CN112704913B

CN112704913B - Affinity chromatographic column and preparation method and application thereof

Info

Publication number: CN112704913B
Application number: CN202011573874.8A
Authority: CN
Inventors: 徐晓勇; 田向荣; 张丰
Original assignee: Hangzhou Huaan Biotechnology Co ltd
Current assignee: Hangzhou Huaan Biotechnology Co ltd
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2022-08-05
Anticipated expiration: 2040-12-25
Also published as: CN112704913A

Abstract

The invention provides an affinity chromatographic column and a preparation method and application thereof, relating to the technical field of biomacromolecule separation and purification. The stationary phase of the affinity chromatographic column is mainly prepared by coupling incubation reaction of biomacromolecules containing amino ligand treated by a denaturant, cyanogen bromide activated agarose chromatographic packing and a surfactant. According to the affinity chromatographic column, the surfactant is added into the fixed phase, so that the coupling of the biomacromolecule containing the amino ligand after the denaturant treatment and the cyanogen bromide activated agarose chromatographic packing can be effectively promoted, and the problems of agglomeration and blocking of a cyanogen bromide activated agarose chromatographic medium caused by the fact that the ligand after the denaturant treatment is easy to precipitate in the preparation process of the existing cyanogen bromide activated agarose affinity chromatographic column are effectively solved.

Description

Affinity chromatographic column and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological macromolecule separation and purification, in particular to an affinity chromatography column and a preparation method and application thereof.

Background

At present, proteins, polypeptides, nucleic acids and other ligands containing amino groups are coupled to a purification medium filler in the market, and cyanogen bromide is mainly used for activating an agarose chromatography medium. Cyanogen bromide activated agarose chromatography medium is an activated intermediate filler, which is activated cyanate ester group generated by reaction of cyanogen bromide and hydroxyl on agarose, the activated cyanate ester group is coupled with molecules with amino group such as protein, polypeptide and amino acid, and the coupling reaction can be carried out in aqueous phase or organic phase.

The cyanogen bromide activated filler in the current market has certain limitation when being coupled and reacted with other ligands containing amino groups, such as protein, polypeptide, nucleic acid and the like: the coupling reaction of the cyanogen bromide activated agarose chromatography medium requires that the ligand to be coupled is in a soluble state, otherwise, effective covalent coupling cannot be carried out. Therefore, during actual coupling, if the ligand to be coupled is denatured and redissolved, for example, 8M urea is used as a denaturant to dissolve the coupled ligands such as protein, polypeptide, and antibody for subsequent coupling, in the actual operation process, since the denatured and dissolved ligands need to be diluted with a certain volume of coupling buffer, the pH range required by the optimal coupling reaction is maintained to ensure the coupling reaction efficiency.

However, in the coupling process, the ligand precipitation phenomenon can occur in the coupling experiment process after the coupling buffer solution is added for dilution, so that cyanogen bromide activates agarose chromatography media to be agglomerated/blocked, and effective coupling can not be carried out. Therefore, it is necessary and urgent to develop a new affinity chromatography column to solve the problem that the ligand treated by the denaturant is easy to precipitate during the preparation of the existing cyanogen bromide activated agarose affinity chromatography column, which further causes the agglomeration/blocking of the cyanogen bromide activated agarose chromatography medium.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide an affinity chromatography column, wherein the stationary phase of the affinity chromatography column is mainly prepared by coupling incubation reaction of biomacromolecules containing amino ligand treated by a denaturant, cyanogen bromide activated agarose chromatography filler and a surfactant. The affinity chromatographic column effectively solves the problem that the ligand treated by the denaturant is easy to generate precipitation in the preparation process of the existing cyanogen bromide activated agarose affinity chromatographic column, so that the cyanogen bromide activated agarose chromatographic medium is agglomerated/blocked.

The second purpose of the invention is to provide a preparation method of the affinity chromatographic column.

