OPTIMIZED METHOD FOR BEVACIZUMAB PURIFICATION

Abstract

The present invention relates to: a method of purifying an antibody, which can prepare a desired antibody with high purity and high quality by removing impurities without using an expensive protein A column, and particularly, can purify an antibody in a high yield while greatly reducing an amount (volume) of a buffer used in an elution process; and an antibody prepared by the method.

Claims

1. A method of purifying an antibody, the method comprising: a step of loading a sample comprising an antibody and one or more host cell proteins (HCPs) and having a pH of 5.5 to 7.0 and a conductivity of 5 mS/cm to 8 mS/cm into an equilibrated cation exchange column, washing the cation exchange column with a washing buffer, and then eluting the antibody bound to the column with an elution buffer, wherein the washing of the cation exchange column with the washing buffer includes: 1) a step of washing the antibody with a buffer comprising a 10 mM to 50 mM phosphate with a pH of 5.5 to 7.0, comprising 0 mM to 38 mM sodium chloride, and the eluting of the antibody bound to the column with the elution buffer includes: 1) a first elution step of eluting the antibody with a buffer comprising a 10 mM to 50 mM phosphate with a pH of 6.0 to 7.0, comprising 38 mM to 50 mM sodium chloride; and 2) a second elution step of eluting the antibody with a buffer comprising a 10 mM to 50 mM phosphate with a pH of 6.0 to 7.0, comprising 50 mM to 60 mM sodium chloride.

2. The method of claim 1, wherein 1) the step of washing the antibody with a buffer comprising a 10 mM to 50 mM phosphate with a pH of 5.5 to 7.0, comprising 0 mM to 38 mM sodium chloride comprises the following three steps: a) a primary washing step of washing the antibody with a washing buffer comprising a 10 mM to 50 mM phosphate having a pH of 5.5 to 7.0 that does not comprise sodium chloride; b) a secondary washing step of washing the antibody with a washing buffer including a 10 mM to 50 mM phosphate having a pH of 5.5 to 7.0 that does not comprise sodium chloride; and c) a tertiary washing step of washing the antibody with a washing buffer comprising a 10 to 7.0 mM phosphate having a pH of 6.0 to 7.0 that comprises 20 to 38 mM sodium chloride.

3. The method of claim 1, wherein in the first elution step, the antibody is treated with 10 to 20 column volumes of the buffer.

4. The method of claim 1, wherein in the second elution step, the antibody is treated with 5 to 12 column volumes of the buffer.

5. The method of claim 1, wherein the antibody has an isoelectric point of 6 to 11.

6. The method of claim 1, wherein the antibody is bevacizumab.

7. The method of claim 1, wherein a functional group of the cation exchange column is selected from the group consisting of carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S).

8. The method of claim 7, wherein a functional group of the cation exchange column is Fractogel™ EMD SO.sub.3.

9. The method of claim 1, further comprising: a step of subjecting a filtrate recovered by the elution step to ultrafiltration and diafiltration; and a step of recovering the filtrate by allowing the filtrate to pass through a multi-layer filtration filter.

10. The method of claim 9, further comprising: removing the host cell protein using an anion exchange column after the step of recovering the filtrate by allowing the filtrate to pass through a multi-layer filtration filter.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0111] FIG. 1 illustrates an elution profile of the cation exchange chromatography step according to an exemplary embodiment of the present invention.

[0112] FIG. 2 is a CE-HPLC analysis graph of an eluate obtained according to an exemplary embodiment of the present invention.

[0113] FIG. 3 is a graph showing the suitable pH conditions of the ultrafiltration and diafiltration according to an exemplary embodiment of the present invention.

MODES OF THE INVENTION

[0114] Hereinafter, the present invention will be described in detail through the Examples. However, the following Examples are only for exemplifying the present invention, and the present invention is not limited by the following Examples.

[0115] Example 1: Bevacizumab Pre-treatment Step

[0116] A bevacizumab antibody was expressed by culturing recombinant CHO cells expressing the bevacizumab antibody. It was confirmed that the bevacizumab antibody according to exemplary embodiments of the present invention had an antibody titer at a level of 2 to 3, and the antibody was used in the experiment of the present invention.

[0117] In order to adsorb the antibody onto a cation exchange column, an experiment was performed by adding a 1 M glycine HCl (pH of 3.0) buffer to a culture solution to adjust the pH to 6.0 and adjusting the conductivity to 6.5 mS/cm to 8.5 mS/cm.

[0118] The amount of 1 M glycine-HCl (pH of 3.0) used for the pre-treatment of the culture solution for each test group, the amount of tertiary purified water added, and the loaded sample conditions after the pre-treatment are as follows in the following Table 1.

