CN117720604A - Oligonucleotide desalination process - Google Patents
Oligonucleotide desalination process Download PDFInfo
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- CN117720604A CN117720604A CN202311510781.4A CN202311510781A CN117720604A CN 117720604 A CN117720604 A CN 117720604A CN 202311510781 A CN202311510781 A CN 202311510781A CN 117720604 A CN117720604 A CN 117720604A
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Abstract
The invention relates to the field of biochemistry and bioengineering, and particularly discloses an oligonucleotide desalting process which comprises centrifugal pretreatment, secondary centrifugation, separation and elution, desalting and purification treatment, further separation and elution and dehydration and drying treatment. The invention uses affinity chromatographic column to desalt and purify, fills magnetic affinity group solid phase adsorption material into the affinity column, removes salt impurity by utilizing the specific combination of oligonucleotide, separates the oligonucleotide target and salt impurity by means of external magnetic field, and finally elutes to obtain purified high concentration oligonucleotide. The process is simple and convenient, can purify the oligonucleotide more efficiently while the oligonucleotide desalting process is simple and convenient, can meet the industrial requirements more accurately, and promotes the development of scientific research and biomedical fields.
Description
Technical Field
The present invention relates to the fields of biochemistry and bioengineering, and more particularly to an oligonucleotide desalting process.
Background
Oligonucleotides are short single-or double-stranded DNA or RNA fragments and find wide application in the fields of biology and genetic engineering. Oligonucleotides are typically synthesized in the laboratory and subjected to multiple purification steps during the course of the experiment to ensure their biological activity and stability. During the synthesis and purification of oligonucleotides, various chemicals, such as salts, organic solvents, etc., are often used. These reagents may negatively affect the biological activity and stability of the oligonucleotide and therefore need to be removed during the course of the experiment. In addition, salts may affect the solubility and stability of the oligonucleotides, and thus removal of salts from the sample is often required before further experiments can be performed. Traditional oligonucleotide desalting techniques include dialysis, gel filtration, ion exchange, and liquid chromatography. These methods each have advantages and disadvantages, such as the long time required for dialysis and gel filtration, and the possible inability to remove all salts; ion exchange requires specific resins and may result in oligonucleotide loss; liquid chromatography requires expensive instrumentation and operating experience. Accordingly, there is a need for a faster, efficient and simple oligonucleotide desalting process that ameliorates the limitations of the prior art. This will help to improve laboratory work efficiency and reduce experimental costs.
Magnetic affinity group desalting oligonucleotides are an efficient technique for separating and purifying oligonucleotide samples that utilize magnetic affinity groups as solid phase adsorption materials with the ability to specifically bind target oligonucleotides. The magnetic affinity group is a magnetic particle or microsphere material containing a ligand of a specific affinity. These affinity groups may interact with specific sequences or structures of the oligonucleotides to achieve highly selective adsorption of the target oligonucleotides. Common affinity groups include DNA or RNA sequences complementary to oligonucleotides, antibodies, proteins, or other biomolecules. The principle of desalting and purifying magnetic affinity group desalting oligonucleotides is based on specific interactions of biomolecules. When the oligonucleotide sample passes through the magnetic affinity group, the target oligonucleotide is specifically bound to the ligand on the affinity group, while other non-target substances are eluted, thereby realizing purification and separation.
This technique is extremely selective in that the affinity groups are highly compatible with a particular oligonucleotide sequence or structure, allowing efficient enrichment of the target oligonucleotide from a complex mixture, reducing the presence of impurities.
Disclosure of Invention
In order to achieve the above purpose, the present invention provides the following technical solutions:
an oligonucleotide desalting process comprising the steps of:
step one, centrifugal pretreatment: dissolving the oligonucleotide sample in deionized water, centrifuging in a centrifuge, and centrifuging at 5000r/h for 15min, wherein the supernatant contains the separated nucleic acid;
step two, secondary centrifugation: transferring the supernatant separated in the centrifugal machine into another clean centrifugal tube, and repeating the operation to perform secondary centrifugation so as to remove particles and impurities;
step three, separating and eluting: eluting the nucleic acid using a low salt buffer to separate the oligonucleotides and other impurities;
step four, desalting and purifying treatment: pouring the treated oligonucleotide sample into a washed affinity chromatographic column, enabling the oligonucleotide sample to be free in a solid phase and a mobile phase in the affinity column, performing chromatographic analysis, and adsorbing the oligonucleotide in the column through the specific binding between a magnetic affinity group in a filling material of the affinity chromatographic column and the oligonucleotide;
step five, further separating and eluting: collecting the effluent containing the purified oligonucleotide after the adsorption of the magnetic affinity group, and performing elution separation treatment;
step six, dehydration and drying treatment: in order to facilitate storage of the purified oligonucleotide, the product needs to be collected and dehydrated.
