CN112574321A

CN112574321A - Affinity purification method for capturing monoclonal antibody-tumor necrosis factor fusion protein

Info

Publication number: CN112574321A
Application number: CN202011611594.1A
Authority: CN
Inventors: 方国波; 蔡则玲; 吴珩
Original assignee: Shanghai Celgen Biopharma Co ltd
Current assignee: Shanghai Celgen Biopharma Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-03-30
Anticipated expiration: 2040-12-30
Also published as: CN112574321B

Abstract

The invention discloses an affinity purification method for capturing antibody-tumor necrosis factor fusion protein, which specifically comprises the following steps: (a) providing a raw material to be purified, wherein the raw material contains an antibody-tumor necrosis factor fusion protein; (b) subjecting the raw material to be purified to affinity chromatography, and collecting eluent containing sample peaks, wherein the filler of the affinity chromatography is selected from the following group: mabselectc Sure, Prism A, Mabcapture A, Amsphere A3, Eshmuno A, Kancap A3G, Unimab 50, or a combination thereof, wherein the eluate is an intermediate product comprising an antibody-tumor necrosis factor fusion protein. The antibody-tumor necrosis factor fusion protein purified by the method has high purity and yield, and the content of the aggregate is extremely low, so the antibody-tumor necrosis factor fusion protein has high safety and wide application prospect.

Description

Affinity purification method for capturing monoclonal antibody-tumor necrosis factor fusion protein

Technical Field

The invention relates to the field of protein purification, in particular to an affinity purification method for capturing monoclonal antibody-tumor necrosis factor fusion protein.

Background

The monoclonal antibody is widely applied to the targeted therapy of tumors, and inhibits the growth of tumor cells by combining with tumor tissue cell surface antigen molecules. In order to enhance the therapeutic effect of the monoclonal antibody, the monoclonal antibody is modified, such as glycosylation modification is changed to increase the ADCC function; the monoclonal antibody is coupled with chemical molecules to increase the killing effect such as ADC molecules; the development of bispecific antibodies which bind different antigen molecules enhances the coordination of drug functions; develop antibody-tumor necrosis factor fusion protein, coordinate the function of immunocyte, and enhance the function of inhibiting tumor cell growth or killing. Due to a unique molecular mechanism, the antibody-tumor necrosis factor fusion protein is developed into a hot spot for developing tumor treatment molecules at present. In the development process of antibody-tumor necrosis factor fusion protein drugs, in addition to the safety and effectiveness of the products, the stability of the molecules, especially the aggregation of the molecules due to tumor necrosis factor, should be of great concern.

In the process of preparing the monoclonal antibody, Protein A chromatography is preferably used for capturing the monoclonal antibody. The Protein A chromatography condition adopts a mode of combining a sample under a neutral pH condition and eluting the sample under an acidic pH condition. Most of the monoclonal antibodies adopt pH3.2-3.8 elution solution, and the eluted protein has high purity and high recovery rate. For the antibody-tumor necrosis factor fusion Protein, because the tumor necrosis factor is fused into the monoclonal antibody structure, the spatial structure of the monoclonal antibody can change to a certain extent, and meanwhile, part of the fusion Protein, such as IL-10, TNF alpha has the characteristic of tending to aggregate, therefore, the adoption of the traditional Protein A chromatography condition to capture the antibody-tumor necrosis factor fusion Protein has certain challenges.

According to the characteristics of the monoclonal antibody-tumor necrosis factor fusion protein, the chromatography medium has a great influence on the purification effect of the monoclonal antibody, for example, Cytiva Capto Q and Tosoh Q-600C are adopted, the intermediate step purification is carried out in a sample flow-through mode, the Capto Q chromatography medium shows a flow-through mode, and the Q-600C is a binding mode. The affinity chromatography medium matrix contains hydrophilic substances such as agarose and cellulose, and hydrophobic substances such as polystyrene divinyl phenyl resin and polymethacrylic resin, and for the affinity chromatography medium of the hydrophobic medium, the reduction of the nonspecific adsorption of protein mainly depends on the preparation process of the medium, the modification degree, the stability and the uniformity of a surface hydrophilic layer. The characteristics of Protein molecules, the characteristics of an affinity chromatography medium matrix and the structural characteristics of bonded Protein A influence the purity and yield of affinity chromatography Protein, and the selection of a proper affinity chromatography medium has certain challenges for the purification of antibody-tumor necrosis factor fusion Protein.