TABLE-US-00001 TABLE 1 Loaded sample after Culture 1M glycine-HCl Tertiary pre-treatment Conductivity solution (pH 3.0) purified water pH Conductivity 6.5 mS/cm 400 mL 17.40 mL 498.15 mL 6.02 6.48 7.0 mS/cm 400 mL 17.40 mL 450.15 mL 6.02 7.01 7.5 mS/cm 400 mL 17.56 mL 354.53 mL 6.00 7.5 8.0 mS/cm 454.97 mL   20.51 mL 363.57 mL 6.01 8.02 8.5 mS/cm 400 mL 17.42 mL 272.99 mL 6.03 8.45

[0119] Example 2: Cation Exchange Column Chromatography

[0120] In the present example, a Fractogel™ EMD SO.sub.3 column was used as a cation exchange column, and the process was as follows.

[0121] For equilibration, a 20 mM phosphate buffer (pH of 6.0) was flowed in an amount of 3 column volumes to equilibrate the column. Then, the supernatant for which the pre-treatment of Example 1 had been completed, was loaded at or below the adsorption capacity of SO.sub.3. The loading amount was 30 g or less of protein per 1 L resin volume, and the loading rate was 150 cm/hr.

[0122] Then, a three-step washing step and a two-step elution step were performed under the conditions shown in the following Table 2.

TABLE-US-00002 TABLE 2 Column Buffer volumes Washing 1 step 20 mM phosphate buffer, pH 6.0, 5 Washing 2 step 20 mM phosphate buffer, pH 6.48, 3 Washing 3 step 20 mM phosphate buffer including 5 37.5 mM NaCl, pH 6.48, 5.8 mS/cm Elution 1 step 20 mM phosphate buffer including 16 40 mM NaCl, pH 6.48, 6.2 mS/cm Elution 2 step 20 mM phosphate buffer including 7 58 mM NaCl, pH 6.48, 7.9 mS/cm

[0123] As can be confirmed above, in the present invention, the column volumes of the elution 1 step and the elution 2 step corresponding to the washing 4 step and the elution 1 step, respectively, of the following Comparative Example were reduced to 16 column volumes and 7 column volumes, respectively.

[0124] Meanwhile, for comparison, a Comparative Example was set under the conditions of the following Table 3 in which the column volumes in the washing 4 step (corresponding to the elution 1 step in the Examples) and the elution 1 step (corresponding to the elution 2 step in the Examples) were set to 30 column volumes and 20 column volumes, respectively.

TABLE-US-00003 TABLE 3 Column Buffer volumes Washing 1 step 20 mM phosphate buffer, pH 6.0, 5 Washing 2 step 20 mM phosphate buffer, pH 6.48, 3 Washing 3 step 20 mM phosphate buffer including 5 37.5 mM NaCl, pH 6.48, 5.8 mS/cm Washing 4 step 20 mM phosphate buffer including 30 40 mM NaCl, pH 6.48, 6.2 mS/cm Elution 1 step 20 mM phosphate buffer including 20 58 mM NaCl, pH 6.48, 7.9 mS/cm

[0125] In particular, in the case of the above Comparative Example, the column volume is used in the same form as the method actually used in the invention of Korean Patent No. 10-1569783.

[0126] Example 3: Confirmation of Yield and Charge Variant Content By Difference In Pre-treatment Conductivity

[0127] The changes in step yield and charge variant content were confirmed by subjecting the pre-treated bevacizumab sample prepared according to the conductivity according to Example 1 to cation exchange chromatography under the conditions of Example 2.

[0128] The results are shown in Tables 4 and 5.

TABLE-US-00004 TABLE 4 Total protein Loaded Eluate recovery Step Conductivity protein amount volume amount yield 6.5 mS/cm 409.5 mg 167.26 mL 348.0 mg 84.9% 7.0 mS/cm 402.7 mg 166.56 mL 389.1 mg 96.7% 7.5 mS/cm 400.7 mg 166.56 mL 373.1 mg 93.2% 8.0 mS/cm 422.1 mg 164.72 mL 388.6 mg 92.0% 8.5 mS/cm 394.8 mg 123.10 mL 117.8 mg 29.8%

TABLE-US-00005 TABLE 5 Experimental Acidic Main Basic Total group peak (%) peak (%) peak (%) basic (%) 6.5 mS/cm 23.1 72.9 1.6 4.0 7.0 mS/cm 20.6 75.8 0.8 2.1 7.5 mS/cm 21.5 74.8 0.9 2.5 8.0 mS/cm 16.7 79.4 1.2 3.0 8.5 mS/cm 12.4 79.4 3.6 8.1

[0129] Table 4 shows the results of confirming the change in yield according to the difference in pre-treatment conductivity. As can be confirmed in Table 4, it could be confirmed that a step yield of 90.0% or more at 7.0 mS/cm to 8.0 mS/cm was exhibited, and it was confirmed that the best step yield was exhibited particularly at 7.0 mS/cm.

