 1: (0.25-0.5), the height of the second filter layer is not more than 50% of the height of the packing layer, and the volume of the packing layer is 20-40% of the volume of the cavity.

4. The syringe-type gravity column according to claim 1, wherein the first and second filter layers are formed of a filter material selected from at least one of porous glass, porous oxide, porous glass fiber, and porous polymer material, preferably a polypropylene compound;

the aperture of the filter material is 15-30 mu m;

the filler is selected from one of affinity filler, ion exchange filler and hydrophobic filler, and is preferably affinity filler.

5. A solid phase extraction system, comprising: a solid phase extractor and a syringe type gravity column according to any one of claims 1 to 4.

6. Use of a syringe-type gravity column according to any one of claims 1 to 4 or a solid phase extraction system according to claim 5 for solid phase extraction.

7. Use according to claim 6, characterized in that the solid phase extracted sample is a protein solution.

8. A method of purification using the solid phase extraction system of claim 5, comprising:

placing the syringe type gravity column of any one of claims 1 to 4 on a chromatographic column frame in a solid phase extraction apparatus, adding a balance liquid, a sample to be detected, a balance liquid, a washing liquid and an eluent into the syringe type gravity column in sequence, pressurizing each time after adding one liquid, and collecting a purified sample flowing out after the last pressurization.

9. The method of claim 8, wherein the sample to be tested is a protein solution;

preferably, the protein is selected from at least one of IgG protein, protein with isoelectric point of 5.0-8.5 or protein with molecular weight of 140-155 kDa.

10. The method according to claim 8, wherein the pressure of the pressure treatment is not higher than 1 bar.

Technical Field

The present invention relates to the field of biological devices. In particular, the invention relates to a solid phase extraction system and a method combining a syringe type gravity column and a solid phase extractor.

Background

The antibody medicine has the characteristics of good curative effect, small side effect and strong targeting property, and is a key point and a hot point in the current medicine research and development. The research and development of a new antibody and fusion protein medicine needs to be carried out through a large amount of research works such as the construction of a stable cell strain, upstream process development, culture medium screening, downstream process development, preparation process development, pilot scale production and the like from the determination of a molecular sequence to the realization of the industrial amplification of the protein, and particularly, the rapid determination of the optimal stable cell strain and culture process conditions in the stages of the construction of the stable cell strain, the upstream process development and the culture medium screening is particularly critical, and the research and development progress of the whole project is directly determined. Because the sample contains a large amount of impurities and related purity indexes cannot be directly detected, the sample can be further detected and analyzed after purification. The rapid, reliable and efficient automatic separation and analysis technology can greatly improve the efficiency and reduce the time and personnel cost. High throughput, small-scale protein production and purification becomes critical with minimal human intervention and high reproducibility, thereby avoiding excessive time waste in protein sample preparation.

At present, Magnetic bead methods (Magnetic Beads), AKTA pure instrument purification methods, gun head extraction methods and manual gravity column methods are common laboratory protein purification methods, but the methods have obvious defects. The paramagnetic particle method generally has a certain flux, but has a limited service life, is expensive relative to consumables, has generally low protein yield and limited adsorption capacity, cannot meet the preparation of a large amount of protein, and cannot be applied in a process development stage. The AKTA pure instrument purification method has high automation purity, low equipment flux (6-8 samples are processed in each machine for 24 hours) and high equipment cost. The gun head type extraction method has small solution usage amount and high flux, but the filling amount of the filler is low and is only about 500 mu l generally, so that the obtained protein sample is less, and the requirements can not be met in the stages of construction of stable cell strains, upstream process development and culture medium screening. The manual syringe type gravity column method is purely manual, cannot carry out data rechecking without online data, is purely manual, has larger flux (6-8 samples in 1 person; 5hr) relative to AKTA, and consumes manpower.

The solid phase extraction instrument is developed by combining liquid-solid extraction and liquid chromatography technologies, utilizes a solid adsorbent to adsorb a target compound in a liquid sample, separates the target compound from a matrix and an interference compound of the sample, and then uses eluent for elution or heating desorption to achieve the purpose of separating and enriching the target compound (namely separation, purification and enrichment of the sample), and has the advantages of high flux, strong accuracy, high sensitivity and the like.

