CN114195885B

CN114195885B - Monoclonal antibody composition purification method

Info

Publication number: CN114195885B
Application number: CN202210032656.6A
Authority: CN
Inventors: 沈文婷; 郑曙剑
Original assignee: Hangzhou Clongene Biotech Co ltd
Current assignee: Hangzhou Clongene Biotech Co ltd
Priority date: 2022-01-12
Filing date: 2022-01-12
Publication date: 2024-04-26
Anticipated expiration: 2042-01-12
Also published as: CN114195885A

Abstract

The application relates to the field of biological pharmacy, in particular to a monoclonal antibody composition purification method, which comprises the following steps: s1, treating a monoclonal antibody composition through mesoporous silica microspheres, and removing lipid substances in the composition to obtain an intermediate; s2, further separating the intermediate by any one of an ammonium sulfate two-step method or an octanoic acid-ammonium sulfate two-step method to obtain a monoclonal antibody; wherein the monoclonal antibody composition comprises water, fat, impurity protein and monoclonal antibody. According to the technical scheme, the fat impurities in the monoclonal antibody composition can be effectively removed through the mesoporous silica microspheres, and the obtained final monoclonal antibody has higher purity and smaller loss.

Description

Monoclonal antibody composition purification method

Technical Field

The application relates to the field of biopharmaceuticals, in particular to a method for purifying a monoclonal antibody composition.

Background

Monoclonal antibodies have been of great use in the relevant fields of biopharmaceutical, biological detection and the like since their development. The preparation method of the monoclonal antibody is generally as follows: immunizing a mouse through an antigen, fusing spleen cells and myeloma cells of the mouse after immunization to obtain hybridoma cells, screening hybridoma cell strains with higher positivity in the hybridoma cells for subcloning culture, screening for multiple times to obtain hybridoma cells with highest positivity, planting the hybridoma cells in abdominal cavities of the mouse, and finally separating and purifying ascites to obtain monoclonal antibodies.

The existing ascites separation method has more types, and the common method is an ammonium sulfate two-step method or an octanoic acid-ammonium sulfate, and the principle is that different components are separated out respectively by controlling the concentration of the ammonium sulfate or the pH value of a system through the difference of isoelectric points of antibodies and impurity proteins, so that the separation is realized. The two methods are simple to operate, the material sources are cheaper, the method is suitable for industrial production, and the method is of great significance in further research at the present that the maximum capacity of the monoclonal antibody is only kilogram level. However, in addition to the impurity protein, there are some lipid substances in the ascites, which are difficult to separate from the antibody, and sometimes interfere with the separation of the impurity protein and the antibody.

Disclosure of Invention

In order to reduce the influence of lipid components in ascites on antibody separation, the application provides a monoclonal antibody composition purification method.

The monoclonal antibody composition purification method provided by the application specifically adopts the following technical scheme:

a method for purifying a monoclonal antibody composition comprising the steps of:

S1, treating a monoclonal antibody composition through mesoporous silica microspheres, and removing lipid substances in the composition to obtain an intermediate;

s2, further separating the intermediate by any one of an ammonium sulfate two-step method or an octanoic acid-ammonium sulfate two-step method to obtain a monoclonal antibody;

Wherein the monoclonal antibody composition comprises water, fat, impurity protein and monoclonal antibody.

The antibody composition can be cultured ascites of mice, or can be other mixed components containing lipid, impurity protein, even extracellular body and vesicle, and the components are complex. The treatment with mesoporous silica can effectively remove lipids from the antibody composition, thereby enhancing the subsequent purification effect.

The mesoporous silica has a surface with a more mesoporous structure, and the mesoporous structure generally has a pore diameter of 2-50 nm, and under the size, the adsorption capacity to antibodies is weaker, and the adsorption capacity to lipid components is stronger, so that the mesoporous silica has no obvious influence on the antibodies under the condition of effectively removing the lipid components, and therefore has a wide application prospect in actual production.

