Chromatographic method for on-column disulfide bond oxidation

Abstract

The invention discloses a chromatographic method for on-column disulfide bond oxidation, and belongs to the technical field of purification. According to the method, for the disulfide bond-containing antibody, a flushing fluid containing cysteine and cystine is used for flushing the affinity filler after the antibody is combined, and finally, the target antibody is eluted and collected. According to the method, broken disulfide bonds can be effectively reoxidized to form complete molecules, meanwhile, HCP and potential viruses can be reduced, the target molecule content is effectively increased, and the fragment content is reduced. The method is not only suitable for laboratory scale, but also can be used as a production process.

Description

Technical Field

[0001] The invention belongs to the technical field of purification, and in particular relates to a chromatography method for disulfide bond oxidation on a column. Background Art

[0002] Reduction of disulfide bonds is a common problem during the manufacturing process of antibodies. This reduction may result in the presence of a large amount of low molecular weight (LMW) species in the drug, which may affect the safety and efficacy of the drug, resulting in the drug failing to meet release specifications.

[0003] A typical antibody has a molecular weight of about 150 kilodaltons (kDa) and consists of two identical light chains and two identical heavy chains, which are connected by disulfide bonds between heavy chain-heavy chain (HH) and heavy chain-light chain (HL). Disulfide bonds connecting two heavy chains or connecting a light chain and a heavy chain are called interchain disulfide bonds. Disulfide bonds connecting two β-sheets in a single domain (constant domain or variable domain) are called intrachain disulfide bonds. In a typical monoclonal antibody, there are 12 intrachain disulfide bonds (one per domain), interchain disulfide bonds between two light chains and heavy chains, and between two and eleven interchain disulfide bonds in heavy chains. Disulfide bonds stabilize proteins thermodynamically and mechanically. Improper disulfide bond formation and disulfide bond reduction can affect process performance, protein stability and biological function.

[0004] Reported studies focusing on preventing interchain disulfide bond cleavage do not always adequately prevent disulfide bond reduction from occurring. In addition, these methods may require additional equipment and increase manufacturing costs. Therefore, it is necessary to develop a method that can effectively reoxidize disulfide bonds in reduced antibody materials to generate intact antibody molecules to address the challenges of disulfide bond reduction. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a chromatography method for disulfide bond oxidation on a column. Protein A affinity chromatography is a separation technology that utilizes the specific affinity between biological molecules. It can bind to proteins with Fc regions, so such proteins are retained on the column, and then by adding additives or changing the pH of the flushing liquid during the flushing process, impurities such as host cell proteins adsorbed on the column are washed off, and finally the target protein is eluted. In the chromatography method of the present invention, after the antibody molecule is bound to the affinity filler, Tris-HCl buffer is used as a flushing liquid, cysteine ​​and cystine are used as redox agents, and 3 to 20 column volumes are flushed, which can achieve an increase in the proportion of intact antibody molecules bound to the affinity chromatography filler.

[0006] The present invention provides a chromatography method for improving the purity of an antibody containing a disulfide bond. In the chromatography method, a Tris-HCl buffer added with cysteine ​​and cystine is used as a flushing liquid; that is, fragments caused by the loss of disulfide bonds are oxidized during the chromatography process, thereby improving the purity of the antibody containing a disulfide bond.

[0007] Furthermore, the pH range of the Tris-HCl buffer in the chromatography method is pH 6.0 to pH 8.0; more preferably pH 7.0 to pH 8.0; most preferably pH 7.2.

[0008] Furthermore, in the chromatography method, the concentration of Tris-HCl in the Tris-HCl buffer is 10 mmol / L to 60 mmol / L; more preferably 15 mmol / L to 35 mmol / L; most preferably 20 mmol / L.

[0009] Furthermore, in the chromatography method, the concentration of cysteine ​​in the Tris-HCl buffer is 0.1 mmol / L to 10 mmol / L; more preferably 0.5 mmol / L to 5 mmol / L.

[0010] Furthermore, in the chromatography method, the concentration of cystine in the Tris-HCl buffer is 0.1 mmol / L to 5 mmol / L; more preferably 0.1 mmol / L to 0.5 mmol / L.

[0011] Specifically, optionally, the concentration of cysteine ​​in the Tris-HCl buffer in the chromatography method is 1 mmol / L.