Therefore, the current solid phase extraction methods, especially those suitable for proteins, are still under investigation.

Disclosure of Invention

The present invention aims to solve, at least to some extent, the technical problems of the prior art. Therefore, the invention provides an improved syringe type gravity column, a solid phase extraction system, application of the improved syringe type gravity column and the solid phase extraction system in solid phase extraction and a method for performing solid phase extraction by using the improved syringe type gravity column and the solid phase extraction system, which can realize high-throughput automatic extraction, improve purification efficiency, save solution usage amount, and have higher target product yield, purity and yield, are especially suitable for protein purification and are beneficial to realizing large-scale research and development application.

In one aspect of the invention, a syringe-type gravity column is provided. According to an embodiment of the invention, the syringe-type gravity column comprises: the cylinder with cavity, the upper end of cylinder is provided with the inlet, the lower extreme of cylinder is provided with the liquid outlet, it has first filter layer, packing layer, buffer layer and second filter layer to connect gradually to fill in the cavity of cylinder from bottom to top.

The inventor combines a syringe type gravity column filled with a filter layer (namely a first filter layer) and a filler layer from bottom to top with a solid phase extractor to purify protein, and finds that macromolecular protein is easy to have filler glue surface inclination under pressurization action compared with small molecular compounds, thereby greatly reducing the protein yield. For this reason, the inventor adds a filter layer (i.e., a second filter layer) above the filler layer, and found that the filler glue surface inclination can be effectively avoided. Further, if the filler layer is attached to the filter layer additionally arranged on the upper layer, liquid in the filler is pressed dry under the action of pressurization, so that subsequent protein solution cannot be adsorbed by the filler, and the experiment fails. Therefore, the inventor adds a buffer layer formed by buffer solution in the filler layer and the second filter layer, and finds that the phenomenon that the filler is pressed dry can be effectively avoided. Therefore, the improved syringe type gravity column can realize high-flux automatic extraction, improve the purification efficiency, save the use amount of solution, has higher yield, purity and acquisition amount of target objects, is particularly suitable for protein purification, and is favorable for realizing large-scale research and development and application.

According to an embodiment of the present invention, the syringe-type gravity column may further have the following additional features:

according to the embodiment of the invention, the buffer layer is formed by a buffer solution, and the buffer solution comprises 10-30 vol% ethanol solution.

According to an embodiment of the present invention, a height ratio of the filler layer to the buffer layer is 1: (0.25-0.5), the height of the second filter layer is not more than 50% of the height of the packing layer, and the volume of the packing layer is 20-40% of the volume of the cavity.

According to an embodiment of the present invention, the first and second filter layers are formed of a filter material selected from at least one of porous glass, porous oxide, porous glass fiber, and porous polymer material, preferably a polypropylene compound; the aperture of the filter material is 15-30 mu m.

According to an embodiment of the invention, the filler is selected from one of an affinity filler, an ion exchange filler, a hydrophobic filler, preferably an affinity filler, in particular a filler coupled to protein a protein.

In another aspect of the invention, a solid phase extraction system is provided. According to an embodiment of the invention, the solid phase extraction system comprises: a solid phase extractor and a syringe type gravity column as described above. The solid phase extraction instrument containing the needle cylinder type gravity column can realize high-flux automatic extraction, improve the purification efficiency, save the solution usage amount, and has higher target object yield, purity and acquisition amount, thereby being particularly suitable for protein purification and being beneficial to realizing large-scale research and development application.

In a further aspect of the invention, the invention provides the use of a syringe-type gravity column or a solid phase extraction system as described above in solid phase extraction. The needle cylinder type gravity column or the solid phase extraction apparatus containing the needle cylinder type gravity column can realize high-flux automatic extraction, improve the purification efficiency, save the solution usage amount, have higher target object yield, purity and acquisition amount, and are beneficial to realizing large-scale research and development and application.

According to an embodiment of the invention, the above-mentioned use may also have the following additional technical features:

according to an embodiment of the invention, the sample of the solid phase extraction is a protein solution.