[0130] Further, Table 5 shows the results of comparing the charge variant content according to the difference in pre-treatment conductivity. As can be confirmed in Table 5, a main peak similar to a control drug Avastin, and acidic isomer and basic isomer peaks at 7.0 mS/cm to 8.0 mS/cm according to the present invention, and particularly, a main peak, which is most similar to a control drug Avastin, and acidic isomer and basic isomer peaks at 7.0 mS/cm were exhibited.

[0131] In particular, when the elution process was performed through the use of a small amount of buffer with the change in conductivity as described above, excellent step yield and low basic isomer content could be confirmed.

[0132] Example 4: Confirmation of Increase In Yield According to Decrease In Column Volume

[0133] Under the conditions of Example 2, cation exchange chromatography was performed on the following sample having a conductivity of 7.0 mS/cm.

[0134] In the case of the pre-treated sample of Example 1, the sample concentration was 1.04 mg/ml, the loading volume was 162 ml, and the total protein dose was 168.8 mg.

[0135] In the purification process, the conditions of CV: 5 mL (pre-packed), bed height: 10.0 cm, flow rate: 0.67 mL/min or 0.84 mL/min were used.

[0136] Meanwhile, changes in yield were confirmed by testing the conditions of the Comparative Example in Table 3 together in the same manner as the above conditions.

[0137] The results are shown in Table 6.

TABLE-US-00006 TABLE 6 Yield Acidic Main Basic Total Process (%) peak (%) peak (%) peak (%) basic (%) Example 2 66.5 8.9 85.3 4.1 5.9 (Repetition 1) Example 2 66.5 9.1 85.1 4.1 5.9 (Repetition 2) Example 2 66.9 8.8 85.3 4.2 5.9 (Repetition 3) Comparative 48.1 6.8 87.2 3.9 6.0 Example

[0138] As confirmed above, the yield during the application of the purification process of the Comparative Example was only about 50%. Meanwhile, when the method according to the present invention was used, a high yield of about 60% or more was shown, although the time of the washing process and the elution process and the amount of buffer used were significantly reduced. In addition, it was confirmed that a charge variant content was exhibited at a level suitable for biosimilar preparation even in terms of charge variant content.

[0139] Example 5: Virus Inactivation

[0140] Viruses were inactivated at a pH of 3.8 for 1 hour by adding a 1 M glycine-HCl (pH of 3.0) buffer to the eluate subjected to the cation exchange chromatography according to Example 2. After the inactivation was completed, the sample was allowed to pass through a 0.2 pm filter, and then the pH of the sample was adjusted to 6.0 by adding a 1 M Tris-HCl (pH of 9.0) buffer.

[0141] Example 6: Ultrafiltration and Diafiltration Process

[0142] In consideration of the sample volume, a micro-centricon (0.5 mL) was used in the ultrafiltration and diafiltration (UF/DF1) process, and the process was performed in triplicate under each condition.

[0143] Amicon Ultra 0.5 mL Ultracel was used as the micro-centricon, and an Eppendorf centrifuge was used as a centrifuge used for concentration and buffer exchange. In order to adjust an initial concentrated concentration of 20 mg/mL, 400 μl (5.13 mg/mL) of UF/DF1 injection sample was put into the micro-centricon, and centrifugation was performed at 5,000 rpm for 10 minutes to concentrate the sample to 100 μl. The sample was diluted 2-fold by adding 100 μl of the buffer at each pH in Table 7 to the sample concentrated to 100 μl, and then centrifuged again at 5,000 rpm for 5 minutes to concentrate the sample to 100 μl. UF/DF1 was completed by performing the above process 7 times.

[0144] For the most accurate experiment, micro-centricon, UF/DF1 injection sample, and post-UF volumes were all measured by weight.

TABLE-US-00007 TABLE 7 Measured values Buffer Cond. Name composition pH (mS/cm) Experiment 1 20 mM Na-phosphate pH 6.0 5.988 1.61 Experiment 2 20 mM Na-phosphate pH 6.5 6.519 1.89 Experiment 3 20 mM Na-phosphate pH 7.0 7.000 2.53 Experiment 4 50 mM Tris-HCl, 6.997 6.36 20 mM NaCl pH 7.0 Experiment 5 50 mM Tris-HCl, 7.504 5.84 20 mM NaCl pH 7.5

[0145] For the application of an integration event for the SE-HPLC analysis, the aggregate content was analyzed by applying the integration event defined in the SOP.

[0146] The results are shown in Table 8.