Optionally, the mesoporous silica microsphere is hollow mesoporous silica, and the average particle size is 50-100 nm.

The hollow mesoporous silica has better adsorptivity, and because the silica has a neutral charge structure, the silica is less prone to adsorb antibody components, and has better antibody purification effect and less loss.

Optionally, step S1 is specifically as follows: mixing mesoporous silica microsphere with monoclonal antibody composition for 5-10 min at 20-37 deg.c, and centrifuging to separate the mesoporous silica microsphere with adsorbed fat.

The whole parameters are selected to have better adsorptivity and less antibody loss, and belong to optimized parameters.

Optionally, the mesoporous silica microsphere is coated with a paramagnetic core structure.

The mesoporous silica particles with the core-shell structure formed by the paramagnetic core mesoporous silica shells can be separated in a magnetic adsorption mode after absorbing the lipid in the system, the separation is more convenient, and the loss of antibodies in the separation process is less. The paramagnetic core can be ferroferric oxide nano-particles or ferroferric oxide nano-particles

Alternatively, the average diameter of the paramagnetic core is 50 to 100nm.

After being coated, paramagnetic particles with the particle size of 5-40 nm have better stability as a whole, and the mesoporous layer has the advantages of better adsorption of lipid, difficult desorption and easier separation.

Optionally, in step S1, the mesoporous silica microspheres and the monoclonal antibody composition are mixed for 15 to 30 minutes at a mixing temperature of 20 to 37 ℃ and then the mesoporous silica microspheres adsorbed with fat are separated by magnetic attraction.

In the technical scheme, the lipid can be fully adsorbed, and the purification of the monoclonal antibody composition is facilitated.

Optionally, in step S1, after mixing mesoporous silica microspheres and monoclonal antibodies, carrying out oscillation treatment, wherein the oscillation frequency is 100-200 r/min, and the oscillation amplitude is 10-20 mm.

Through vibration, the adsorption of the antibody on the surface of the mesoporous silica microsphere is reduced, in the vibration process, the thermomechanical equilibrium state is easier to occur because of more sufficient contact, and in the thermomechanical equilibrium state, the adsorption of the lipid has higher priority, so that the loss of the antibody is further reduced.

Optionally, in step S1, a nonionic surfactant is added, wherein the mass of the nonionic surfactant is 0.5 to 1 time of the mass of the mesoporous silica microspheres.

In the technical scheme, the dispersibility of the mesoporous silica microspheres in the system can be improved by adding the nonionic surfactant, and the subsequent antibody sedimentation is not obviously affected.

Optionally, in the step S1, citric acid is also added, wherein the adding mass of the citric acid is 0.02-0.1 times of the mass of the mesoporous silica microspheres.

On the one hand, the addition of the citric acid provides a certain oxidation resistance, reduces the loss of the monoclonal antibody in the purification process, and meanwhile, the addition of a small amount of the citric acid does not have adverse effect on the subsequent separation. In addition, the citric acid can further improve the adsorption performance of the mesoporous silica microsphere on lipid and improve the separation effect.

Optionally, in step S1, a buffer solution is added, the pH value is controlled to be 6.8-7.4, and after the buffer solution is added, the mass concentration of the mesoporous silica microspheres in step S1 is controlled to be 2-10 mg/mL.

In the technical scheme, the whole pH value is adjusted through the buffer solution, so that on one hand, the stability of a system in the separation process can be improved, and on the other hand, after the pH value is controlled, the precipitation of the antibody can be reduced, and the loss of the antibody caused by adsorption or agglomeration can be reduced.

In summary, the application at least comprises the following beneficial effects:

1. In the application, the lipid in the monoclonal antibody composition is separated by adding mesoporous silica microspheres and utilizing the adsorption capacity of the mesoporous silica microspheres on the lipid, so that the purity of the monoclonal antibody after separation is improved, and the method is suitable for industrial production of the monoclonal antibody.

2. In the further arrangement of the application, hollow mesoporous silica is selected, so that the adsorption performance on lipid is better, the separation is more thorough, the subsequent residual lipid is less, and the required treatment time is less.

