NON-PROTEIN A PURIFICATION METHOD FOR ADALIMUMAB

Abstract

The present invention relates to a method of preparing a population of antibodies, whereby a desired high-purity and high-quality population of antibodies can be prepared by removing impurities without using an expensive protein A column, and in particular, production costs can be significantly reduced while achieving process automation; and a population of antibodies prepared thereby.

Claims

1. A method of preparing a population of antibodies, the method comprising: (a) a step of removing a host cell protein (HCP) and an isomeric antibody from a sample comprising a mixed solution of antibodies, comprising loading the sample comprising the mixed solution of antibodies into an equilibrated cation exchange column, washing the cation exchange column, and then eluting antibodies bound to the column with an elution buffer; (b) a step of removing the host cell protein (HCP) and the residual DNA from an antibody eluate, comprising loading a sample obtained by mixing a salt with an antibody eluate eluted in Step (a) into a hydrophobic interaction column and eluting antibodies bound to the column with an elution buffer; (c) a step of removing the host cell protein (HCP) and the residual DNA, including collecting a flow-through by allowing an antibody eluate from which the host cell protein (HCP) and the residual DNA in Step (b) have been removed to pass through an anion exchange column; (d) a step of removing viruses by allowing the flow-through in Step (c) to pass through a virus filter; and (e) a step of concentrating the antibody eluate eluted in Step (d), performing buffer exchange and preparing a population of antibodies containing the residual DNA and the host cell protein at a concentration of 10 ppb and 10 ppm or less, respectively.

2. The method of claim 1, wherein a sample comprising a mixed solution of antibodies in Step (a) is prepared by a method comprising a step of removing a precipitated precipitate by adjusting the pH of a culture supernatant to 4 to 6.

3. The method of claim 1, wherein the sample comprising the mixed solution of antibodies in Step (a) has a conductivity of 5 mS/cm to 7 mS/cm.

4. The method of claim 1, wherein the antibody has an isoelectric point of 7 to 10.

5. The method of claim 1, wherein the antibody is adalimumab.

6. The method of claim 1, wherein the antibody eluate eluted in Step (a) comprises 60% or more of a main active antibody, 20% or less of an acidic isomeric antibody, and 20% or less of a basic isomeric antibody.

7. The method of claim 1, wherein the step of loading the sample comprising the mixed solution of antibodies into an equilibrated cation exchange column comprises a step of loading the sample comprising the mixed solution of antibodies into a Fractogel COO.sup.- column equilibrated with an equilibration buffer having a pH of 4.5 to 5.5 and comprising 15 mM to 30 mM acetate and 35 mM to 45 mM sodium chloride.

8. The method of claim 1, wherein the step of washing the cation exchange column comprises: 1) a first washing step of washing the column with a buffer having a pH of 4.5 to 5.5 and comprising 15 mM to 30 mM acetate and 35 mM to 45 mM sodium chloride; 2) a second washing step of washing the column with a buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate and 55 to 59 mM sodium chloride.

9. The method of claim 8, wherein the buffer in the second washing step is prepared so as to have a sodium chloride molar concentration of 55 to 59 mM by mixing a buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate and a buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate and sodium chloride having a predetermined molar concentration.

10. The method of claim 1, wherein the step of eluting the antibody bound to the column with the elution buffer comprises: 1) a first elution step of eluting an antibody with a buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate and 57 to 63 mM sodium chloride; 2) a second elution step of eluting the antibody with a buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate and 67 to 73 mM sodium chloride; and 3) a third elution step of eluting the antibody with a buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate and 77 to 83 mM sodium chloride.

11. The method of claim 10, wherein the buffers in the first, second and third elution steps are prepared so as to have predetermined sodium chloride molar concentrations in the first, second and third elution steps by mixing a buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate and a buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate and sodium chloride having a predetermined molar concentration.