In yet another aspect of the invention, the invention provides a method of purification using the solid phase extraction system described above. According to an embodiment of the invention, the method comprises: and placing the syringe type gravity column on a chromatographic column frame in a solid phase extraction instrument, sequentially adding a balance liquid, a sample to be detected, the balance liquid, a washing liquid and an eluent into the syringe type gravity column, pressurizing each time after adding one liquid, and collecting a purified sample flowing out after pressurizing for the last time. Therefore, high-throughput automatic extraction can be realized, the purification efficiency is improved, the solution usage amount is saved, the target product yield, the purity and the yield are high, and the large-scale research and development application is facilitated.

According to an embodiment of the present invention, the above-mentioned method of solid phase extraction may further have the following additional technical features:

according to an embodiment of the present invention, the sample to be tested is a protein solution.

According to an embodiment of the present invention, the protein is selected from at least one of IgG protein, protein having isoelectric point of 5.0-8.5, or protein having molecular weight of 140-155 kDa.

According to an embodiment of the invention, the pressure of the pressure treatment is not higher than 1 bar.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a schematic view of a syringe-type gravity column according to one embodiment of the present invention;

FIG. 2 is a schematic diagram of a solid phase extraction system incorporating a cartridge gravity column in accordance with one embodiment of the present invention;

FIG. 3 shows a schematic diagram of a chromatography column scaffold according to an embodiment of the invention;

FIG. 4 shows a CE purity analysis spectrum of a purified sample obtained in example 1 according to the present invention;

FIG. 5 shows a SEC purity analysis profile of a purified sample obtained according to example 1 of the present invention;

FIG. 6 shows a CE purity analysis spectrum of a purified sample obtained in comparative example 1 according to the present invention;

FIG. 7 shows a SEC purity analysis profile of a purified sample obtained according to comparative example 1 of the present invention;

FIG. 8 shows a CE purity analysis spectrum of a purified sample obtained in comparative example 2 according to the present invention;

FIG. 9 shows a SEC purity analysis profile of a purified sample obtained according to comparative example 2 of the present invention;

figure 10 shows a schematic diagram of a syringe-type gravity column structure without a buffer layer and a second filter layer, according to one embodiment of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

The invention provides a syringe type gravity column, a solid phase extraction system, application of the syringe type gravity column or the solid phase extraction system in solid phase extraction, and a method for purifying by using the solid phase extraction system, which are respectively described in detail below.

Syringe type gravity column

In one aspect of the invention, a syringe-type gravity column is provided. According to an embodiment of the present invention, referring to fig. 1, the syringe-type gravity column includes a cylinder having a cavity, an upper end of which is provided with a liquid inlet 100 and a lower end of which is provided with a liquid outlet 200. Thus, liquid can enter the cavity from the liquid inlet, and unadsorbed substances can flow out from the liquid outlet. Specifically, a protective cover can be additionally arranged on the liquid inlet, and a protective cover can be additionally arranged on the liquid outlet, so that the cylinder and the internal liquid are prevented from being polluted or flowing out.

According to the embodiment of the present invention, the first filter layer 300, the filler layer 400, the buffer layer 500 and the second filter layer 600 are sequentially filled in the hollow space of the column from bottom to top. The first filter layer plays a role in supporting the filler, and the filler is prevented from flowing out of the liquid outlet under the pressurizing effect. The second filter layer can effectively avoid the phenomenon that the rubber surface of the filler is inclined under the action of pressurization, and the buffer layer can effectively avoid the filler from being pressed dry under the action of pressurization, so that the yield, the purity or the yield of a target object is improved, and the buffer layer is particularly suitable for protein.

According to an embodiment of the present invention, the buffer layer is formed of a buffer solution including 10 to 30 vol% ethanol solution. Thus, the packing can be effectively prevented from being pressed dry under the action of pressure, and the target object, especially the protein, can not be interfered with to be adsorbed and desorbed in the packing.

According to the embodiment of the invention, the height ratio of the filler layer to the buffer layer is 1: (0.25 to 0.5), whereby the yield, purity or yield of the target substance can be effectively improved, and the method is particularly suitable for proteins. If the height of the buffer layer is too large, the dead volume above the packing layer is easily caused to be too large, so that the concentration of the solution is diluted after the solution is added, the protein content in the final eluted effluent liquid is low, the subsequent operation is not facilitated, or the subsequent process also needs concentration, and the workload and the cost are increased.