3. In another scheme of the application, mesoporous silica with a core-shell structure is selected, and paramagnetic materials are used as cores, so that magnetic adsorption separation can be realized, and the separation is more convenient.

4. In the present application, nonionic surfactant and citric acid may be added to further enhance the separation effect.

Detailed Description

The present application will be described in further detail with reference to examples.

In the following examples and comparative examples, the sources of some of the raw materials are shown in Table 1.

TABLE 1 partial Material data sheet

In the following examples, the monoclonal antibodies used were prepared by the following procedure.

1. The mice were immunoreacted.

CA125 antigen (1 mg/mL) was combined with Freund's complete adjuvant (see Table 1) at a ratio of 1:1 by a syringe, and emulsifying completely, selecting 6-8 week old BALB/c female mice for first subcutaneous immunization, immunizing 4 mice, and injecting 100ul of emulsified antigen into each mouse. Re-immunization with CA125 antigen (1 mg/mL) Freund's incomplete adjuvant at 1:1, and emulsifying to completely immunize the mice, wherein the immunization is performed once every 15 days, the subcutaneous and the intraperitoneal are alternately performed, the immunization is enhanced after the immunization is performed 3 times, and the cell fusion is performed on the 3 rd day.

2. Spleen cell and myeloma cell fusion

2.1 Preparation of feeder cells: the method comprises the steps of (1) taking eyeballs from mice without immune reaction, carrying out blood discharge and sacrifice, soaking in 75% alcohol for sterilization for 5min, fixing on a foam plate, cutting off the outer skin of the abdomen of the mice, exposing the peritoneum of the mice, injecting 5ml of DMEM serum-free culture medium preheated at 37 ℃ into the abdominal cavity, lightly rubbing the abdominal cavity of the mice for 1 min, suspending abdominal cavity cells, and sucking abdominal cavity liquid; cutting the chest of the mouse, grinding the thymus, filtering, collecting suspension, combining with peritoneal fluid, centrifuging, and re-suspending the precipitate with HAT complete culture solution to obtain feeder cell suspension.

2.2 Culture of mouse myeloma cells SP 2/0: the mouse myeloma cells SP2/0 are subjected to subculture by using a DMEM medium (formula shown in table 1) containing 10% FBS by volume fraction, the mouse myeloma cells SP2/0 are ensured to be in the logarithmic growth phase before cell fusion, and the liquid is changed on the previous day to ensure that the growth state is good for cell fusion.

2.3 Spleen cell preparation: the immunized BALB/c female mice were sacrificed, sterilized in 75% alcohol for 5min, and then the spleens were aseptically removed. Washing with DMEM serum-free medium (formula shown in Table 1) once, placing on stainless steel screen in a plate, grinding into cell suspension with syringe needle core, centrifuging after copper screen filtration, discarding supernatant, and centrifuging again with serum-free culture medium for use.

2.4 Spleen cells fused with myeloma cells: the spleen cells and the mouse myeloma cells SP2/0 are uniformly mixed, the cells are centrifugally washed, the supernatant is removed, the mixture is placed in a water bath with the temperature of 37 ℃ for preheating for 1min, 1ml of PEG 4000 with the temperature of 37 ℃ for 90s are added, and then the mixture is left to stand for 1min. Adding 1ml of DMEM serum-free culture solution within 1min, and directly adding the system into the DMEM serum-free culture solution to terminate PEG action; centrifuging, and discarding the supernatant; the pellet was then resuspended in HAT selection medium (see table 1) and added to feeder cell suspension, inoculated into 96 well cell culture plates, plated for 12 pieces, and incubated in a 5% CO ₂ incubator at 37 ℃. After the HAT selection medium was maintained for one week, HT medium (see table 1) was used instead, and the culture was continued for one week, DMEM medium was used instead, and the culture was continued for the fusion cells.