12. The method of claim 1, wherein Step (a) comprises: 1) a step of loading a mixed solution of antibodies into a Fractogel COO.sup.- cation exchange column equilibrated with an equilibration buffer having a pH of 4.5 to 5.5 and comprising 15 mM to 30 mM acetate and 35 mM to 45 mM sodium chloride; 2) a first washing step of washing the column with a buffer having a pH of 4.5 to 5.5 and comprising 15 mM to 30 mM acetate and 35 mM to 45 mM sodium chloride; 3) a second washing step of washing the column with a buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate and 55 mM to 59 mM sodium chloride; 4) a first elution step of eluting an antibody with a buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate and 57 to 63 mM sodium chloride; 5) a second elution step of eluting the antibody with a buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate and 67 to 73 mM sodium chloride; and 6) a third elution step of eluting the antibody with a buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate and 77 to 83 mM sodium chloride.

13. The method of claim 1, wherein Step (b) elutes the antibody by a concentration gradient method.

14. The method of claim 13, wherein the concentration gradient method comprises a step of eluting an antibody by loading a sample in which the antibody eluate eluted in Step (a) is adjusted to a citrate concentration that is the same as that of an equilibration buffer into a hydrophobic interaction column equilibrated with an equilibration buffer comprising 25 mM to 35 mM acetate (pH of 5.5 to 6.5) and 0.3 M to 1.0 M citrate and applying an elution buffer having a pH of 5.5 to 6.5 and comprising 25 mM to 35 mM acetate in a concentration gradient manner.

15. The method of claim 1, wherein the hydrophobic interaction column in Step (b) is a phenyl sepharose column.

16. The method of claim 1, wherein the anion exchange column in Step (c) is equilibrated with an equilibration buffer having a pH of 7.0 to 8.0 before injection of the sample.

17. The method of claim 16, wherein the equilibration buffer comprises Tris-HCI having a pH of 7.0 to 8.0.

18. The method of claim 1, wherein the anion exchange column in Step (c) is a Q Fast Flow column.

19. A population of antibodies prepared by the method of claim 1, wherein the population of antibodies comprises 60% or more of a main active antibody.

20. A population of antibodies prepared by the method of claim 1, wherein a concentration of the residual DNA and the host cell protein is 0.1 ppb and 5 ppm or less, respectively.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0121] FIG. 1 is a flow chart illustrating a process of preparing an adalimumab antibody according to the present invention.

[0122] FIG. 2 illustrates the results of cation exchange chromatography performed by a method similar to the method described in Korean Patent No. 10-1498771.

[0123] FIG. 3 illustrates the results of performing cation exchange chromatography according to Example 2-1 of the present invention.

[0124] FIG. 4 illustrates the results of performing cation exchange chromatography according to Example 2-2 of the present invention.

[0125] FIG. 5 illustrates the results of performing hydrophobic interaction chromatography according to the present invention.

[0126] FIG. 6 illustrates the results of performing anion exchange resin chromatography according to the present invention.

MODES OF THE INVENTION

[0127] 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.

[0128] Hereinafter, the flow chart of the antibody preparation process according to exemplary embodiments is specifically shown in FIG. 1. Specifically, a culture solution sample including the antibody according to the present invention is recovered, filtered, and then allowed to pass through a cation exchange resin. Then, the sample is subjected to virus inactivation and allowed to pass through a hydrophobic interaction resin. Next, the sample is subjected to primary ultrafiltration, and then allowed to pass through an anion exchange resin. Finally, the sample is allowed to pass through a virus filter and subjected to secondary ultrafiltration, and then formulation is performed. The specific processes briefly described above will be specifically described in the following examples.

Example 1: Pre-Treatment Method of Culture Solution for Antibody Purification

[0129] After an adalimumab antibody was expressed by culturing recombinant CHO cells expressing the adalimumab antibody, pH was lowered to 6 or less in order to adsorb the antibody onto a cation exchange column.

[0130] In the present example, the degree of removal of impurities was confirmed by the pre-treatment method of a culture solution.

[0131] In the present example, a method of preparing a sample for injecting into a cation exchange column through a filtration filter after lowering the pH to 5 in a state of a culture solution including cells was used, and the specific conditions thereof are shown in Table 1.