Protein A chromatography is applied to the primary purification of antibody-tumor necrosis factor fusion Protein, is mainly limited by the selection of chromatography packing and chromatography conditions, and needs to maintain the balance between Protein purity and yield.

Therefore, there is a strong need in the art to develop a new method for significantly reducing the content of the polymer without affecting the yield.

Disclosure of Invention

The object of the present invention is a novel process for significantly reducing the content of oligomers without affecting the yield.

In a first aspect of the present invention, there is provided a method for purifying an antibody-tumor necrosis factor fusion protein, comprising the steps of:

(a) providing a raw material to be purified, wherein the raw material contains an antibody-tumor necrosis factor fusion protein;

(b) subjecting the raw material to be purified to affinity chromatography, and collecting eluent containing sample peaks, wherein the filler of the affinity chromatography is selected from the following group: mabselectc Sure, Prism A, Mabcapture A, Amsphere A3, Eshmuno A, Kancap A3G, Unimab 50, or a combination thereof, wherein the eluate is an intermediate product comprising an antibody-tumor necrosis factor fusion protein.

In another preferred embodiment, the antibody-tumor necrosis factor fusion protein comprises an anti-Her-2 humanized monoclonal antibody-tumor necrosis factor a fusion protein.

In another preferred embodiment, the filler is selected from the group consisting of: kancap a 3G, Unimab 50, or a combination thereof, preferably, Unimab 50.

In another preferred example, the method further comprises the following sterilization steps: the chromatography column is sterilized with 2 to 10 column volumes (preferably, 3 to 7 column volumes) of an alkaline solution.

In another preferred embodiment, the concentration of the alkaline solution is 0.02 to 1mol/L, preferably 0.05 to 0.8mol/L, more preferably 0.08 to 0.5 mol/L.

In another preferred embodiment, the method comprises at least one (preferably 2) sterilization step.

In another preferred embodiment, the alkaline solution is selected from the group consisting of: sodium hydroxide, potassium hydroxide, or a combination thereof.

In another preferred example, the step (b) further includes:

(b1) leaching the chromatographic column by using a leaching buffer solution; and/or

(b2) Balancing the chromatographic column by using a balance buffer solution; and

(b3) eluting with an elution buffer solution, and collecting an eluent containing a sample peak, wherein the eluent is an intermediate product containing the antibody-tumor necrosis factor fusion protein.

In another preferred embodiment, in step (b1), the washing buffer is selected from the group consisting of: acetic acid-sodium acetate buffer, Tris-hydrochloric acid buffer, citric acid buffer, phosphate buffer, glycine-hydrochloric acid buffer, or a combination thereof.

In another preferred embodiment, the pH of the elution buffer is between 4 and 8, more preferably between 4.5 and 5.5.

In another preferred embodiment, the volume of the elution buffer is 2 to 20 column volumes, preferably 3 to 10 column volumes, more preferably 5 to 8 column volumes.

In another preferred embodiment, the concentration of the elution buffer is 10-100 mmol/L, preferably 10-80mmol/L, more preferably 20-50 mmol/L.

In another preferred embodiment, the elution buffer further comprises sodium chloride.

In another preferred embodiment, the concentration of sodium chloride is 0.1 to 2mol/L, preferably 0.2 to 1.5mol/L, more preferably 0.3 to 1 mol/L.

In another preferred embodiment, in step (b2), the equilibration buffer is selected from the group consisting of: a phosphate buffer, a Tris-hydrochloric acid buffer, or a combination thereof.

In another preferred embodiment, the pH of the equilibration buffer is neutral.

In another preferred embodiment, the pH of the equilibration buffer is between 6 and 9, preferably between 6 and 8, more preferably between 6.5 and 7.5.

In another preferred embodiment, the volume of the equilibration buffer is 3-10 column volumes, preferably 3-8 column volumes, more preferably 3-5 column volumes.