3. Selection of hybridoma cells

3.1 Primary screening

The fused cells are half-changed once after 7 days, the growth condition of the fused cells in a 96-hole cell culture plate is observed, when the cells grow to cell clusters (the size of the cells occupies 1/3 of the visual field under the observation of a 16-time objective lens and a 10-time eyepiece), the culture supernatant of the fused cells is sucked, and the positive clones are screened by adopting an indirect ELISA method.

3.2 Double sieves

And (3) re-screening the screened positive cell strains by an ELISA method, and screening the hybridoma cell strains with higher positive values for subcloning culture.

3.3 Cloning of hybridoma cell lines

The cloning culture of hybridoma cell lines is carried out according to a limiting dilution method, cells are accurately counted, DMEM culture medium containing 20% FBS is used for diluting the cells into 4 cell suspensions per ml, then 200 mu l of diluted cell suspensions per hole are inoculated into a 96-hole cell culture plate, after 7 days, the growth condition of the cells is observed, the antibody level of cell culture supernatant is detected, 3 monoclonal cells with the highest titer are selected for cloning culture until the detection positive rate of monoclonal cell antibodies reaches 100% for many times; finally, a monoclonal antibody hybridoma cell with high sensitivity and good specificity is obtained, and the monoclonal antibody hybridoma cell is subjected to expansion culture.

4. Ascites antibody culture

4.1 Cell transplantation: prestimulation of 6-8 week old BALB/c females with pristane was performed 14 days in advance, monoclonal antibody hybridoma cells were observed to ensure a viable cell proportion of higher than 90%, then hybridoma cells were diluted to 10 ⁶/mL with DMEM medium, and then the above dilutions were injected into the peritoneal cavity of BALB/c females in an amount of 0.5 mL/min, and the health status and ascites production status of females were observed.

4.2 Collection of ascites: after 7-10 d of hybridoma cell injection (specific time is determined according to the ascites condition of mice), puncture and drainage are carried out, and ascites of female mice are collected, after one puncture is finished, the female mice are moved back to a mouse cage and observed, if the sliding is not obvious under the health condition, the mice can be transplanted again after two days, and at most two punctures are carried out on transplanted mice, and the obtained ascites is split-packed and stored in an ultralow temperature refrigerator at-80 ℃.

Preparation examples 1 to 5, mesoporous silica microspheres are obtained by coating mesoporous silica on the surface of ferroferric oxide nano particles, and the specific preparation method is as follows:

Dispersing 0.1g of ferroferric oxide nano particles in 1mL of chloroform, dissolving 0.5gCTAB in 20mL of aqueous solution, adding and ultrasonically dispersing uniformly the chloroform dispersion of the ferroferric oxide, keeping stirring, heating to 60 ℃ to evaporate chloroform (evaporation time is 30 min), then reducing the cost of the system to a solution containing 150mL of sodium hydroxide with concentration of 2mol/L, adding a specific amount of TEOS and ethyl acetate, stirring at a speed of 500rpm for 3min, continuing to react at a speed of 200rpm for 6h, separating the nano particles by using a magnet, washing with water and ethanol for three times respectively, adding the obtained nano particles into 200mL of ethanol solution with mass fraction of 5%, heating to reflux, reacting for 6h, separating the nano particles by using a magnet, washing with ethanol, and drying to obtain the ferroferric oxide nano particles.

Among them, in preparation examples 1 to 5, the particle diameters of the ferroferric oxide nanoparticles, the amounts of TEOS and ethyl acetate used, and the average particle diameters of the finally produced nanoparticles (measured by dynamic light scattering) are shown in Table 2.

Table 2, amounts of materials used in preparation examples 1 to 5

The above-mentioned preserved ascites was separated by the mesoporous silica microspheres prepared in preparation examples 1 to 5 and other mesoporous silica microspheres purchased, to obtain the following examples.

In the following examples and comparative examples, the purity of antibodies was determined by CE-SDS with the following specific parameters: the total length of the capillary is 30.2cm, the inner diameter is 50 mu m, the effective length is 20cm, and the temperature is 25 ℃; the detector is a PDA detector, and the detection wavelength is 220nm.