TABLE-US-00001 Procedure Method 1 Culture solution 2 Supernatant is recovered by removing cells with a first filtration filter (depth Filter) and a second filtration filter (sterilization filter) 3 Adjust conductivity 4 pH is lowered by adding 10% acetic acid to supernatant 5 Stir at low speed at room temperature (about 25° C.) for 1 hour 6 Precipitate is removed and bacteria are sterilized with filtration and sterilization filters

[0132] The culture solution is pre-treated by the above method, and turbidities of the culture solution of Procedure (1), the supernatant of Procedure (2), the culture solution after acid treatment of Procedure (4), and the pre-treatment culture solution after filtration and sterilization filters of Procedure (6) were analyzed. As a result, in the case of the culture solution of (1), the turbidity was confirmed to be 6810 NTU, and in the case of the supernatant of (2), the turbidity was confirmed to be 2.98 NTU. Further, in the case of the culture solution after acid treatment of (4), the turbidity was confirmed to be 5475 NTU, and in the case of the pre-treated culture solution after filtration and sterilization filters of final (6), the turbidity was confirmed to be 5.13 NTU.

[0133] In addition, it could be confirmed that the average flux showed a large filtration capacity around 150 LMH in the filtration step, and thus loss was small in the pre-treatment stage of the culture solution.

[0134] It was confirmed that a pre-treated culture solution suitable for use in the subsequent steps was prepared by showing a turbidity removal rate of about 99% or more after the filtration treatment at each step.

[0135] The results show that a method of performing a process after primary removal of cells with an initial filtration filter as the pre-treatment method of the present invention is suitable. In addition, the results showed that when the precipitate was removed by lowering the pH to 6 or less (preferably pH 5), a culture supernatant having a much higher purity than the initial culture solution could be obtained.

Example 2: Cation Exchange Chromatography

[0136] Among the cation exchange columns capable of replacing a column process using Protein A, Fractogel COO.sup.- was selected as a column having a functional group advantageous in terms of purity and yield.

[0137] An experiment was performed to adjust an isomeric antibody using the cation exchange column set above, and the process thereof is as follows.

[0138] For equilibration, the column was equilibrated (6.0 mS/cm) by flowing an amount of 14 column volumes of a buffer having a pH of 5.0 and including 20 mM and 40 mM sodium chloride, and then the pre-treated supernatant was loaded below the adsorption capacity of CM (25 mg/mL column).

[0139] After loading, a washing 1 step of performing treatment with a washing 1 buffer (buffer having a pH of 5.0 and including 20 mM acetate and 40 mM sodium chloride, 6.0 mS/cm) in an amount of 5 column volumes to attach the antibody which was not attached to the column and washing the remaining supernatant was performed.

[0140] Then, a washing 2 step was performed by setting a first buffer (pH 6.0, 30 mM acetate, 2.4 mS/cm) and a second buffer (buffer having a pH of 6.0 and including 30 mM acetate and 100 mM sodium chloride, 12.5 mS/cm) to a total amount of 15 column volumes to perform treatment with the first buffer and the second buffer at a ratio of 43 wt% and 57 wt%.

[0141] Here, a washing 2 step was performed in the form of a buffer in which the first buffer and the second buffer was mixed at a ratio of 43% and 57%, respectively.

[0142] Then, in order to perform a desorption step, the desorption step was performed while decreasing and increasing the first buffer and the second buffer in three steps of 40 wt%, 30 wt% and 20 wt%; and 60 wt%, 70 wt% and 80 wt%, respectively.

[0143] After each mixing was performed preferentially as in the above washing 2 step, such as two stages of gastric lavage, a three-step elution (desorption) step was performed using the mixed buffer.

[0144] Unlike a general method of the existing cation exchange chromatography, in the present invention, the washing step was significantly reduced to two steps compared to the technique in the related art, while simplifying the types of buffer solutions to three.