In another preferred embodiment, the concentration of the equilibration buffer is 10-300mmol/L, preferably 20-100mmol/L, more preferably 20-50 mmol/L.

In another preferred embodiment, the equilibration buffer further comprises sodium chloride.

In another preferred embodiment, the concentration of sodium chloride is 0.1 to 300mmol/L, preferably 1 to 200mmol/L, more preferably 10 to 80mmol/L, more preferably 50 to 80 mmol/L.

In another preferred embodiment, in step (b3), the elution buffer is selected from the group consisting of: acetic acid-sodium acetate buffer, glycine-hydrochloric acid buffer, citric acid buffer, arginine-hydrochloric acid buffer, or a combination thereof.

In another preferred embodiment, the pH of the elution buffer is 3 to 4.5, preferably 3.2 to 4.5, more preferably 3.5 to 4.2, more preferably 3.8 to 4.

In another preferred embodiment, the concentration of the elution buffer is 10 to 300mmol/L, preferably 10 to 200mmol/L, more preferably 20 to 100 mmol/L.

In another preferred embodiment, in the step (b3), the pH of the eluent is 3.2-5, preferably 3.8-4.

In another preferred embodiment, the method further comprises the step of preserving the chromatography column with a preservation solution.

In another preferred example, the preservation solution is selected from the group consisting of: ethanol, benzyl alcohol, or combinations thereof.

In another preferred embodiment, the antibody-tumor necrosis factor fusion protein is selected from the group consisting of: an anti-Her-2 humanized monoclonal antibody heavy chain-tumor necrosis factor a fusion protein (Herceptin HC-TNFa), an anti-Her-2 humanized monoclonal antibody light chain (Herceptin LC), or a combination thereof.

In another preferred embodiment, the aggregate content in the eluent is less than or equal to 10%, preferably less than or equal to 5%, more preferably less than or equal to 2%.

In another preferred example, the purification method further comprises:

(c) and refining the eluent of the intermediate product containing the monoclonal antibody-tumor necrosis factor fusion protein obtained in the last step to obtain a purified antibody-tumor necrosis factor fusion protein product.

In another preferred embodiment, the purity is calculated as the ratio of peak areas of antibody-tumor necrosis factor fusion protein monomer peaks on an HPLC chart.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows an affinity chromatography media eluent SEC-HPLC separation profile; wherein the content of the first and second substances,

1.KanCapA 3G pH3.8；2.Mabcapture A pH3.6；3.ESHMUNO A pH3.8；4.Amsphere A3 pH3.6；5.Prism A pH3.8；6.Unimab 50pH3.8 7.Mabselect SuRe pH3.8；

and, the above-mentioned numbers 1 to 7 correspond to the numbers 1 to 7 in example 1.

Detailed Description

The present inventors have conducted extensive and intensive studies and, as a result of extensive research on the production process, have unexpectedly developed a novel method for purifying an antibody-tnf fusion protein to significantly reduce the content of the dimer in the antibody-tnf fusion protein without affecting the yield for the first time. The method of the invention can prepare the antibody-tumor necrosis factor fusion protein with high yield, and can also obviously reduce the content of the polymer in the antibody-tumor necrosis factor fusion protein. Based on the above findings, the inventors have completed the present invention.

Antibody-tumor necrosis factor fusion protein

The antibody-tumor necrosis factor fusion protein comprises an anti-Her-2 humanized monoclonal antibody-tumor necrosis factor a fusion protein or other monoclonal antibodies-tumor necrosis factor fusion proteins.

Purification method

The invention provides a method for purifying an antibody-tumor necrosis factor fusion protein based on a specific chromatographic medium, which comprises the following steps:

In a preferred embodiment of the present invention, the method comprises:

step 1, processing affinity filler, and loading;

step 2, leaching, namely leaching the chromatographic column by using a leaching buffer solution 2 of acetic acid-sodium acetate buffer solution, Tris-hydrochloric acid buffer solution, citric acid buffer solution or phosphate buffer solution with the pH value of 4.0-8.0;

step 3, balancing after washing, and selecting a neutral pH phosphate buffer solution for balancing;

step 4, eluting the product by using an acetic acid-sodium acetate buffer solution with the pH value of 3.0-4.0, a glycine-hydrochloric acid buffer solution and a citric acid buffer solution, and collecting the product;

and 5, CIP (cleaning in situ), and using NaOH solution to disinfect the chromatographic column.