As a further scheme of the invention, the step four of desalination and purification treatment adopts a filling material which is an affinity group solid-phase adsorption material of a composite affinity chromatographic column formed by a ligand combined with DNA and RNA target molecules complementary with oligonucleotides, the filling concentration is 35-50%, and a matrix solidified by the filling material is magnetic ferrite particles; the hydrophobicity of the surface is increased through a surface coating process, and the coating material is polyethyleneimine; the mobile phase in the affinity column is isopropanol solvent with concentration of 20%, and the addition volume ratio of the solid phase to the mobile phase is 6:4.
As a further scheme of the invention, the magnetic microsphere particles are introduced with carboxyl, thio, ketone group and other functional groups by adding pentanedione as an activating agent; adding bamboo charcoal as blocking agent to reduce nonspecific nucleic acid binding; the addition amount accounts for 0.5 percent and 0.1 percent of the mass of the mixed solution respectively.
As a further scheme of the invention, the outer part of the column structure of the selected affinity chromatographic column is made of cylindrical glass material; the inside of the affinity column takes injection-molded silicon-based materials as carriers, and is generally silica gel.
As a further scheme of the invention, the step three separation elution and the step five separation elution are carried out, the low-salt buffer solution for elution is specifically Tris-HCL solution, the concentration of the solution is 20mM to 50mM, the pH value is 7.8-8.9, and the elution step is carried out under the room temperature condition.
As a further scheme of the invention, the reaction time of the desalination and purification treatment in the step four comprises the balance time and the combination time, and the reaction time and the combination time are carried out at the room temperature of 25 ℃ for 0.5 to 1.5 hours and the PH value of 7.0 to 8.0.
As a further scheme of the invention, the step five further separation elution and the step six dehydration drying treatment are specifically operated as follows:
step Q1, attracting the magnetic adsorption material and the oligonucleotides attached thereon to the wall of the centrifuge tube or the side wall of the vessel using an external magnetic field (magnet), separating out the mixture together, centering the supernatant (containing salts and other ions), removing the supernatant from the affinity column, leaving the oligonucleotides attached to the magnetic particles;
step Q2, eluting the target object again, and extracting the oligonucleotides on the magnetic adsorption material to remove any residual salt and other impurities, thereby obtaining an oligonucleotide solution with higher purity;
and Q3, collecting the eluted oligonucleotide solution, placing the solution in a vacuum concentrator, removing water under reduced pressure, purifying the desalted oligonucleotide, adding the fluorescent-labeled primer into the purified oligonucleotide, and storing the oligonucleotide under the conditions of drying, pH value of 7-8, no illumination and low temperature.
As a further aspect of the present invention, the affinity chromatography column instrument functions further include data collection and purity detection functions.
The oligonucleotide desalting process has the beneficial effects that:
1. the method can efficiently separate the oligonucleotide and the salt, and ensure the high purity of the oligonucleotide. The affinity groups on the magnetic material bind to the oligonucleotides, allowing for rapid, selective removal of salts from the oligonucleotides. Methods using magnetic chromatography affinity columns are generally milder and do not cause degradation or damage to the oligonucleotides, helping to reduce cross-reactions with other impurities.
2. The desalting of the magnetic material can be completed in a short time, and is therefore suitable for high-throughput experiments. The rapid separation properties of the magnetic material help to improve the efficiency of the experiment. Desalting of magnetic materials generally results in less waste and reduced environmental impact relative to single use column chromatography columns.
Drawings
FIG. 1 is a process flow diagram of an oligonucleotide desalting process according to the invention.