[0145] Furthermore, the step for performing desorption also enabled automation and simplification of the entire step by performing the reaction while adjusting only the ratio of the solution of the first buffer and the second buffer.

[0146] Further, in the cation exchange chromatography according to the present example, a flow rate which was 1.5-fold faster than the 180 cm/hr of the conventionally known methods was used. In the case of such a high flow rate, it is possible to have a higher production rate over time than that of the existing method.

[0147] Hereinafter, Table 2 specifically describes the process of purifying the isomeric antibody using the cation exchange resin Fractogel COO.sup.- according to the present invention.

TABLE-US-00002 Procedure Buffer step Example 2-1 Example 2-2 Equiliabration pH 5.0, 20 mM acetate and 40 mM sodium chloride 14 column volumes 14 column volumes Loading Conductivity Con 6.4 mS/cm or less, adsorption capacity: 25 mg/column mL or less Washing 1 pH 5.0, 20 mM acetate and 40 mM sodium chloride 5 column volumes 5 column volumes Washing 2 pH 6.0, 30 mM acetate (43%); pH 6.0, 30 mM acetate and 100 mM sodium chloride (57%) 15 column volumes 15 column volumes Desorption 1 pH 6.0, 30 mM acetate (40%); pH 6.0, 30 mM acetate and 100 mM sodium chloride (60%) 15 column volumes 8 column volumes Desorption 2 pH 6.0, 30 mM acetate (30%); pH 6.0, 30 mM acetate and 100 mM sodium chloride (70%) 9 column volumes 15 column volumes Desorption 3 pH 6.0, 30 mM acetate (20%); pH 6.0, 30 mM acetate and 100 mM sodium chloride (80%) 14 column volumes 9 column volumes Strip 2 M NaCl 2 column volumes 2 column volumes Column regeneration 1 M NaOH 3 column volumes 3 column volumes

[0148] Meanwhile, for comparison, elution was performed by adding a NaCl-free equilibration step in a manner similar to the prior Korean Patent No. 10-1498771. Specifically, treatment was performed with pH 5.0, 20 mM acetate and 40 mM sodium chloride in a total amount of 5 column volumes in washing 1 step; with pH 6.0, 30 mM acetate in a total amount of 10 column volumes in washing 2 step; with pH 6.0, 30 mM acetate and 50 mM sodium chloride in a total amount of 10 column volumes in desorption 1 step; with pH 6.0, 30 mM acetate in an amount of 1.5 column volumes in desorption 2 step; with pH 6.0, 30 mM acetate and 80 mM sodium chloride in an amount of 8 column volumes in desorption 3 step (Comparative Example).

[0149] The experimental results are shown in FIGS. 2 to 4.

[0150] FIG. 2 illustrates the results of cation exchange chromatography according to the Comparative Example, FIG. 3 illustrates the results of Example 2-1 according to the present invention, and FIG. 4 illustrates the results of Example 2-2 according to the present invention.

[0151] The ratios and yields of the acidic isomeric antibody, the main active antibody and the basic isomeric antibody confirmed through the cation exchange chromatography are shown in the following Table 3.

TABLE-US-00003 Example 2-1 Example 2-2 Comparative Example Acidic isomeric antibody peak (%) 16 to 19 14 to 19 15 to 16 Main active antibody peak (%) 66 to 69 65 to 69 68 Basic isomeric antibody peak (%) 13 to 15 13 to 19 16 Yield (%) 60 to 67 67 to 89 52 to 56

[0152] The purification process using the conditions of the cation exchange column as described above significantly increased the content of the main active antibody and also significantly increased the yield. In addition, process automation was made possible by using only three types of buffer solutions in collecting the protein to be eluted. Such advantages show an additional advantage of reducing the costs of buffer preparation and footprinting during antibody production.