Specifically, the specific operation method in the step 1 is as follows:

1a, filling different affinity fillers, and disinfecting the chromatographic column by using NaOH solution with the volume of 3-5 times of the column volume;

1b, balancing the chromatographic column by using 3-5 times of column volume of balancing buffer solution;

1c, loading, and allowing the sample to remain on the column for 2-10 minutes.

Specifically, the affinity filler ligand is Protein A. The affinity fillers include, but are not limited to, Mabselectc Sure, Prism A, Merck's Eshmuno A, Pall's Kancap A3G, nanomolar Unimab 50, Cytiva.

Specifically, the specific operation method of step 2 is:

2a, balancing the chromatographic column by using 3-5 times of column volume of balancing buffer solution;

2b, eluting the chromatographic column by using elution buffer solution with the volume of 5-8 times of the column volume;

specifically, the specific operation method of step 3 is: the column is equilibrated with 3-5 column volumes of equilibration buffer.

Specifically, the specific operation method of step 4 is: eluting with 3-10 column volumes of elution buffer.

Specifically, the specific operation method in step 5 is as follows:

5a, balancing the chromatographic column by using 3-5 times of column volume of balancing buffer solution;

5b, disinfecting the chromatographic column by using NaOH solution with the volume of 3-5 times of the column volume;

5c, balancing the chromatographic column by using 3-5 times of column volume of balancing buffer solution;

and 5d, preserving the chromatographic column by using 3-5 times of preservation solution of the column volume.

Preferably, the chromatographic medium screened in step 1a is Mabselect Sure, Prism A, Mabcapture A, Unimab 50, Amsphere A3, Eshmuno A, Kancap A3G, and the optimal chromatographic medium is Unimab 50, Kancap A3G.

Preferably, the equilibration buffer used in the steps 1b, 2a, 2c, 4a and 4c is phosphate buffer or Tris-hydrochloric acid buffer, the pH value is 6.5-7.5, and the salt concentration is 0.15 mol/L.

Preferably, the elution buffer used in step 2b is an acetic acid-sodium acetate buffer, a Tris-hydrochloric acid buffer, a citric acid buffer or a phosphate buffer, the pH value of the elution buffer is 4.0-8.0, and the concentration of the elution buffer is 20-50 mmol/L. The elution buffer may also contain sodium chloride as an additive, wherein the concentration of sodium chloride is 0-2 mol/L. The pH of the elution buffer is most preferably 4.5-5.5.

Preferably, the equilibration buffer used in step 3 is phosphate buffer with pH of 6.0-8.0 and concentration of 20-50 mmol/L. The elution buffer may also contain sodium chloride as an additive, wherein the concentration of sodium chloride is 0-200 mMol/L. The pH of the elution buffer is most preferably 6.5-7.5, and the NaCl concentration is 50-150 mMol/L.

Preferably, the elution buffer used in the step 4 is an acetic acid-sodium acetate buffer, a glycine-hydrochloric acid buffer and a citric acid buffer, the pH value of the elution buffer is 3.0-4.0, and the concentration of the elution buffer is 20-100 mmol/L. The pH of the elution buffer is most preferably 3.80-4.00.

Preferably, the concentration of the NaOH solution used in steps 1a and 5b is 0.1 mol/L.

Preferably, the preservation solution in the 5d step is 20% ethanol.

In addition, affinity chromatography is performed on an AKTA explorer 100 instrument, and the assays involved include:

(1) protein concentration: detected by the absorption value of UV 280;

(2) monomer purity: detection by size exclusion high performance liquid chromatography (SEC-HPLC);

the affinity medium used in the invention is a chromatography medium in which Protein A ligand is crosslinked to a matrix including, but not limited to, agarose, hydroxylated polyether resin, polyacrylic resin, polystyrene divinyl phenyl resin, polymethacrylic resin, polystyrene resin, hydroxyapatite, glass and the like. Preferred affinity fillers are Cytiva Mabselectc Sure, Merck's Eshmuno A, Pall's Kancap A3G, nanomolar Unimab 50.