The third objective of the present invention is to provide an application of an affinity chromatography column, which can be widely applied in the separation and purification process of antibodies or proteins.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the stationary phase of the affinity chromatographic column is mainly prepared by coupling incubation reaction of biomacromolecules containing amino ligand treated by a denaturant, cyanogen bromide activated agarose chromatographic packing and a surfactant.

Further, the surfactant comprises sodium lauryl sarcosinate;

preferably, the surfactant is a solution of sodium lauryl sarcosinate with a mass concentration of 0.5 wt%.

Furthermore, the mass of the cyanogen bromide activated agarose chromatography filler in the fixed phase of the affinity chromatography column is 0.1-0.3g, and the mass concentration of the sodium dodecyl sarcosinate solution is 0.5-1%.

Further, the amino ligand-containing biomacromolecule comprises at least one of a protein, a polypeptide and an antibody.

Further, the denaturant-treated denaturant includes at least one of urea, guanidine hydrochloride, and dimethylformamide.

Furthermore, the dosage of biomacromolecules containing amino ligands after being treated by a denaturant in a fixed phase of the affinity chromatography column is 1-5 mg, and the dosage of the cyanogen bromide activated agarose chromatography filler is 0.1-0.3 g.

The invention provides a preparation method of the affinity chromatographic column, which comprises the following steps:

performing coupling incubation reaction on the biomacromolecule containing the amino ligand, the cyanogen bromide activated agarose chromatographic packing and the surfactant which are treated by the denaturant in a chromatographic column hollow column to obtain a reaction column with a stationary phase, and then sequentially washing and sealing the reaction column to obtain the affinity chromatographic column.

Further, the coupling incubation reaction comprises the step of mixing the biomacromolecule containing the amino ligand treated by the denaturant, the cyanogen bromide activated agarose chromatography filler and the surfactant uniformly and then carrying out a shaking reaction for 1-2 hours.

Further, the blocking solution for blocking treatment is 0.1M Tris-HCl solution with pH of 8.0.

The invention provides an application of the affinity chromatographic column in separation and purification of antibodies or proteins.

Compared with the prior art, the invention has the beneficial effects that:

the stationary phase of the affinity chromatographic column is mainly prepared by coupling incubation reaction of biomacromolecules containing amino ligand treated by a denaturant, cyanogen bromide activated agarose chromatographic packing and a surfactant. The affinity chromatographic column is characterized in that a surfactant is added into a fixed phase, and sodium dodecyl sarcosinate is a strong anion denaturant and can denature protein at high concentration, but can be used as a molecular chaperone to prevent reaggregation and promote folding at low concentration to play a role in solubilization, so that the coupling of biomacromolecules containing an amino ligand treated by the denaturant and cyanogen bromide activated agarose chromatographic packing can be effectively promoted, and the problem of agglomeration/blocking of cyanogen bromide activated agarose chromatographic media caused by the fact that the ligand treated by the denaturant is easy to precipitate in the preparation process of the existing cyanogen bromide activated agarose affinity chromatographic column is effectively solved.

The preparation method of the affinity chromatographic column provided by the invention comprises the steps of carrying out coupling incubation reaction on the biomacromolecule containing the amino ligand, the cyanogen bromide activated agarose chromatographic packing and the surfactant which are treated by the denaturant in a chromatographic column hollow column to obtain a reaction column with a stationary phase, and then sequentially washing and sealing the reaction column to obtain the affinity chromatographic column. The preparation method has the advantage of easy preparation operation.

The affinity chromatographic column provided by the invention can be widely applied to the separation and purification process of biological macromolecules, such as antibodies or proteins.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

According to one aspect of the invention, the stationary phase of the affinity chromatography column is mainly prepared from biomacromolecules containing amino ligand treated by a denaturant, cyanogen bromide activated agarose chromatography filler and a surfactant through coupling incubation reaction.