[0153] The biggest advantage is that the convenience of obtaining antibodies in high yield is enhanced under established conditions, which can be suitably applied to process automation because the above three types of buffers are sequentially injected and proteins are collected based on column volume during elution,

Example 3: Virus Inactivation

[0154] Viruses were inactivated at a pH of 3.8 for 1 hour by adding a 1 M citric acid buffer to the primary eluates obtained in Examples 2-1 and 2-2. After the inactivation was completed, the pH of the sample was adjusted to 6.0 by adding a 2 M Trizma base buffer. The virus-inactivated sample was allowed to pass through a 0.2-.Math.m filter and filtered.

Example 4: Hydrophobic Interaction Resin

[0155] A process of increasing the purity of the antibody during antibody purification was performed using Phenyl Sepharose Fast Flow, which is a type of hydrophobic interaction chromatography (HIC).

[0156] Specifically, each eluate prepared by the method of Example 3 was used in order to perform HIC.

[0157] A method of performing elution by adsorbing the acetate at a concentration of 0.6 M citrate based on pH 6.0, and giving the elution buffer a concentration gradient of up to 5 column volumes during elution was used.

[0158] The base buffer in the cation exchange resin uses acetate with a pH of 6.0 or less because the use of a pH of 6.0 or less has an advantage in that the preparation process can be simplified in terms of buffer preparation.

[0159] In the elution method, the 5-column volume concentration gradient method showed excellent results in terms of yield, and in the case of a buffer, an acetate buffer with a pH of 6.0 was also excellent in terms of yield and antibody pH stability.

[0160] Specific conditions for the above hydrophobic interaction chromatography (HIC) are shown in the following Table 4.

TABLE-US-00004 HIC loading solution: Fractogel COO.sup.- (M) eluate+ Eluate and equal volume of buffer including 60 mM acetate with pH 6.0 and 1.2 M citrate Equilibration buffer: 30 mM acetate with pH 6.0 + 0.6 M citrate Elution condition: 30 mM acetate (5 column volume gradient elution)

[0161] Hydrophobic interaction chromatography was performed under the above conditions, and the results are shown in FIG. 5.

Example 5: Primary Ultrafiltration

[0162] A Pellicon 3 Cassette (membrane) was equilibrated with a 25 mM Tris-HCl buffer (pH 7.5, 2.0 mS/cm) under conditions of Feed pressure ≤ 1 bar, and a process solution was concentrated to 10 mg/mL. Thereafter, the primary ultrafiltration was performed by performing buffer exchange such that the pH and conductivity of the process solution were 7.5 and ≤ 3.0 mS/cm.

Example 6: Anion Exchange Resin Chromatography

[0163] In the preparation method of the present invention, a process of preparing a population of antibodies having a higher purity was investigated using anion exchange resin chromatography.

[0164] Specifically, since the anion exchange column adsorbs cation-bearing proteins at the isoelectric point or higher, in the case of an antibody with an isoelectric point of 7 or higher (for example, in the case of adalimumab, the isoelectric point is 7 to 10, and in the case of Humira, the isoelectric point is approximately 8.4), when a neutral pH buffer is used, this antibody escapes to the flow-through without being attached to the anion exchange resin. Thus, the following experiments were conducted in order to investigate the conditions of the anion exchange resin and the buffer solution suitable for the preparation process of the present invention.

[0165] Specifically, in the present example, purification was performed using quaternary amine series Q Fast Flow (QFF, GE), which is frequently used as an anion exchange resin on a production scale. First, as a sample preparation for loading into the anion exchange resin, suitable conductivity and pH were prepared by substituting the buffer in a culture supernatant through a cation exchange column, a hydrophobic interaction column (HIC) and primary ultrafiltration. The purity, host cell protein content, residual DNA content and yield were confirmed under the conditions of 25 mM Tris HCl with a pH of 7.5 as a buffer.

[0166] The results of chromatography using an anion exchange resin are shown in FIG. 6.

[0167] In the prepared population of antibodies, the purity was 100%, the HCP content was 6.8 ng/mg, the residual DNA content was 0.04 pg/mg, and the yield was 94 to 97%. As a result, it was suggested that the buffer in the anion exchange resin chromatography was a buffer including Tris HCl, and that the pH of 7 to 8 was advantageous for the preparation of the population of antibodies of the present invention.