According to an embodiment of the invention, the height of the second filter layer is not more than 50% of the height of the filler layer. The inventor obtains the preferable height ratio through a large number of experiments, thereby effectively supporting the packing layer and avoiding the inclination.

According to the embodiment of the invention, the volume of the filler layer is 20-40% of the volume of the cavity. This makes it possible to sufficiently bind the target substance to the filler and to improve the purification efficiency.

According to an embodiment of the present invention, the first filter layer and the second filter layer are formed of a filter material selected from at least one of porous glass, porous oxide, porous glass fiber, and porous polymer material, preferably a polypropylene compound. Therefore, the filter can play a certain role in filtering and supporting a sample to be detected, can avoid the filler layer from falling to a liquid outlet and also can avoid the rubber surface of the filler layer from inclining, and meanwhile, the characteristics of the sample to be detected, especially protein, cannot be changed.

According to the embodiment of the invention, the aperture of the filter material is 15-30 μm. Therefore, protein can pass through, and the inclination of the rubber surface of the filler layer or the leakage of the filler caused by the overlarge pore diameter can be avoided.

According to an embodiment of the invention, the filler is selected from affinity fillers, more preferably fillers coupled to proteinA protein. Protein A (protein A) is derived from a strain of Staphylococcus aureus and contains 5 domains that specifically bind to the Fc region of antibodies and fusion proteins. So about 70-80% of the antibody purification uses an affinity filler coupled to the proteinA protein to capture the protein.

Affinity fillers include, but are not limited to fortuitum r-proteinA, Mabselect SuRe LX, prism a; merck's Eshmumo a; TOYOPEARL AF-rprotien A-650F from Tosoh; MabApture A flying by Saimer; nanomicro Unimab Pro, Nmab; diamond plus of bocolon; blantt PRAESTO Jetted a 50; mabpurix A45 and Mabpurix P45.

Solid phase extraction system

In another aspect of the invention, a solid phase extraction system is provided. According to an embodiment of the invention, a solid phase extraction system comprises: a solid phase extractor and a syringe type gravity column as described above. The solid phase extraction instrument (see figure 2) is combined with the syringe type gravity column, the operations of high flux balance, washing, elution and the like can be automatically completed by injecting solution from the top of the syringe type gravity column and positively pressurizing, the automatic purification can be completed without other auxiliary instrument equipment, and the purpose of high flux rapid purification is achieved, and the device is particularly suitable for protein.

It will be appreciated by those skilled in the art that the features and advantages described above with respect to the cartridge-type gravity column are equally applicable to the solid phase extraction system and will not be described in further detail herein.

Application of syringe type gravity column or solid phase extraction system in solid phase extraction

In a further aspect of the invention, the invention proposes the use of a syringe-type gravity column or a solid phase extraction system in solid phase extraction. The needle cylinder type gravity column or the solid phase extraction apparatus containing the needle cylinder type gravity column can realize high-flux automatic extraction, improve the purification efficiency, save the solution usage amount, have higher target object yield, purity and acquisition amount, and are beneficial to realizing large-scale research and development and application.

According to an embodiment of the invention, the sample of the solid phase extraction is a protein solution. The needle cylinder type gravity column or the solid phase extraction system can effectively purify protein and has the advantages of high purification efficiency, high protein yield, high purity, high yield and the like.

It will be appreciated by those skilled in the art that the features and advantages described above in relation to the cartridge-type gravity column and the solid phase extraction system are equally applicable to this application and will not be described in further detail herein.

Method for purifying by using solid phase extraction system

In one aspect of the invention, a method of purification using a solid phase extraction system is provided. According to an embodiment of the invention, the method comprises: and placing the syringe type gravity column on a chromatographic column frame in a solid phase extraction instrument, sequentially adding a balance liquid, a sample to be detected, the balance liquid, a washing liquid and an eluent into the syringe type gravity column, pressurizing each time after adding one liquid, and collecting a purified sample flowing out after pressurizing for the last time.