The stationary phase of the affinity chromatographic column is mainly prepared by coupling incubation reaction of biomacromolecules containing amino ligands, cyanogen bromide activated agarose chromatographic packing and a surfactant which are treated by a denaturant. The affinity chromatographic column is characterized in that a surfactant is added into a fixed phase, and sodium dodecyl sarcosinate is a strong anion denaturant and can denature protein at high concentration, but can be used as a molecular chaperone to prevent reaggregation and promote folding at low concentration to play a role in solubilization, so that the coupling of biomacromolecules containing an amino ligand treated by the denaturant and cyanogen bromide activated agarose chromatographic packing can be effectively promoted, and the problem of agglomeration/blocking of cyanogen bromide activated agarose chromatographic media caused by the fact that the ligand treated by the denaturant is easy to precipitate in the preparation process of the existing cyanogen bromide activated agarose affinity chromatographic column is effectively solved.

In a preferred embodiment of the present invention, the surfactant comprises sodium lauryl sarcosinate;

In the preferred embodiment, the cyanogen bromide activated Sepharose chromatography packing is generally used in an amount of 0.1-0.3g in the stationary phase of the affinity column, since it is in a dry powder state, and after weighing, the Sepharose is first swollen to a homogeneous state by water absorption with 1mM dilute hydrochloric acid, and then replaced with a coupling buffer, generally a coupling buffer plus the protein solution to be coupled in a volume of about 10ml, and then sarcosyl is added to the column to a final concentration of 0.5-1%.

In a preferred embodiment of the present invention, the amino ligand-containing biological macromolecule comprises at least one of a protein, a polypeptide, and an antibody.

In a preferred embodiment of the present invention, the denaturant treated with the denaturant treatment includes at least one of urea, guanidine hydrochloride, and dimethylformamide.

In a preferred embodiment, the biomacromolecule having a part of the amino group-containing ligand is not in a soluble state, and when the biomacromolecule is used as a ligand for preparing an affinity chromatography column, it is necessary to dissolve the biomacromolecule using a denaturing agent and then prepare the biomacromolecule.

In a preferred embodiment of the invention, the dosage of the biomacromolecule containing the amino ligand after the treatment of the denaturant in the fixed phase of the affinity chromatography column is 1-5 mg, and the dosage of the cyanogen bromide activated agarose chromatography filler is 0.1-0.3 g.

According to an aspect of the present invention, a method for preparing the above affinity chromatography column comprises the following steps:

In a preferred embodiment of the invention, the coupling incubation reaction comprises mixing the biomacromolecule containing the amino ligand treated by the denaturant, the cyanogen bromide activated agarose chromatography filler and the surfactant uniformly, and then performing a shaking reaction for 1-2 hours.

In a preferred embodiment of the present invention, the blocking solution for the blocking treatment is a 0.1M Tris-HCl solution with pH 8.0.

As a preferred embodiment, the above 0.1M Tris-HCl solution with pH8.0 can block active groups in the column packing to reduce nonspecific binding of the affinity column.

According to one aspect of the invention, the affinity chromatography column is used for separating and purifying antibody or protein.

The affinity chromatographic column provided by the invention can be widely applied to the separation and purification process of antibodies or proteins.

The technical solution of the present invention will be further described with reference to examples and comparative examples.

Example 1

Firstly, preparing a solution:

1. amino group-containing ligand solution: the denaturant used in this example was 4M urea, and the amount of protein used was 2mg, at a concentration of 1 mg/ml.

The preparation method comprises the following steps: the concentration of the amino ligand-containing protein to be coupled is 2mg/ml, the dosage is 2ml, the protein buffer solution is 4M urea, and the pH value is 7.4.

2. 1mM hydrochloric acid solution at pH 3.0: 0.1mL of concentrated hydrochloric acid is added with 1.1mLUP of water and mixed evenly to prepare 1M dilute hydrochloric acid. Then, 1mL of the diluted hydrochloric acid was diluted to 1L to prepare 1mM diluted hydrochloric acid, and the pH was measured to be 3.0.