The chromatography comprises the following specific steps:

step 1a, filling different affinity fillers, and disinfecting the chromatographic column by using NaOH solution with the volume of 3-5 times of the column volume.

Step 1b, equilibrating the chromatography column with 3-5 column volumes of equilibration buffer.

Step 1c, load, let sample on the column for 2-10 minutes.

Step 2a, equilibrating the chromatography column with 3-5 column volumes of equilibration buffer.

And 2b, eluting the chromatographic column by using elution buffer with 5-8 times of column volume.

Step 2c, equilibrating the chromatography column with 3-5 column volumes of equilibration buffer.

And 3, eluting the product by using elution buffer solution with 3-5 times of column volume.

Step 4a, equilibrating the chromatography column with 3-5 column volumes of equilibration buffer.

And 4b, disinfecting the chromatographic column by using NaOH solution with the volume being 3-5 times of the column volume.

Step 4c, equilibrating the chromatography column with 3-5 column volumes of equilibration buffer.

And 4d, preserving the chromatographic column by using 3-5 times of preservation solution of the column volume.

The equilibrium buffer used in steps 1b, 2a, 2c, 4a, 4c is phosphate buffer or Tris-hydrochloric acid buffer, the pH is 6.5-7.5, and the salt concentration is 0.15 mol/L.

The elution buffer used in the step 2b is an acetic acid-sodium acetate buffer, a Tris-hydrochloric acid buffer, a citric acid buffer or a phosphate buffer, the pH value of the elution buffer is 4.0-8.0, and the concentration of the elution buffer is 20-50 mmol/L. The elution buffer may also contain sodium chloride as an additive, wherein the concentration of sodium chloride is 0-2 mol/L. The pH of the elution buffer is most preferably 4.5-5.5.

The elution buffer solution used in step 3 is acetic acid-sodium acetate buffer solution, glycine-hydrochloric acid buffer solution and citric acid buffer solution, the pH value is 3.0-4.0, and the concentration is 20-100 mmol/L. The pH of the elution buffer is most preferably 3.80-4.00.

The concentration of the NaOH solution used in steps 1a, 4b was 0.1 mol/L.

And 4d, the preservation solution is 20% ethanol.

The chromatography medium and the purification conditions used by the affinity purification process are suitable for capturing the antibody-tumor necrosis factor fusion protein with low pH and poor stability.

Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein may be made and used without departing from the spirit and scope of the invention.

The reagents adopted by the invention are all common commercial products and can be purchased in the market.

The main advantages of the invention include:

(1) the invention screens out the chromatography medium suitable for affinity purification of the antibody-tumor necrosis factor fusion protein, in particular to the chromatography medium suitable for the relatively high pH elution condition.

(2) The purification method can prepare the antibody-tumor necrosis factor fusion protein with high yield, the yield is up to more than 80%, and the purity is up to more than 84%.

(3) The aggregate content of the antibody-tumor necrosis factor fusion protein obtained by the method is very low and is lower than 5%.

(4) The invention screens the primary capture affinity medium of the preparation process of the antibody-tumor necrosis factor fusion protein and optimizes proper chromatographic conditions, thereby obviously reducing the content of the polymer on the premise of not influencing the yield.

(5) The method selects proper affinity chromatography packing, changes the pH value of the elution step, uses mild elution conditions, and inhibits the increase of polymers, thereby playing a role in improving the purity of the sample.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

The fusion protein is obtained from Shanghai seiki biological medicine company, and consists of 2 pieces of anti-Her-2 humanized monoclonal antibody heavy chain-tumor necrosis factor a fusion protein (Herceptin HC-TNFa) and 2 pieces of anti-Her-2 humanized monoclonal antibody light chain (Herceptin LC), and adopts the structure of the monoclonal antibody to add a tumor necrosis factor a fusion protein sequence at the C-end of the heavy chain, wherein the amino acid sequence of the anti-Her-2 humanized monoclonal antibody heavy chain-tumor necrosis factor a fusion protein (Herceptin HC-TNFa) is shown in SEQ ID NO.1, and the amino acid sequence of the anti-Her-2 humanized monoclonal antibody light chain (Herceptin LC) is shown in SEQ ID NO. 2.