Referring to fig. 2 and 3, the chromatographic column holder contains a plurality of holes, the syringe type gravity column is placed in the holes, and then the chromatographic column holder is placed in the corresponding position in the solid phase extractor. And then, adding a balance liquid into the column to balance the column, adding a sample to be detected, enabling a target object in the sample to be detected to be combined on the filler, then sequentially enabling the balance liquid, a washing liquid and an eluent to pass through the column, eluting the target object combined on the filler, and flowing out to finally obtain a purified sample. Therefore, high-throughput automatic extraction can be realized, the purification efficiency is improved, the solution usage amount is saved, the target product yield, the purity and the yield are high, and the large-scale research and development application is facilitated.

According to an embodiment of the present invention, the sample to be tested is a protein solution. Specifically, the protein is selected from at least one of IgG protein, protein with isoelectric point of 5.0-8.5 or protein with molecular weight of 140-155 kDa. The needle cylinder type gravity column or the solid phase extraction system can effectively purify protein and has the advantages of high purification efficiency, high protein yield, high purity, high yield and the like.

According to an embodiment of the invention, the pressure of the pressure treatment is not higher than 1 bar. Therefore, the liquid flowing speed can be increased, and the extraction efficiency is improved.

It will be appreciated by those skilled in the art that the features and advantages described above in relation to the cartridge-type gravity column and the solid phase extraction system apply equally to this method and will not be described in further detail herein.

Advantageous effects

Compared with the prior art, the syringe type gravity column, the solid phase extraction system or the purification method can be more convenient, faster and more automatic, can effectively remove other impurities in a sample, saves the sample preparation time and effectively reduces the dosage of a solvent, and is particularly suitable for proteins such as antibodies, fusion proteins and the like. A plurality of samples (2ml of filler can be used for treating 48 samples, and each sample can be used for preparing not less than 60mg of protein to meet the requirement of subsequent analysis and detection samples) can be purified simultaneously in a short time, belonging to a protein purification process with large preparation quantity, rapidness and high flux, and being suitable for large-scale research and development and application.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The% referred to in the following examples are the peak area percentages determined in SEC-HPLC, nRCE.

Example 1

1. Sample acquisition

Constructing an expression vector, introducing an exogenous gene GLP-1-Fc fusion gene, and transferring recombinant protein GLP-1-Fc (dulaglutide) obtained by expression of CHO (Chinese hamster ovary cells), which comprises the following specific steps: cell strain recovery, passage amplification, bioreactor culture, cell culture fluid harvesting, centrifugation, and filtration by using a sterilization filter cup to obtain culture supernatant containing recombinant protein.

2. Purification of

Step 1: a protective cap was placed over the outlet at the bottom of a syringe-type gravity column (empty column, maximum volume 10mL), and then a polypropylene material was sequentially added to the inner chamber to form a 0.2cm first filter layer, 2mL MabSelect Sure from Stutovan to form a 1cm filler layer, 1mL of a 0.2 vol% ethanol solution to form a 5cm buffer layer, and a polypropylene material was added to form a 0.2cm second filter layer. The loaded column was placed in the well of the chromatographic column holder, which was then placed in the appropriate position in the solid phase extractor (Resolvex A100).

Step 2: filling 6 pipelines of the solid phase extraction apparatus with six solutions A-E, and putting a chromatographic column frame placed in a gravity column in place; 5ml of equilibration solution (50mM Tris-HAc +150mM NaCl, pH 7.4) was added to the syringe gravity column, then pressurized and the procedure repeated once. Then adding 8ml of sample, and pressurizing; 5ml of an equilibrium solution (50mM Tris-HAc +150mM NaCl, pH 7.4) was added thereto, followed by pressurization; then 5ml of washing solution (20mM Na-citrate, pH5.5) was added and pressurized; then, 5ml of an eluate (20mM Na-citrate, pH3.5) was added thereto, and the mixture was pressurized to collect a purified sample. After adding corresponding solution each time, pressurizing by an automatic solid phase extractor, and controlling the operation pressure below 1 bar.

Samples were purified and submitted for SEC, nrCE mass analysis.

Comparative example 1

1. Sample acquisition

The same as in example 1.

2. Purification of

Step 1: a protective cap was placed over the outlet at the bottom of a syringe-type gravity column (empty column, maximum volume 10mL), and then a polypropylene material was sequentially added to the inner chamber to form a 0.2cm first filter layer and 2mL MabSelect Sure from Situofen to form a 1cm filler layer. And (3) placing the assembled column into a hole of a chromatographic column frame, and then placing the chromatographic column frame into a corresponding position in a solid phase extraction instrument.