Detailed Description
The technical solutions of the present embodiment will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
An oligonucleotide desalting process comprising the steps of:
step one, centrifugal pretreatment: dissolving the oligonucleotide sample in deionized water, centrifuging in a centrifuge, and centrifuging at 5000r/h for 15min, wherein the supernatant contains the separated nucleic acid;
step two, secondary centrifugation: transferring the supernatant separated in the centrifugal machine into another clean centrifugal tube, and repeating the operation to perform secondary centrifugation so as to remove particles and impurities;
step three, separating and eluting: eluting the nucleic acid using a low salt buffer to separate the oligonucleotides and other impurities;
step four, desalting and purifying treatment: pouring the treated oligonucleotide sample into a washed affinity chromatographic column, enabling the oligonucleotide sample to be free in a solid phase and a mobile phase in the affinity column, performing chromatographic analysis, and adsorbing the oligonucleotide in the column through the specific binding between a magnetic affinity group in a filling material of the affinity chromatographic column and the oligonucleotide;
step five, further separating and eluting: collecting the effluent containing the purified oligonucleotide after the adsorption of the magnetic affinity group, and performing elution separation treatment;
step six, dehydration and drying treatment: in order to facilitate storage of the purified oligonucleotide, the product needs to be collected and dehydrated.
The fourth step of desalting and purifying treatment, namely selecting a filling material which is a ligand combined with DNA and RNA target molecules complementary with the oligonucleotides to form an affinity group solid-phase adsorption material of the composite affinity chromatographic column, wherein the filling concentration is 35%, and a matrix solidified by the filling material is magnetic ferrite particles; the hydrophobicity of the surface is increased through a surface coating process, and the coating material is polyethyleneimine; the mobile phase in the affinity column is isopropanol solvent with concentration of 20%, and the addition volume ratio of the solid phase to the mobile phase is 6:4.
Wherein, the magnetic microsphere particles are introduced with carboxyl, sulfenyl, ketone group and other functional groups by adding pentanedione as an activating agent; adding bamboo charcoal as blocking agent to reduce nonspecific nucleic acid binding; the addition amount accounts for 0.5 percent and 0.1 percent of the mass of the mixed solution respectively.
The step four is desalting and purifying treatment, and the outside of the column structure of the selected affinity chromatographic column is made of cylindrical glass; the inside of the affinity column takes injection-molded silicon-based materials as carriers, and is generally silica gel.
The low-salt buffer solution for elution is specifically Tris-HCL solution, the concentration of the solution is 30mM, the pH value is 7.8, and the elution step is carried out under the condition of room temperature.
The reaction time of the desalination and purification treatment in the step four comprises the balance time and the combination time, and the desalination and purification treatment is carried out at the room temperature of 25 ℃ and the pH value of 7.8 in 0.5 hour.
Wherein, the fifth step further separates and elutes and step six dehydrates the dry treatment, the concrete operation is as follows:
step Q1, attracting the magnetic adsorption material and the oligonucleotides attached thereon to the wall of the centrifuge tube or the side wall of the vessel using an external magnetic field (magnet), separating out the mixture together, centering the supernatant (containing salts and other ions), removing the supernatant from the affinity column, leaving the oligonucleotides attached to the magnetic particles;
step Q2, eluting the target object again, and extracting the oligonucleotides on the magnetic adsorption material to remove any residual salt and other impurities, thereby obtaining an oligonucleotide solution with higher purity;
and Q3, collecting the eluted oligonucleotide solution, placing the solution in a vacuum concentrator, removing water under reduced pressure, purifying the desalted oligonucleotide, adding the fluorescent-labeled primer into the purified oligonucleotide, and storing the oligonucleotide under the conditions of drying, pH value of 7.8, no illumination and low temperature. The purity of the finally detected oligonucleotide after desalination and purification is as follows: 97.3%.