Herceptin HC-TNFa：

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL(SEQ ID NO.:1)

Herceptin LC：

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO.:2)

Antibody-tumor necrosis factor fusion protein the following experiment was performed using an anti-Her-2 humanized monoclonal antibody-tumor necrosis factor a fusion protein as an example.

EXAMPLE 1 Effect of affinity media on the purification Effect of antibody-TNF fusion proteins

Using 20mmol/L phosphate buffer solution, 150mmol/L NaCl, pH7.0, as the balance buffer solution used in steps 1b, 2a, 2c, 4a, 4 c; 20mmol/L citric acid buffer solution, 500mmol/L NaCl and pH5.2 as the leaching buffer solution in the step 2 b; using 0.1mol/L NaOH solution as the disinfectant in the steps 1a and 4 b; 20% ethanol is used as the preservation solution in the step 4 d; the load was 10mg/ml using a Protein A column (Kancapa A3G, 10 ml; Unimab 50, 10 ml; Mabselect Sure, 10ml, Eshmuno A, 10ml, etc.).

Sterilizing the chromatography column with 5 column volumes of NaOH solution (step 1 a); equilibrating the chromatography column with 5 column volumes of equilibration buffer (step 1 b); loading, leaving the sample on the column for 5 minutes (step 1 c); equilibrating the column with 5 column volumes of equilibration buffer to wash out unbound fermentation broth (step 2 a); then eluting the chromatographic column with 5 times of elution buffer solution (step 2b), and balancing the chromatographic column with 5 times of equilibration buffer solution (step 2 c); eluting the product with 3 column volumes of elution buffer and collecting the product based on UV280 (step 3); equilibrating the chromatography column with 3 column volumes of equilibration buffer (step 4 a); sterilizing the chromatography column with 3 column volumes of NaOH solution (step 4 b); the column was preserved with 3 column volumes of preservation solution (step 4 d).

For different comparative experiments, the fillers in step 1a were Mabselect Sure, Prism A, Mabcapture A, Unimab 50, Amsphere A3, Eshmuno A, Kancap A3G, respectively.

Elution samples were collected for determination of concentration to calculate yield, and product purity was measured (SEC-HPLC). The antibody-tumor necrosis factor fusion protein, chromatography media Amsphere A3 and Mabcapture A, can not be eluted under the condition of pH3.8, and are easy to generate aggregation characteristics under the condition of low pH (less than 3.8), and Table 1 can show that the content of the polymer with pH lower than 3.6 is greatly increased, and the yield is less than 50% when the pH is higher than 3.6 (except KanCap A/Unimab 50). This shows that the affinity chromatography packing aglucon, medium production technology, and different matrix types affect the acting force between Protein and Protein A, thus leading to the difference of Protein elution, and the chromatography packing KanCap A and Unimab 50 can increase the elution pH to 3.8, and can ensure higher yield at the same time. After the elution of the Protein A chromatography is finished, the pH of the obtained eluent is increased to about pH4.2 by balancing again by adopting a phosphoric acid buffer solution under a neutral pH condition, so that the Protein is further stabilized. Therefore, Protein A chromatographic packing KanCap A and Unimab 50 can adopt a mild pH elution condition, so that the purity of a sample can be improved, and the yield of Protein can be ensured.

TABLE 1 purification of antibody-TNF fusion protein Effect by chromatography packing

Protein purity of each chromatography medium affinity eluate is detected by SEC-HPLC, and the chromatographic column is TSK G3000SW_XLThe mobile phase is 100mmol/L phosphate buffer solution/150 mmol/L NaCl (pH7.0), and the protein component map is shown in FIG. 1, as shown in the figure: the sample has high polymer content under the elution condition of pH3.6 (figure 1-2 (figure 1, No. 2), figure 1-4 (figure 1, No. 4)), the sample has relatively low polymer content under the elution condition of pH3.8, the chromatographic medium with polymer content lower than 5% in the eluent has KanCapA 3G, Unimab 50, Prism A, and the sample with highest purity is KanCapA 3G (figure 1-1 (figure 1, No. 1), figure 1-3 (figure 1, No. 3), figure 1-5 (figure 1, No. 5), figure 1-6 (figure 1, No. 6), figure 1-7 (figure 1, No. 7)), the results show that the affinity elution solution pH and the chromatography medium have great influence on the chromatography effect, the purity of the binding protein and the sample yield (Table 1) are obtained, the KanCapA 3G, Unimab 50 chromatography medium is selected, and the elution solution is not lower than pH3.8 for subsequent process optimization.