Step 2: 10ml of equilibration solution (50mM Tris-HAc +150mM NaCl, pH 7.4) was added manually to the syringe-type gravity column, and the column was filled to above the filtration membrane and the procedure repeated once, waiting for the liquid to flow out automatically. Then adding 8ml of sample, staying for 5min for sufficient adsorption, waiting for the liquid to automatically flow out, and enabling the liquid level in the column to be above the filtering membrane; adding 10ml of balance solution (50mM Tris-HAc +150mM NaCl, pH 7.4), and waiting for the liquid to automatically flow out, wherein the liquid level in the column is above the filter membrane; then 10ml of washing solution (20mM Na-citrate, pH5.5) is added, the liquid is waited to flow out automatically, and the liquid level in the column is up to the upper part of the filter membrane; then 10ml of eluent (20mM Na-citrate, pH3.5) was added, and the mixture was left for 5min for sufficient elution, and a purified sample was collected.

The purified sample was simultaneously subjected to SEC-HPLC and nrCE-SDS quality analysis.

Comparative example 2

The sample was purified according to the method of example 1, except that step 2 was as follows:

10ml of equilibration solution (50mM Tris-HAc +150mM NaCl, pH 7.4) was added manually to the syringe-type gravity column, and the column was filled to above the filtration membrane and the procedure repeated once, waiting for the liquid to flow out automatically. Then adding 8ml of sample, staying for 5min for sufficient adsorption, waiting for the liquid to automatically flow out, and enabling the liquid level in the column to be above the filtering membrane; adding 10ml of balance solution (50mM Tris-HAc +150mM NaCl, pH 7.4), and waiting for the liquid to automatically flow out, wherein the liquid level in the column is above the filter membrane; then 10ml of washing solution (20mM Na-citrate, pH5.5) is added, the liquid is waited to flow out automatically, and the liquid level in the column is up to the upper part of the filter membrane; then 10ml of eluent (20mM Na-citrate, pH3.5) was added, and the mixture was left for 5min for sufficient elution, and a purified sample was collected.

Comparative example 3

The sample was purified according to the method of example 1 except that 0.2 vol% ethanol solution was not filled in step 1.

Comparative example 4

The sample was purified by the method of example 1 except that the amount of the ethanol solution added in step 1 was 2mL and the height of the buffer layer formed was 10 mm.

The protein recovery rate and the target protein main peak result obtained by different chromatography modes are compared and shown in table 1, and the analytical maps of example 1, comparative example 1 and comparative example 2 are shown in fig. 4-9. See table 2 for a comparison of the solution volumes consumed for example 1 and comparative example 2.

TABLE 1 results of the experiment

TABLE 2 statistics of solution consumption

If no second filter layer is arranged above the filler, the liquid glue surface in each step is seriously inclined (see figure 10), and the yield is lower by about 60 percent; the upper part of the filler is tightly attached to the second filter layer, so that liquid in the filler is pressed dry in the pressurizing process, subsequent protein solution cannot be adsorbed to the filler, and the experiment fails; if the buffer reaches 1mL or more, for example, 2mL (the height of the buffer layer is 10mL), this may result in an excessively large dead volume above the filler, so that a sample cannot be normally obtained by dilution of the solution concentration after addition of the solution; adding 0.5-1ml of buffer solution (corresponding to the height of 2.5-5 mm) above the filler can keep buffer during positive pressure operation, prevent failure of a filler pressure drying experiment and improve the yield and purity of protein to the maximum extent; the use of combining automatic solid phase extraction appearance can further improve protein purification yield purity, can practice thrift solution use amount 50 moreover, practices thrift the cost, therefore the volume of buffer solution, high optimization and the second filter layer addition, especially are fit for the syringe formula gravity column and combine automatic solid phase extraction appearance to use.

The results show that the protein purity and yield obtained by the purification method of the invention are obviously superior to those of the conventional manual syringe filter (example 1) which only comprises a filter layer and a filler layer. The invention provides a high-flux method, which improves the protein purification efficiency, saves the solution consumption, can completely meet the purification requirements of the yield and the purity of the protein and the obtained protein amount, and still ensures the purity and the yield of the protein on the premise of improving the purification efficiency.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.