3. 0.1M Tris-HCl blocking solution at pH 8.0: 6.057g Tris was weighed into 400mL UP water and adjusted to pH8.0 with HCl to 500 mL.

4. Circulating buffer solution a: 4.1g of anhydrous CH are weighed ₃ COONa, 14.61g NaCl is dissolved in 490mLUP water, the pH value is adjusted to 4.0 by glacial acetic acid, and the volume is adjusted to 500mL after uniform mixing.

5. Circulating buffer solution B: 6.057g Tris and 14.61g NaCl are weighed and dissolved in 490mLUP water, the pH value is adjusted to 8.0 by hydrochloric acid, and the volume is adjusted to 500ml after uniform mixing.

6. Coupling buffer: 8.4g of sodium bicarbonate and 29.22g of sodium chloride, and dissolving and adjusting the pH value to 1L.

7. Preparation of cyanogen bromide activated agarose gel: 0.2g of cyanogen bromide activated Sepharose dry powder was weighed into 15mL of a dry clean conical centrifuge tube. To 0.2g of the weighed dry powder, 10mL of 1mM HCl pH3.0 was added and placed on a rotary homogenizer to be shaken for 10min to be completely dissolved.

Secondly, preparing an affinity chromatographic column:

A. transferring the completely dissolved cyanogen bromide activated agarose gel to a 3mL chromatographic column, washing the gel column by using 3 column volumes of coupling buffer solution, emptying the liquid in the gel column, and plugging a sealing plug at the upper end and the lower end of the gel column. Mixing the prepared ligand solution containing amino to be coupled with cyanogen bromide activated agarose gel (the used cyanogen bromide activated agarose gel is 0.2g, the 2ml of the ligand solution containing amino to be coupled and the amount of protein are 2mg), placing the mixture on a rotary mixer for gentle shaking for 1 hour, adding a sodium dodecyl sarcosinate solution with the final concentration of 0.5% (namely, 0.05g of sodium dodecyl sarcosinate is added into 10ml of the solution) to promote the dissolution of the ligand to be coupled, and continuing incubating and coupling for 1 hour after the agglomerated gel is dispersed into suspended particles.

B. Washing: the gel column was washed with 3 column volumes of coupling buffer.

C. And (3) sealing: blocking with 0.1M Tris-HCl, pH8.0, and placing on a rotary mixer to shake gently for 2 h. The sealing liquid is replaced every half hour, and then sealing is continued to ensure complete sealing.

D. Ligand cleaning: weak ligand binding was removed by washing with cycling buffers A, B for a total of 3 cycles. After the end of the cycle wash the gel was washed with 3 column volumes of coupling buffer.

E. And (3) column preservation: draining the gel column, adding 1mL of 20% ethanol into the gel column, and storing at 4 deg.C to obtain affinity chromatography column.

Comparative example 1

The comparative example, except for the preparation of affinity chromatography column, step a was:

"transfer the fully dissolved cyanogen bromide activated Sepharose to a 3mL chromatography column, wash the gel column with 3 column volumes of coupling buffer, empty the gel column, and plug the lower end of the stopper. Mixing the prepared amino-containing ligand solution to be coupled with cyanogen bromide activated agarose gel (0.2 g of cyanogen bromide activated agarose gel, 2ml of amino-containing ligand solution to be coupled and 2mg of protein), and placing the mixture on a rotary mixer for incubation and coupling for 2h with gentle shaking. "in addition, the rest of the examples are the same.

The present comparative example differs from example 1 in that the fixed phase of the affinity chromatography column of the present comparative example does not contain a solution of sarcosyl.