Wherein, in examples 1 to 3, the same batch of ascites was selected, namely, the same mice were obtained with the ascites batch number 021090505

Example 1, a monoclonal antibody composition separation method, in which ascites prepared as described above is treated with hollow mesoporous silica, specifically comprises the following steps.

S1, diluting ascites with PBS buffer solution (concentration is 20mM, pH=7.4) according to volume concentration of 1:3, adding mesoporous silica nano-microspheres, wherein the dosage of the ascites is 1mL, the dosage of the mesoporous silica microspheres is 20mg, after adding, carrying out oscillation treatment on a constant-temperature shaking table, wherein the oscillation frequency is 100 revolutions per minute, the oscillation amplitude is 10mm, the oscillation time is 5min, the treatment temperature is 37 ℃, centrifuging at 2000rpm after the treatment is finished, and removing bottom sediment to obtain an intermediate;

s2, further separating the intermediate by an ammonium sulfate two-step method, wherein the specific operation is as follows:

Preparing saturated ammonium sulfate aqueous solution, sucking 10mL of the intermediate into a centrifuge tube, adding 5mL of the saturated ammonium sulfate solution while stirring, placing in a refrigerator at 4 ℃ for 2 hours, centrifuging at 10000rpm for 30 minutes, removing supernatant, adding 1mL of distilled water and 3mL of the saturated ammonium sulfate solution into the precipitate, uniformly mixing, placing in the refrigerator at 4 ℃ for 2 hours, centrifuging at 10000rpm for 30 minutes, removing supernatant, dissolving the bottom precipitate into 1mLPBS buffer solution, dialyzing with 100 times volume of PBS buffer solution at 4 ℃ for 24 hours by a dialysis bag, replacing dialysate once every 4 hours, and detecting that no yellow substance is formed in lipid dialysate by using a Nahner reagent.

In example 1, the average particle diameter of the hollow mesoporous silica was 100nm.

Example 2, a method for separating a monoclonal antibody composition, is different from example 1 in that mesoporous silica microspheres are equal in mass to common mesoporous silica, and the average particle size is 100nm.

Example 3a method for isolation of a monoclonal antibody composition based on example 1 using mesoporous silica nanoparticles coated with a core of ferroferric oxide of preparation example 3, step S1 was specifically adapted as follows:

S1, diluting the ascites with PBS buffer solution (concentration is 20mM, pH=7.4) according to the volume concentration of 1:3, adding mesoporous silica nano-microspheres, wherein the dosage of the ascites is 1mL, the dosage of the mesoporous silica microspheres is 20mg, carrying out oscillation treatment on a constant-temperature shaking table after adding, wherein the oscillation frequency is 100 revolutions per minute, the oscillation amplitude is 10mm, the oscillation time is 15min, the treatment temperature is 37 ℃, and removing bottom sediment through magnet adsorption after the treatment is finished.

In comparative examples 1 to 2, the same batch of ascites as in examples 1 to 3 was treated.

Comparative example 1 differs from example 1 in that step S1 was not performed and ascites was directly treated in step S2.

Comparative example 2 differs from example 1 in that in step S1, ascites is treated with equal mass of mesoporous-free silica particles (average particle diameter 100 nm).

In examples 1 to 3 and comparative examples 1 to 2, the purity of the antibodies and the total amount of the monoclonal antibodies isolated are shown in Table 3.

Table 3, experimental results for examples 1 to 3 and comparative examples 1 to 2

Numbering device	Monoclonal antibody purity (%)	Monoclonal antibody content (mg/mL)
			Example 1	98.0	6.8
Example 2	95.9	7.2
			Example 3	97.3	7.3
Comparative example 1	89.6	7.3
			Comparative example 2	92.0	7.1

In general, it is considered that the monoclonal antibody can exert a better effect when the purity of the antibody is higher than 95%, and it is found from the above experimental results that the use of mesoporous silica nanoparticles in the present application contributes to the improvement of the purity of the final antibody of the antibody as compared with the one having no mesoporous silica structure. Meanwhile, in the two methods, a certain amount of antibody is lost by adopting hollow mesoporous silica, but the final purity of the monoclonal antibody is higher. The mesoporous silica coated with the ferroferric oxide has slightly lower purity, but the loss of the antibody is smaller.