Example 2
An oligonucleotide desalting process comprising the steps of:
step one, centrifugal pretreatment: dissolving the oligonucleotide sample in deionized water, centrifuging in a centrifuge, and centrifuging at 5000r/h for 15min, wherein the supernatant contains the separated nucleic acid;
step two, secondary centrifugation: transferring the supernatant separated in the centrifugal machine into another clean centrifugal tube, and repeating the operation to perform secondary centrifugation so as to remove particles and impurities;
step three, separating and eluting: eluting the nucleic acid using a low salt buffer to separate the oligonucleotides and other impurities;
step four, desalting and purifying treatment: pouring the treated oligonucleotide sample into a washed affinity chromatographic column, enabling the oligonucleotide sample to be free in a solid phase and a mobile phase in the affinity column, performing chromatographic analysis, and adsorbing the oligonucleotide in the column through the specific binding between a magnetic affinity group in a filling material of the affinity chromatographic column and the oligonucleotide;
step five, further separating and eluting: collecting the effluent containing the purified oligonucleotide after the adsorption of the magnetic affinity group, and performing elution separation treatment;
step six, dehydration and drying treatment: in order to facilitate storage of the purified oligonucleotide, the product needs to be collected and dehydrated.
The fourth step of desalting and purifying treatment, namely selecting a filling material which is an affinity group solid-phase adsorption material of a composite affinity chromatographic column formed by a ligand combined with DNA and RNA target molecules complementary with the oligonucleotides, wherein the filling concentration is 50%, and a matrix solidified by the filling material is magnetic ferrite particles; the hydrophobicity of the surface is increased through a surface coating process, and the coating material is polyethyleneimine; the mobile phase in the affinity column is isopropanol solvent with concentration of 20%, and the addition volume ratio of the solid phase to the mobile phase is 6:4.
Wherein, the magnetic microsphere particles are introduced with carboxyl, sulfenyl, ketone group and other functional groups by adding pentanedione as an activating agent; adding bamboo charcoal as blocking agent to reduce nonspecific nucleic acid binding; the addition amount accounts for 0.5 percent and 0.1 percent of the mass of the mixed solution respectively.
The step four is desalting and purifying treatment, and the outside of the column structure of the selected affinity chromatographic column is made of cylindrical glass; the inside of the affinity column takes injection-molded silicon-based materials as carriers, and is generally silica gel.
The low-salt buffer solution for elution is specifically Tris-HCL solution, the concentration of the solution is 50mM, the pH value is 8.0, and the elution step is carried out under the condition of room temperature.
The reaction time of the desalination and purification treatment in the step four comprises the balance time and the combination time, and the desalination and purification treatment is carried out at the room temperature of 25 ℃ and the pH value of 8.0 in 1.5 hours.
Wherein, the fifth step further separates and elutes and step six dehydrates the dry treatment, the concrete operation is as follows:
step Q1, attracting the magnetic adsorption material and the oligonucleotides attached thereon to the wall of the centrifuge tube or the side wall of the vessel using an external magnetic field (magnet), separating out the mixture together, centering the supernatant (containing salts and other ions), removing the supernatant from the affinity column, leaving the oligonucleotides attached to the magnetic particles;
step Q2, eluting the target object again, and extracting the oligonucleotides on the magnetic adsorption material to remove any residual salt and other impurities, thereby obtaining an oligonucleotide solution with higher purity;
and Q3, collecting the eluted oligonucleotide solution, placing the solution in a vacuum concentrator, removing water under reduced pressure, purifying the desalted oligonucleotide, adding the fluorescent-labeled primer into the purified oligonucleotide, and storing the oligonucleotide under the conditions of drying, pH value of 8.0, no illumination and low temperature. The purity of the finally detected oligonucleotide after desalination and purification is as follows: 98.6%.
Example 3
An oligonucleotide desalting process comprising the steps of:
step one, centrifugal pretreatment: dissolving the oligonucleotide sample in deionized water, centrifuging in a centrifuge, and centrifuging at 5000r/h for 15min, wherein the supernatant contains the separated nucleic acid;
step two, secondary centrifugation: transferring the supernatant separated in the centrifugal machine into another clean centrifugal tube, and repeating the operation to perform secondary centrifugation so as to remove particles and impurities;
step three, separating and eluting: eluting the nucleic acid using a low salt buffer to separate the oligonucleotides and other impurities;
step four, desalting and purifying treatment: pouring the treated oligonucleotide sample into a washed affinity chromatographic column, enabling the oligonucleotide sample to be free in a solid phase and a mobile phase in the affinity column, performing chromatographic analysis, and adsorbing the oligonucleotide in the column through the specific binding between a magnetic affinity group in a filling material of the affinity chromatographic column and the oligonucleotide;
step five, further separating and eluting: collecting the effluent containing the purified oligonucleotide after the adsorption of the magnetic affinity group, and performing elution separation treatment;
step six, dehydration and drying treatment: in order to facilitate storage of the purified oligonucleotide, the product needs to be collected and dehydrated.