Example 2 Effect of high pH Range elution conditions on the purification Effect of antibody-TNF fusion protein

Using 20mmol/L phosphate buffer solution, 150mmol/L NaCl, pH7.0, as the balance buffer solution used in steps 1b, 2a, 2c, 4a, 4 c; 20mmol/L citric acid buffer solution, 500mmol/L NaCl and pH5.2 as the leaching buffer solution in the step 2 b; using 0.1mol/L NaOH solution as the disinfectant in the steps 1a and 4 b; 20% ethanol is used as the preservation solution in the step 4 d; the sample was loaded on a Protein A column (Kancapa A3G) with a loading of 20 mg/ml.

According to the stability of the sample and the elution efficiency of the chromatographic medium, the elution condition of the chromatographic medium should maintain the balance of the protein purity and the elution yield. For Protein a affinity chromatography, lowering the elution solution pH provides monoclonal antibody recovery, but at the same time reduces Protein purity, primarily due to low pH which causes the aggregates in the cell harvest to elute or during elution, the proteins aggregate. Therefore, it is important to select a suitable pH of the elution solution. As can be seen from the results in Table 2, the elution conditions at pH3.75-3.90 have a better inhibitory effect on the production of the antibody-TNF fusion protein. For the antibody-tumor necrosis factor fusion protein with poor stability, the elution condition with pH value of 3.75-3.90 can be used as an option for optimizing affinity chromatography.

TABLE 2 detection results of pH antibody-TNF fusion protein

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Saiki biomedical Co Ltd

<120> an affinity purification method for capturing monoclonal antibody-tumor necrosis factor fusion protein

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

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Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

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Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

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Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

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Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

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Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

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Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

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Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His

450 455 460

Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg

465 470 475 480

Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln

485 490 495

Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu

500 505 510

Phe Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr

515 520 525

Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser

530 535 540

Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala

545 550 555 560

Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu

565 570 575

Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp

580 585 590

Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu

595 600 605

<210> 2

<211> 214

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims

1. A method for purifying an antibody-tumor necrosis factor fusion protein, comprising the steps of:

2. The method of claim 1, wherein said antibody-tumor necrosis factor fusion protein comprises an anti-Her-2 humanized monoclonal antibody-tumor necrosis factor a fusion protein.

3. The method of claim 1, wherein the filler is selected from the group consisting of: kancap a 3G, Unimab 50, or a combination thereof.

4. The method of claim 1, wherein step (b) further comprises:

5. The method of claim 4, wherein in step (b1), the wash buffer is selected from the group consisting of: acetic acid-sodium acetate buffer, Tris-hydrochloric acid buffer, citric acid buffer, phosphate buffer, glycine-hydrochloric acid buffer, or a combination thereof.

6. The method of claim 4, wherein in step (b2), the equilibration buffer is selected from the group consisting of: a phosphate buffer, a Tris-hydrochloric acid buffer, or a combination thereof.

7. The method of claim 4, wherein in step (b3), the elution buffer is selected from the group consisting of: acetic acid-sodium acetate buffer, glycine-hydrochloric acid buffer, citric acid buffer, arginine-hydrochloric acid buffer, or a combination thereof.

8. The method of claim 4, wherein the concentration of the elution buffer is 10-300mmol/L, preferably 10-200 mmol/L, more preferably 20-100 mmol/L.

9. The method of claim 4, wherein in step (b3), the pH of the eluent is 3.2-5, preferably 3.8-4.

10. The method of claim 1, wherein the elution solution has an aggregate content of 10% or less, preferably 5% or less, more preferably 2% or less.