[0012] Specifically, optionally, the concentration of cysteine ​​in the Tris-HCl buffer in the chromatography method is 0.3 mmol / L.

[0013] The present invention also provides an affinity chromatography method for increasing the content of intact antibody molecules, comprising the following steps:

[0014] (1) equilibrating an affinity chromatography column using an affinity equilibration solution;

[0015] (2) loading the carrier with the antibody containing disulfide bonds onto an affinity chromatography column;

[0016] (3) equilibrating the affinity chromatography column using an affinity equilibration solution;

[0017] (4) washing the affinity chromatography column with a flushing solution;

[0018] (5) using an eluent to elute the main peak of the antibody and recover it;

[0019] (6) Use regeneration solution to elute the regeneration peak.

[0020] In one embodiment of the present invention, the affinity equilibration solution in step (1) and step (3) is Tris-HCl buffer.

[0021] In one embodiment of the present invention, the concentration of Tris-HCl in the Tris-HCl buffer is preferably 10 mmol / L to 60 mmol / L.

[0022] Preferably, the concentration of Tris-HCl in the Tris-HCl buffer is 15 mmol / L to 35 mmol / L.

[0023] Specifically, optionally, the concentration of Tris-HCl in the Tris-HCl buffer is 20 mmol / L.

[0024] In one embodiment of the present invention, the pH range of the Tris-HCl buffer is 6.5-7.5.

[0025] Preferably, the pH of the Tris-HCl buffer is 7.0-7.5.

[0026] Specifically, optionally, the pH of the Tris-HCl buffer is 7.2.

[0027] In one embodiment of the present invention, the Tris-HCl buffer further contains 100-200 mmol / L of sodium chloride.

[0028] In one embodiment of the present invention, the amount of affinity equilibration solution used in step (1) is 3 to 10 column volumes.

[0029] Specifically, the amount of affinity equilibration solution used in step (1) is 5 column volumes.

[0030] In one embodiment of the present invention, the column volume is the volume of an affinity chromatography column.

[0031] Specifically, the volume of the affinity chromatography column is 5 to 20 mL.

[0032] In one embodiment of the present invention, the carrier in step (2) is an affinity filler containing Protein A ligand.

[0033] Specifically, the vector is Cytiva Mabseclect PrismA.

[0034] In one embodiment of the present invention, the disulfide bond-containing antibody in step (2) includes a disulfide bond-containing antibody having a PI (isoelectric point) value of about 6.5 to 8.0.

[0035] In one embodiment of the present invention, the loading amount of the disulfide bond-containing antibody on the carrier in step (2) is 10 g / L to 70 g / L.

[0036] In one embodiment of the present invention, the amount of affinity equilibration solution used in step (3) is 3 to 10 column volumes.

[0037] Specifically, the amount of affinity equilibration solution used in step (3) is 3 column volumes.

[0038] In one embodiment of the present invention, the amount of the washing solution used in step (4) is 3 to 20 column volumes.

[0039] Specifically, the amount of flushing liquid used in step (4) is 12 column volumes.

[0040] In one embodiment of the present invention, the washing solution in step (4) is a Tris-HCl buffer to which cysteine ​​and cystine are added.

[0041] In one embodiment of the present invention, the pH range of the flushing solution is 6.0-8.0.

[0042] Preferably, the pH range of the flushing solution is 7.0-8.0.

[0043] Specifically, optionally, the pH value of the flushing liquid is 7.2.

[0044] In one embodiment of the present invention, the concentration of Tris-HCl in the flushing solution in step (4) is 10 mmol / L to 60 mmol / L.

[0045] Preferably, the concentration of Tris-HCl in the washing solution in step (4) is 15 mmol / L to 35 mmol / L.

[0046] Specifically, optionally, the concentration of Tris-HCl in the flushing solution in step (4) is 20 mmol / L.

[0047] In one embodiment of the present invention, the concentration of cysteine ​​in the flushing solution in step (4) is 0.1 mmol / L to 10 mmol / L.

[0048] Preferably, the concentration of cysteine ​​in the flushing solution in step (4) is 0.5 mmol / L to 5 mmol / L.

[0049] Specifically, optionally, the concentration of cysteine ​​in the flushing solution in step (4) is 1 mmol / L.

[0050] In one embodiment of the present invention, the concentration of cystine in the flushing solution in step (4) is 0.1 mmol / L to 5 mmol / L.