Experimental example 1

In order to show that the affinity chromatography column prepared by the method can effectively solve the problem that the ligand treated by the denaturant is easy to precipitate in the preparation process of the existing cyanogen bromide activated agarose affinity chromatography column to cause the cyanogen bromide activated agarose chromatography medium to agglomerate/block, the affinity chromatography columns prepared by the embodiment 1 and the comparative example 1 are adopted to carry out chromatographic separation on serum and a purified IgG antibody, and carry out ELISA detection on the separated and purified serum and IgG antibody, and the method specifically comprises the following steps:

(1) and a sample acquisition method: providing amino-containing ligand with protein concentration of 1mg/ml, taking 0.5ml, and mixing with immune adjuvant (complete adjuvant for rabbit first immunization and incomplete adjuvant for later immunization) in a volume ratio of 1:1 after mixing, immunization was performed by subcutaneous multiple injections in rabbits.

The dosage of the first immune protein solution is 0.5ml, and the volume of the rest 3 immune protein solutions is 0.25 ml.

After 4 times of immunization, rabbit serum is collected, the serum titer is detected, and the qualified rabbit serum is used as a serum sample to be purified.

(2) And an antibody purification method:

A. column balancing: the affinity column was washed thoroughly with 20ml of equilibration buffer (0.01M PBS, pH 7.4).

B. Sample treatment: taking 6ml of a sample to be purified in a centrifuge tube, carrying out suction filtration by using a filter membrane of 0.45um, and then adding an equilibrium buffer solution with the same volume as that of the sample to be purified for dilution.

C. Loading: the diluted sample is mixed with agarose gel coupled with ligand, and incubated on a shaking bed for 30-45 min.

D. Collecting antibodies: after the incubation was complete, the agarose gel was transferred to an empty column of the chromatography column, which was washed with 20ml of equilibration buffer. The antibody was then eluted with elution buffer (pH 2.7,0.2M glycine) by adding 2ml of each eluent to the column, incubating for 5min and collecting the antibody for a total of 3 elutions.

E. Adjusting the pH value to 7.0 +/-0.5 in time by using 1M Tris-HCl in the antibody collection process. After antibody collection, the pH was adjusted to 7.5 and stored at 4 ℃.

F. Washing and preserving the affinity column: the affinity column was washed with 20ml of equilibration buffer (0.01M PBS, pH 7.4), 3 column volumes of 1M NaCl, then 3 column volumes of equilibration buffer (0.01M PBS, pH 7.4), after which 1ml of 20% ethanol was added, the seal was sealed and the column was stored in a refrigerator at 4 ℃.

(3) ELISA detection of purified antibodies:

A. coating: the protein solution containing the amino ligand was diluted to 1. mu.g/mL with the coating solution, and 50. mu.l/well was added to the microplate, covered with a lid, and coated overnight at 4 ℃.

B. And (3) sealing: and (3) drying the liquid in the hole, adding 1% BSA (bovine serum albumin) into the enzyme label plate in an amount of 150. mu.l/well, covering the cover, and placing the enzyme label plate into an electric heating constant temperature incubator at 37 ℃ for constant temperature sealing for 1 h.

C. Adding a primary antibody: and (3) drying liquid in the holes, diluting the purified antibody with 1% BSA according to the ratio of 1:250, 1:1000, 1:4000, 1:16000, 1:64000, 1:256000 and 1:1024000, sequentially adding the antibody into the same column of the 1 st to 6 th holes in the ELISA plate in the amount of 50. mu.l/well, adding 1% BSA into the 7 th hole as a negative control, repeating the holes for 1 time for each dilution gradient, covering the cover, and putting the cover in a constant-temperature incubator at 37 ℃ for incubation for 45 min.

D. Adding a secondary antibody: spin-drying the primary antibody, adding a washing solution (1 XTSST) into the ELISA plate according to the amount of 180 microliter/well, and washing the ELISA plate for 2 times. GAR-HRP was diluted to working concentration with 1% BSA (1:25000), 50. mu.l/well was added to the microplate, the lid was closed, and the plate was incubated in a 37 ℃ incubator for 30 min.