TABLE-US-00008 TABLE 8 SE-HPLC (%) Name No. Aggregate AVG. Aggregate 50 mM Tris-HCl, 1 5.24 5.44 20 mM NaCl pH 7.5 2 4.76 3 6.33 50 mM Tris-HCl, 1 4.82 4.68 20 mM NaCl pH 7.0 2 4.64 3 4.59 20 mM Na-phosphate 1 3.95 3.70 pH 7.0 2 3.57 3 3.54 20 mM Na-phosphate 1 3.09 2.98 pH 6.5 2 2.92 3 2.95 20 mM Na-phosphate 1 1.82 1.83 pH 6.0 2 1.83 3 1.84

[0147] As a result of the aggregation content analysis under each pH condition, it was confirmed that the aggregation content increased with the change in pH as can be confirmed in Table 8.

[0148] In addition, as can be confirmed in FIG. 3, as a result of confirming the linearity with respect to an independent variable (pH) in order to confirm the significance of the increase in aggregate content in the UF/DF1 process according to the increase in pH, a linear equation of Y=2.4x-12.6 was confirmed, and R2 was confirmed to be 0.88. Since the slope value (2.4) has a positive value, it was confirmed that the dependent variable (aggregate content) on the Y-axis increases as the independent variable (pH) on the X-axis increases.

[0149] Through the results, it was determined that it is difficult for the conditions of pH 7.5 or higher using a Tris-HCl buffer to be applied to the process because the aggregate content increases in the UF/DF1 step. In contrast, it was confirmed that it was suitable to apply the condition of 20 mM Na-phosphate at pH 6.0.

[0150] Example 7: Multi-layer Filtration Filtering Process

[0151] The multi-layer filtration filter may reduce host-derived DNA (HCD), host-derived protein (HCP), and the like due to its electrostatic properties and the like. Thus, an XOHC-type multi-layer filtration filter was prepared, and tertiary purified water and a 20 mM sodium phosphate (pH of 6.0) buffer were flowed for substitution and equilibration. Then, after viruses were inactivated, the sample of Example 6 that had been subjected to a filtration process was substituted with a 20 mM sodium phosphate (pH of 6.0) buffer, and then filtered at a flow rate of 100 LMH(liter/m2/hour) by flowing the substituted sample into the multi-layer filtration filter. All the filtered samples were recovered and used as samples for the following anion exchange column chromatography purification.

[0152] Example 8: Anion Exchange Column Chromatography

[0153] Since the anion exchange column adsorbs anion-bearing proteins at the isoelectric point or more, in the case of an antibody with an isoelectric point of 7 or less (in the case of bevacizumab, its isoelectric point is 8.3), when a buffer with a neutral pH is used, this antibody does not adhere to the anion exchange resin and escapes into the flow-through. Thus, the following experiments were conducted in order to investigate the anion exchange resin and buffer solution conditions suitable for the preparation process of the present invention.

[0154] Specifically, in the present example, purification was performed using quaternary amine series Q sepharose™ Fastflow, which is frequently used as an anion exchange resin on a production scale.

[0155] First, a sample for loading into the anion exchange resin was prepared to have a pH of 6.0 and a conductivity of 1.4 mS/cm by completing cation exchange column chromatography for a culture supernatant and virus inactivation as in the Examples, subjecting the sample to primary ultrafiltration, and then substituting the sample with a 50 mM sodium phosphate (pH of 6.0) equilibration buffer.

[0156] The loading amount was 30 g or less of protein per 1 L resin volume, the loading and elution rate was 150 cm/hr, and a column flow-through was collected when the absorbance at A280 nm increased. The column was regenerated with 1 M NaCl and then equilibrated with an equilibration buffer.

[0157] The step yield under each pH condition was confirmed by subjecting the bevacizumab sample prepared according to Example 7 to anion exchange chromatography by the method as described above.

[0158] The results are shown in Table 9.

TABLE-US-00009 TABLE 9 pH Condition Step yield (%) 6.0 96.6% 7.0 95.4% 7.5 93.2% 8.0 90.3% 8.5 62.8%

[0159] Table 9 shows the results of confirming the change in step yield according to the difference in pH condition. As can be confirmed in Table 9, it could be confirmed that when anion exchange chromatography was performed, a step yield of 90.0% or more in a pH range of 6.0 to 8.0 was exhibited, and particularly, it was confirmed that the best step yield was exhibited at a pH of 6.0.

[0160] From the foregoing description, it will be understood by those skilled in the art to which the present invention pertains that the present invention can be implemented in other concrete forms without modifying the technical spirit or essential features of the present invention. In this regard, it should be understood that the above-described embodiments are only exemplary in all aspects and are not restrictive. The scope of the present invention is represented by the claims to be described below rather than the detailed description, and it should be interpreted that the meaning and scope of the claims and all the changes or modified forms derived from the equivalent concepts thereto fall within the scope of the present invention.