[0051] Preferably, the concentration of cystine in the flushing solution in step (4) is 0.1 mmol / L to 0.5 mmol / L.

[0052] Specifically, optionally, the concentration of cystine in the flushing solution in step (4) is 0.3 mmol / L.

[0053] In one embodiment of the present invention, the elution method in step (5) is isocratic elution.

[0054] In one embodiment of the present invention, the elution volume is 4 to 6 column volumes.

[0055] Specifically optionally, the elution volume is 5 column volumes.

[0056] In one embodiment of the present invention, the eluent in step (5) is acetic acid-sodium acetate buffer.

[0057] In one embodiment of the present invention, the concentration of sodium acetate in the eluent in step (5) is 20 to 100 mmol / L.

[0058] Preferably, the concentration of sodium acetate in the eluent in step (5) is 30 to 60 mmol / L.

[0059] In one embodiment of the present invention, the pH of the eluent in step (5) is 2-4.

[0060] In one embodiment of the present invention, the regeneration solution in step (6) is acetic acid or citrate buffer.

[0061] In one embodiment of the present invention, the pH range of the regeneration liquid in step (6) is pH 1.5 to pH 3.0.

[0062] In one embodiment of the present invention, when the regeneration liquid is acetic acid, the concentration of acetic acid is 0.5 mol / L to 3 mol / L.

[0063] In one embodiment of the present invention, when the regeneration solution is a citrate buffer, the concentration of citric acid is 0.5 mol / L to 1 mol / L.

[0064] In one embodiment of the present invention, the amount of regeneration liquid used in step (6) is 2 to 5 column volumes.

[0065] In one embodiment of the present invention, the amount of regeneration liquid used in step (6) can be specifically selected to be 3 column volumes.

[0066] Beneficial Effects

[0067] Other methods for redox of intact antibody molecules in vitro require the addition of oxidants in vitro to promote disulfide bond formation, thereby forming intact antibody molecules, and then ultrafiltration is required to remove the oxidants. Compared with the in vitro method, the present invention can remove host protein residues and potential viruses by adding redox agents during flushing. The biggest advantage is that disulfide bonds can be efficiently reoxidized to form intact molecules, thereby increasing the intact molecule content (95.9%), and no ultrafiltration step is required. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is the affinity chromatography spectrum of Example 1.

[0069] Figure 2 This is the CE-SDS (NR) test result of the untreated sample.

[0070] Figure 3 This is the CE-SDS (NR) test result of Example 1.

[0071] Figure 4 This is the CE-SDS (NR) test result of Comparative Example 1. DETAILED DESCRIPTION

[0072] The following examples are only provided to further illustrate the present invention and should not be construed as any limitation to the present invention.

[0073] Source of raw materials

[0074] CHO cells were purchased from ECACC; the preparation method of the harvest fluid of CHO cell culture expression: monoclonal CHO cells containing the target protein were cultured for 15 days in Hycell CHO culture medium at 25°C, and then centrifuged to retain the supernatant to obtain the harvest fluid of CHO cell culture expression.

[0075] Example 1

[0076] 1. Affinity chromatography:

[0077] Chromatography medium: Cytiva Mabseclect PrismA

[0078] Chromatography column: Column height 12.5cm, column volume 9.81mL

[0079] Chromatography equipment: AKTA Pure 150

[0080] Chromatography sample: harvested liquid from CHO cell culture expression (protein content is about 5.30 mg / mL)

[0081] Balance solution: 20mmol / L Tris-HCl, 150mmol / L NaCl, pH 7.2

[0082] Flushing solution: 20mmol / L Tris-HCl, 1mmol / L cysteine, 0.3mmol / L cystine, pH 7.2