Examples 4 to 14 were obtained by adjusting the particle size and the reaction conditions of the mesoporous silica based on example 1.

The ascites treated in examples 4 to 14 were the same batch of ascites, and the batch number of ascites was 021090507.

Wherein, example 4 was identical to example 1 except for ascites.

Example 5, a method for isolating a monoclonal antibody composition, differs from example 4 in that the hollow mesoporous silica has an average particle size of 50nm.

Example 6, a monoclonal antibody composition separation method, differs from example 4 in that the hollow mesoporous silica has an average particle size of 200nm.

Example 7, a method for isolating a monoclonal antibody composition, differs from example 4 in that in step S1, the temperature upon shaking is 25℃and the time is 8min.

Example 8, a method for isolating a monoclonal antibody composition, differs from example 4 in that in step S1, the temperature upon shaking is 20℃and the time is 10min.

Example 9, a method for isolating a monoclonal antibody composition, is different from example 4 in that in step S1, the amount of mesoporous silica microspheres is 50mg, the oscillation frequency of the constant temperature shaking table is 200 rpm, the oscillation amplitude is 10mm, and the oscillation time is 5min.

Example 10, a monoclonal antibody composition isolation method, differs from example 4 in that in step S1, the amount of mesoporous silica microspheres used is 100mg.

Example 11, a method of isolation of a monoclonal antibody composition, differs from example 4 in that in step S1, 10mg of sorbitol ester-20 (nonionic surfactant) is also added.

Example 12, a method of isolating a monoclonal antibody composition, differs from example 11 in that sorbitol ester-20 is added in an amount of 20mg.

Example 13, a monoclonal antibody composition isolation method, differs from example 11 in that in step S1, citric acid having a mass of 0.4mg is also added.

Example 14, a monoclonal antibody composition isolation procedure, differs from example 13 in that citric acid is added in an amount of 2mg.

The monoclonal antibody production and purity in examples 4 to 14 are shown in Table 4.

Table 4, results of experiments in examples 4 to 14

Examples 4 to 6 are different in that the hollow mesoporous silica has a larger particle diameter, and when the particle diameter is smaller, the surface effect is stronger, and the antibody has a certain flocculation property, so that the antibody is easy to settle together in the centrifugation process, and thus the purity of example 5 is improved and the antibody content is reduced compared with example 4. In example 6, the hollow mesoporous silica had an excessively large particle size, and was poor in dispersibility and adsorptivity, resulting in a certain loss of antibody purity. It should be noted that, in the ascites of this batch, the antibody content is lower than that of examples 1 to 3, which is the influence of the constitution of the mice producing the antibodies, and similar phenomena will be generated when other batches of ascites are replaced later, and the details will not be repeated.

In examples 8 to 10, the phenomenon that the purity was improved and the yield of antibody was lowered was also observed when the amount of hollow mesoporous silica was increased, and it was also proved that the amount of silica was controlled within a certain range as much as possible, and that the increase of the amount of hollow mesoporous silica, although being capable of better adsorbing lipid substances and some other components in the system, resulted in the loss of some monoclonal antibodies, and the need to put the same into practical experiments.

In examples 11 to 12, the addition of the nonionic surfactant further improved the purity of the antibodies, and it is possible that the nonionic surfactant could improve the dispersibility of the mesoporous silica in the system and reduce the agglomeration of the mesoporous nanosilicon. Citric acid also has the effect of reducing antibody loss and improving antibody purity.