E. Color development, stop and read: discarding liquid in the hole, adding a washing solution into the ELISA plate according to the amount of 180 mul/well, and washing the ELISA plate for 3 times; adding 50 μ l of newly prepared TMB chromogenic substrate into each reaction hole, and incubating at constant temperature of 37 ℃ for 10 min; the reaction was then stopped by the addition of 90. mu.l/well of stop solution and the OD at 450nm was determined on a microplate reader.

Specific results are shown in tables 1 and 2 below.

Table 1 shows the results of titer test of "purified IgG antibody and unpurified serum" using the affinity chromatography resin prepared in example 1 of the present invention.

Table 1:

table 2 is a table showing the results of titer test of "purified IgG antibody and unpurified serum" using the affinity chromatography resin prepared in comparative example 1 of the present invention.

Table 2:

as is apparent from the data of tables 1 and 2 above, when the purified antibody titer was measured in the purification using the affinity chromatography columns of comparative example 1 and example 1, it was revealed that only a small amount of antigen was covalently coupled to cyanogen bromide-activated agarose gel during the preparation of the affinity chromatography column of comparative example 1 due to the occurrence of the agglomeration phenomenon, and thus only a small amount of antibody was bound during the purification.

While the affinity chromatographic column of example 1, when the clumping phenomenon occurs, added with a final concentration of 0.5% sarcosyl, promoted protein refolding and prevented aggregation, increased the solubility of ligand protein containing amino groups, improved the ratio of protein covalent coupling to cyanogen bromide activated agarose gel, thus in the affinity purification process, can bind more antibodies.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An affinity chromatographic column is characterized in that a stationary phase of the affinity chromatographic column is mainly prepared by coupling incubation reaction of biomacromolecules containing amino ligand treated by a denaturant, cyanogen bromide activated agarose chromatographic packing and a surfactant;

the surfactant is a sodium dodecyl sarcosinate solution with the mass concentration of 0.5-1%;

the dosage of the cyanogen bromide activated agarose chromatography filler in the fixed phase of the affinity chromatography column is 0.1-0.3g, because the agarose chromatography filler is in a dry powder state, after the agarose chromatography filler is weighed, 1mM dilute hydrochloric acid is firstly used for enabling agarose to absorb water and swell into a uniform state, then the agarose is replaced into a coupling buffer solution, the volume of the coupling buffer solution and a protein solution to be coupled is 10ml, then sodium dodecyl sarcosinate is added, and the final concentration of the sodium dodecyl sarcosinate is 0.5-1%.

2. The affinity chromatography column of claim 1, wherein the amino ligand-containing biomacromolecule comprises at least one of a protein, a polypeptide, and an antibody.

3. The affinity chromatography column of claim 1, wherein the denaturant-treated denaturant comprises at least one of urea, guanidine hydrochloride, and dimethylformamide.

4. The affinity chromatography column of claim 1, wherein the amount of biomacromolecule containing amino ligand after denaturant treatment in the stationary phase of the affinity chromatography column is 1-5 mg, and the amount of cyanogen bromide activated agarose chromatography filler is 0.1-0.3 g.

5. A method for preparing an affinity chromatography column according to any one of claims 1 to 4, comprising the steps of:

6. The preparation method of the affinity chromatography column according to claim 5, wherein the coupling incubation reaction comprises mixing the denaturant-treated biological macromolecule containing the amino ligand, the cyanogen bromide-activated agarose chromatography filler and the surfactant uniformly, and then performing a shaking reaction for 1-2 hours.

7. The method for preparing an affinity chromatography column according to claim 6, wherein the blocking solution for blocking is Tris-HCl solution.

8. Use of an affinity chromatography column according to any one of claims 1 to 4 for the separation and purification of biological macromolecules.

9. Use of an affinity chromatography column according to claim 8 for the separation and purification of biological macromolecules, wherein the biological macromolecules comprise antibodies or proteins.