[0083] Eluent: 50mmol / L acetic acid-sodium acetate pH 3.5

[0084] Regeneration liquid: 1mol / L acetic acid

[0085] Disinfectant: 0.1 mol / L sodium hydroxide

[0086] Sample conditioning solution: 2mol / L Tris

[0087] Experimental operation process: use 5 column volumes of equilibration solution to rinse the affinity chromatography column for equilibration, then load the harvested liquid expressed by CHO cell culture onto the affinity chromatography column (loading capacity is 50 g / L), then use 3 column volumes of equilibration solution to rinse the affinity chromatography column for equilibration, then use 12 column volumes of rinsing solution to rinse the affinity chromatography column, after the rinsing, rinse the affinity chromatography column with 5 column volumes of eluent to prepare for elution, and use 5 column volumes of eluent for isocratic elution. After the elution starts, UV280 is used as the judgment standard. When the UV280 absorbance value rises to 100 mAu, start collecting, and when the UV280 absorbance value drops to 100 mAu, stop collecting. The collection liquid is named affinity collection liquid, and the pH of the affinity collection liquid is adjusted to 6.0 with 2 mol / L Tris. After elution, use 3 column volumes of regeneration solution to elute the regeneration peak, then use 3 column volumes of equilibrium solution to rinse the regeneration solution, and then use 3 column volumes of disinfectant to rinse the chromatography column, and then store it.

[0088] The detection equipment used in the present invention mainly includes:

[0089] SEC-HPLC liquid phase detection equipment: liquid chromatography system (Thermo UltiMate3000), chromatographic column (ThermoMAbPac SEC-1), mobile phase 20mM PB+200mM Nacl pH 6.8; flow rate 0.5mL / min

[0090] CE-SDS (NR) capillary electrophoresis detection equipment: Capillary electrophoresis equipment: Beckman PA800 plus, Uncoated Capillary: SCIEX-338451; Sample buffer: Citric acid-phosphate pH 6.5 + 10% SDS, 250mM NEM (N-ethylmaleimide). Injection voltage -5kV, injection time 20s, separation voltage -15kV, separation time 40min.

[0091] Complete molecular weight detection equipment: Mass spectrometer: Thermo Q Exactive, chromatographic column: Waters AcquityBEH C4; glycosidase: PNGase F

[0092] Table 1 Affinity chromatography quality data of Example 1

[0093]

[0094] The fragment content in the initial antibody was 9.7%. Some disulfide bonds were broken during the culture process. After flushing, the disulfide bonds were reoxidized to form complete molecules, increasing the content of complete molecules from 90.0% to 95.9%.

[0095] Comparative Example 1

[0096] The in vitro redox procedure for intact antibody molecules is:

[0097] 1. In vitro redox

[0098] Experimental operation process: Add cysteine ​​and cystine with a final concentration of 1 mmol / L to the harvested liquid of CHO cell culture expression, and then stir and react at 18-26°C for 5 hours to obtain the oxidized harvested liquid. After the reaction is completed, immediately load it to affinity chromatography to remove cystine.

[0099] 2. Affinity chromatography:

[0100] Chromatography medium: Cytiva Mabseclect PrismA

[0101] Chromatography column: Column height 12.5cm, column volume 9.81mL

[0102] Chromatography equipment: AKTA Pure 150

[0103] Chromatographic sample: Harvest solution after oxidation (protein content is about 4.77 mg / mL)

[0104] Balance solution: 20mmol / L Tris-HCl, 150mmol / L NaCl, pH 7.2

[0105] Eluent: 50mmol / L acetic acid-sodium acetate pH 3.5

[0106] Regeneration liquid: 1mol / L acetic acid

[0107] Disinfectant: 0.1 mol / L sodium hydroxide

[0108] Sample conditioning solution: 2mol / L Tris

[0109] Experimental operation process: Use 5 column volumes of balance solution to wash the affinity chromatography column for balance, then load the oxidized harvest liquid onto the affinity chromatography column (load capacity is 50g / L), then use 3 column volumes of balance solution to wash the affinity chromatography column for balance, and immediately elute after balance. The elution uses 5 column volumes of eluent for isocratic elution. After the elution begins, use UV280 as the judgment standard. When the UV280 absorption value rises to 100mAu, start collecting, and when the UV280 absorption value drops to 100mAu, stop collecting. The collection liquid is named affinity collection liquid, and the pH of the affinity collection liquid is adjusted to 6.0 with 2mol / L Tris. After the elution is completed, use 3 column volumes of regeneration liquid to elute the regeneration peak, then use 3 column volumes of balance liquid to wash the regeneration liquid, and then use 3 column volumes of disinfectant to wash the chromatography column, and then store it.