In addition, the following examples were set on the basis of example 3, and parameters and overall processing conditions of the mesoporous silica microspheres were adjusted to obtain examples 15 to 23. In examples 15 to 23, the same batch of ascites was used, and the batch number of the ascites was 021100601.

Example 15, a monoclonal antibody composition isolation procedure, differs from example 3 only in the treated ascites batch,

Example 16, a method for isolating a monoclonal antibody composition, differs from example 15 in that the mesoporous silica microspheres prepared in preparation example 1 were selected for treatment.

Example 17, a method for isolating a monoclonal antibody composition, differs from example 15 in that the mesoporous silica microspheres prepared in preparation example 2 were selected for treatment.

Example 18, a method for isolating a monoclonal antibody composition, differs from example 14 in that the mesoporous silica microspheres prepared in preparation example 4 were selected for treatment.

Example 19a method for isolating a monoclonal antibody composition differs from example 15 in that mesoporous silica microspheres prepared in preparation example 5 are selected for treatment.

Example 20, a method for isolating a monoclonal antibody composition, differs from example 15 in that the mesoporous silica microspheres are added in an amount of 50mg.

Example 21, a method for isolating a monoclonal antibody composition, differs from example 15 in that the mesoporous silica microspheres are added in an amount of 100mg.

Example 22, a method of isolation of a monoclonal antibody composition, differs from example 15 in that 10mg of sorbitol ester-20 (nonionic surfactant) is also added in step S1.

Example 23, a monoclonal antibody composition isolation method, differs from example 22 in that in step S1, citric acid having a mass of 0.4mg is further added in step S1.

The monoclonal antibody production and purity of examples 15 to 23 are shown in Table 5.

Table 5, results of experiments in examples 15 to 23

Numbering device	Monoclonal antibody purity (%)	Monoclonal antibody content (mg/mL)
			Example 15	97.3	7.7
Example 16	97.8	6.9
			Example 17	97.4	7.2
Example 18	96.7	7.8
			Example 19	97.4	7.7
Example 20	97.7	7.5
			Example 21	98.0	7.2
Example 22	97.9	7.8
			Example 23	98.6	8.1

According to the experimental data, the mesoporous silica microspheres coated with the ferroferric oxide have similar trend on the whole as that of the hollow mesoporous silica microspheres, and the separation can be carried out through magnet adsorption, so that the operation is more convenient, but the purity is slightly lower than that of the embodiment adopting the hollow mesoporous silica microspheres.

The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.

Claims

1. A method for purifying a monoclonal antibody composition comprising the steps of:

wherein the monoclonal antibody composition is cultured ascites of the mouse;

The mesoporous silica microsphere is coated with a paramagnetic core structure;

The average diameter of the paramagnetic core is 50-100 nm;

the paramagnetic core structure is ferroferric oxide nano particles.

2. The method of claim 1, wherein in step S1, the mesoporous silica microspheres and the monoclonal antibody composition are mixed for 15-30 min at 20-37 ℃ and then the mesoporous silica microspheres adsorbed with fat are separated by magnetic attraction.

3. The method for purifying a monoclonal antibody composition according to any one of claims 1 to 2, wherein in step S1, after mixing mesoporous silica microspheres with monoclonal antibodies, the mixture is subjected to a shaking treatment, the shaking frequency is 100 to 200 rpm, and the shaking amplitude is 10 to 20mm.

4. The method for purifying a monoclonal antibody according to any one of claims 1 to 2, wherein in step S1, a nonionic surfactant is added, the mass of the nonionic surfactant being 0.5 to 1 times the mass of the mesoporous silica microspheres.

5. The method according to claim 4, wherein citric acid is added in the step S1, wherein the added mass of the citric acid is 0.02-0.1 times of the mass of the mesoporous silica microspheres.

6. The method for purifying a monoclonal antibody composition according to any one of claims 1 to 2, wherein in step S1, a buffer solution is added, the pH value is controlled to be 6.8 to 7.4, and after the buffer solution is added, the mass concentration of the mesoporous silica microspheres in step S1 is controlled to be2 to 10mg/mL.