The fourth step of desalting and purifying treatment, namely selecting a filling material which is an affinity group solid-phase adsorption material of a composite affinity chromatographic column formed by a ligand combined with DNA and RNA target molecules complementary with the oligonucleotides, wherein the filling concentration is 50%, and a matrix solidified by the filling material is magnetic ferrite particles; the hydrophobicity of the surface is increased through a surface coating process, and the coating material is polyethyleneimine; the mobile phase in the affinity column is isopropanol solvent with concentration of 20%, and the addition volume ratio of the solid phase to the mobile phase is 6:4.
Wherein, the magnetic microsphere particles are introduced with carboxyl, sulfenyl, ketone group and other functional groups by adding pentanedione as an activating agent; adding bamboo charcoal as blocking agent to reduce nonspecific nucleic acid binding; the addition amount accounts for 0.5 percent and 0.1 percent of the mass of the mixed solution respectively.
The step four is desalting and purifying treatment, and the outside of the column structure of the selected affinity chromatographic column is made of cylindrical glass; the inside of the affinity column takes injection-molded silicon-based materials as carriers, and is generally silica gel.
The low-salt buffer solution for elution is specifically Tris-HCL solution, the concentration of the solution is 20mM, the pH value is 8.0, and the elution step is carried out under the condition of room temperature.
The reaction time of the desalination and purification treatment in the step four comprises the balance time and the combination time, and the desalination and purification treatment is carried out at the room temperature of 25 ℃ and the pH value of 8.0 in 0.5 hour.
Wherein, the fifth step further separates and elutes and step six dehydrates the dry treatment, the concrete operation is as follows:
step Q1, attracting the magnetic adsorption material and the oligonucleotides attached thereon to the wall of the centrifuge tube or the side wall of the vessel using an external magnetic field (magnet), separating out the mixture together, centering the supernatant (containing salts and other ions), removing the supernatant from the affinity column, leaving the oligonucleotides attached to the magnetic particles;
step Q2, eluting the target object again, and extracting the oligonucleotides on the magnetic adsorption material to remove any residual salt and other impurities, thereby obtaining an oligonucleotide solution with higher purity;
and Q3, collecting the eluted oligonucleotide solution, placing the solution in a vacuum concentrator, removing water under reduced pressure, purifying the desalted oligonucleotide, adding the fluorescent-labeled primer into the purified oligonucleotide, and storing the oligonucleotide under the conditions of drying, pH value of 8.0, no illumination and low temperature. The purity of the finally detected oligonucleotide after desalination and purification is as follows: 96.5%.
According to the above examples, it was found that the purity of the purified oligonucleotide was affected by the solid phase packing concentration, buffer concentration, reaction time and pH during desalting purification at 25℃at room temperature. Lower packing concentrations may provide more separation capacity, but may also reduce column capacity, reducing throughput; higher packing concentrations may increase sample capacity but may decrease separation efficiency. Lower concentrations of buffer may result in retention of some impurities during elution, affecting purity; higher concentrations of buffer generally help to elute the target oligonucleotide more efficiently, but care is taken to avoid excessive concentrations leading to non-specific elution. At the same time, the pH of the reaction time also affects the degree of completion of adsorption of the oligonucleotide by the affinity group. Table 1 shows the purity of the oligonucleotides obtained under different conditions in the examples of the invention.
Table 1 purity of oligonucleotides obtained under different conditions
In general, the oligonucleotide desalting process has the advantages of high efficiency, high selectivity and convenient separation, and is suitable for separating and purifying oligonucleotide samples to obtain high-quality oligonucleotides. Meanwhile, the performance and purity of the product can be further improved by improvement and optimization.