[0110] The detection equipment used in the present invention mainly includes:

[0111] SEC-HPLC liquid phase detection equipment: liquid chromatography system (Thermo UltiMate3000), chromatographic column (ThermoMAbPac SEC-1), mobile phase 20mM PB+200mM Nacl pH 6.8; flow rate 0.5mL / min

[0112] CE-SDS (NR) capillary electrophoresis detection equipment: Capillary electrophoresis equipment: Beckman PA800 plus, Uncoated Capillary: SCIEX-338451; Sample buffer: Citric acid-phosphate pH 6.5 + 10% SDS, 250mM NEM (N-ethylmaleimide). Injection voltage -5kV, injection time 20s, separation voltage -15kV, separation time 40min.

[0113] Complete molecular weight detection equipment: Mass spectrometer: Thermo Q Exactive, chromatographic column: Waters AcquityBEH C4; glycosidase: PNGase F

[0114] Table 2 Comparative Example 1 Affinity Chromatography Quality Data

[0115]

[0116]

[0117] The fragment content in the initial antibody was 9.7%, and some disulfide bonds were broken during the culture process. After the oxidant was removed by in vitro oxidation reaction, the complete molecule content increased from 90.0% to 93.4%, which was lower than that in Example 1 (the complete molecule content in Example 1 was 95.9%).

[0118] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A chromatography method for improving the purity of an antibody containing a disulfide bond, characterized in that: In the chromatography method, Tris-HCl buffer to which cysteine ​​and cystine are added is used as a washing solution; The pH range of the Tris-HCl buffer is pH 6.0 to pH 8.0; the concentration of Tris-HCl in the Tris-HCl buffer is 10 mmol / L to 60 mmol / L, the concentration of cysteine ​​is 0.1 mmol / L to 10 mmol / L, and the concentration of cystine is 0.1 mmol / L to 5 mmol / L.

2. The chromatography method according to claim 1, characterized in that The concentration of cysteine ​​in the Tris-HCl buffer is 0.5 mmol / L to 5 mmol / L, and the concentration of cystine is 0.1 mmol / L to 0.5 mmol / L.

3. An affinity chromatography method for increasing the content of intact antibody molecules, characterized in that: The following steps are involved: (1) equilibrating an affinity chromatography column using an affinity equilibration solution; (2) loading the carrier with the antibody containing disulfide bonds onto an affinity chromatography column; (3) equilibrating the affinity chromatography column using an affinity equilibration solution; (4) washing the affinity chromatography column with a flushing solution; (5) using an eluent to elute the main peak of the antibody and recover it; (6) Use regeneration solution to elute the regeneration peak.

4. The affinity chromatography method according to claim 3, characterized in that: The affinity balancing solution in step (1) and step (3) is a Tris-HCl buffer; the Tris-HCl concentration in the Tris-HCl buffer is preferably 10 mmol / L to 60 mmol / L; the pH range of the Tris-HCl buffer is 6.5 to 7.5; and the Tris-HCl buffer further contains 100 to 200 mmol / L of sodium chloride.

5. The affinity chromatography method according to claim 3, characterized in that: The amount of affinity equilibration solution used in step (1) is 3 to 10 column volumes.

6. The affinity chromatography method according to claim 3, characterized in that: In step (2), the carrier is an affinity filler containing a Protein A ligand; the loading amount of the disulfide bond-containing antibody on the carrier is 10 g / L to 70 g / L.

7. The affinity chromatography method according to claim 3, characterized in that: The amount of affinity equilibration solution used in step (3) is 3 to 10 column volumes.

8. The affinity chromatography method according to claim 3, characterized in that: The amount of the flushing solution in step (4) is 3 to 20 column volumes; the flushing solution in step (4) is a Tris-HCl buffer added with cysteine ​​and cystine; the pH range of the flushing solution is 6.0 to 8.0; the concentration of Tris-HCl in the flushing solution is 10 mmol / L to 60 mmol / L; the concentration of cysteine ​​in the flushing solution is 0.1 mmol / L to 10 mmol / L; and the concentration of cystine in the flushing solution is 0.1 mmol / L to 5 mmol / L.

9. The affinity chromatography method according to claim 3, characterized in that: In step (5), the elution method is isocratic elution or linear elution; the eluent is acetic acid-sodium acetate buffer; the concentration of sodium acetate in the eluent is 20 to 100 mmol / L.

10. The affinity chromatography method according to claim 3, characterized in that: In step (6), the regeneration liquid is acetic acid or citric acid buffer; the pH range of the regeneration liquid is pH 1.5 to pH 3.0; and the amount of the regeneration liquid is 2 to 5 column volumes.

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