The foregoing is merely specific embodiments of the present application, but the scope of the present application is not limited thereto, and any person skilled in the art can easily think about changes or substitutions within the technical scope of the present application, and the changes and substitutions are intended to be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Finally: the foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (8)
1. An oligonucleotide desalting process, comprising the steps of:
step one, centrifugal pretreatment: dissolving the oligonucleotide sample in deionized water, centrifuging in a centrifuge, and centrifuging at 5000r/h for 15min, wherein the supernatant contains the separated nucleic acid;
step two, secondary centrifugation: transferring the supernatant separated in the centrifugal machine into another clean centrifugal tube, and repeating the operation to perform secondary centrifugation so as to remove particles and impurities;
step three, separating and eluting: eluting the nucleic acid using a low salt buffer to separate the oligonucleotides and other impurities;
step four, desalting and purifying treatment: pouring the treated oligonucleotide sample into a washed affinity chromatographic column, enabling the oligonucleotide sample to be free in a solid phase and a mobile phase in the affinity column, performing chromatographic analysis, and adsorbing the oligonucleotide in the column through the specific binding between a magnetic affinity group in a filling material of the affinity chromatographic column and the oligonucleotide;
step five, further separating and eluting: collecting the effluent containing the purified oligonucleotide after the adsorption of the magnetic affinity group, and performing elution separation treatment;
step six, dehydration and drying treatment: in order to facilitate storage of the purified oligonucleotide, the product needs to be collected and dehydrated.
2. The process for desalting oligonucleotides according to claim 1, wherein said step four of desalting and purifying treatment comprises selecting a packing material as an affinity group solid-phase adsorption material for forming a composite affinity chromatographic column by using a ligand binding with DNA and RNA target molecules complementary to the oligonucleotides, wherein the packing concentration is 35% -50%, and the matrix solidified by the packing material is magnetic ferrite particles; the hydrophobicity of the surface is increased through a surface coating process, and the coating material is polyethyleneimine; the mobile phase in the affinity column is isopropanol solvent with concentration of 20%, and the addition volume ratio of the solid phase to the mobile phase is 6:4.
3. The oligonucleotide desalting process according to claim 2, wherein the magnetic microsphere particles are introduced with carboxyl, thio and ketone groups by adding pentanedione as an activator; adding bamboo charcoal as blocking agent to reduce nonspecific nucleic acid binding; the addition amount accounts for 0.5 percent and 0.1 percent of the mass of the mixed solution respectively.
4. The oligonucleotide desalting process according to claim 1, wherein the outer part of the column structure of the selected affinity chromatographic column is made of cylindrical glass; the inside of the affinity column takes injection-molded silicon-based material as a carrier.
5. The process according to claim 1, wherein the step three separation and elution and the step five separation and elution are performed in a low salt buffer, specifically Tris-HCL solution, at a concentration of 20mM to 50mM and a ph of 7.8 to 8.9, and the elution step is performed at room temperature.
6. The process according to claim 1, wherein the reaction time of the purification treatment is 0.5 to 1.5 hours, at room temperature of 25℃and pH 7.0 to 8.0.
7. The oligonucleotide desalting process according to claim 1, wherein the step five further separation elution and the step six dehydration drying treatment are specifically performed as follows:
step Q1, attracting the magnetic adsorption material and the oligonucleotides attached on the magnetic adsorption material to the wall of a centrifugal tube or the side wall of a container by using an external magnetic field, separating out a mixture together, centering the supernatant, removing the supernatant in an affinity column, and leaving the oligonucleotides attached on the magnetic particles;
step Q2, eluting the target object again, and extracting the oligonucleotides on the magnetic adsorption material to remove any residual salt and other impurities, thereby obtaining an oligonucleotide solution with higher purity;
and Q3, collecting the eluted oligonucleotide solution, placing the solution in a vacuum concentrator, removing water under reduced pressure, purifying the desalted oligonucleotide, adding the fluorescent-labeled primer into the purified oligonucleotide, and storing the oligonucleotide under the conditions of drying, pH value of 7-8, no illumination and low temperature.
8. The oligonucleotide desalting process according to claim 1, wherein the affinity chromatography column instrument function further comprises a data collection and purity detection function.
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