Example 7: Virus Filtering, Secondary Ultrafiltration and Diafiltration

[0168] The sample subjected to the anion exchange resin chromatography of Example 6 was filtered using a virus filter Modus 1.3 (Merck).

[0169] Then, secondary ultrafiltration was performed using a Pellicon 3 Cassette (membrane) filter. Specifically, a Pellicon 3 Cassette (membrane) was equilibrated with a 14 mM phosphate buffer (pH 5.2, 12.0 mS/cm) under conditions of Feed pressure ≤ 1 bar, and a process solution was concentrated to 55.5 mg/mL. Thereafter, buffer exchange was performed such that the pH and conductivity of the process solution were 5.2 and ≤ 11.0 mS/cm.

[0170] The virus filtering as described above performs virus filtering before the secondary ultrafiltration is performed, unlike the method in the existing prior patent Korean Patent No. 10-1498771. In the aforementioned prior patent, it is difficult to prepare a high-concentration drug because virus filtering is performed after performing primary ultrafiltration and secondary ultrafiltration. In contrast, the method according to the present invention has an advantage in that by first performing virus filtering before secondary ultrafiltration to reduce the loading amount in the secondary ultrafiltration step, protein quality is secured and simultaneously the yield loss is lowered, and a high-concentration drug is easily prepared.

Example 8: Confirmation of Changes in Host-Derived Protein (HCP) and DNA Content According to Entire Process in Large Batch

[0171] According to the procedures of Examples 1 to 7, the host-derived protein and DNA contents in the entire process were confirmed, and the results thereof are shown in the following Table 5.

[0172] In the case of the samples shown in the following Table 5, the results are for the samples obtained by the preparation process according to Example 2-2.

TABLE-US-00005 Adalimumab Test Item Volume (kg) Concentration (mg/ml) HCP content (ng/mg) DNA content (pg/mg) Day 13 of main culture HCP, endotoxin, microorganisms 4.945268 17.3 N/A After main culture depth filter HCP, endotoxin, microorganisms 187.2 3.746587 20.5 N/A After main culture micro filtration HCP, endotoxin, microorganisms 217.9 3.788743 20.3 N/A CEX Pool (Example 2) HCP, residual DNA 624.7 0.7 N/A 0.1 Virus Inactivation (Example 3) HCP 655.8 0.7 N/A 0.73 HIC process eluate (Example 4) HCP, Residual DNA 172.6 2.4 29.4 0.09 UF/DF1 process solution (Example 5) HCP, Residual DNA 48 8.1 N/A 0.11 AEX eluate (Example 6) HCP, residual DNA 56.1 6.5 6.8 0.04 VF process solution (Example 7 Virus filter) HCP, residual DNA 56.8 6.2 8.1 0.03 UF/DF2 process solution (Example 7 Ultrafiltration) HCP, residual DNA 6.4 54.4 1.7 0.02 DS (Example 7 Final population of antibodies) 7 49.2 1.6 0

[0173] As can be seen in Table 5, the HCP content and DNA content were confirmed for each step. As a result, the content of host cell-derived DNA was significantly reduced through the cation exchange chromatography described in Example 2-2. Then, virus inactivation was preferentially performed, and protein quality was secured and yield loss was lowered at the same time through ultrafiltration/diafiltration in a step immediately before formulation, facilitating high-concentration drug preparation. Furthermore, it was possible to prepare a population of antibodies in which the contents of host-derived protein and DNA were minimized through the above series of steps.

Example 9: Confirmation of Virus Removal Ability According to Entire Process in Large Batch

[0174] Among the entire process performed according to the procedures of Examples 1 to 7, the virus removal rate in the low pH treatment, anion exchange chromatography, and virus reduction filtration processes was confirmed, and the results thereof are shown in the following Table 6.

TABLE-US-00006 LOG REDUCTION FACTORS LOW pH TREATMENT RUN 1 RUN 2 Murine leukemia virus (MLV) 6.57 ± 0.35 log.sub.10 6.61 ± 0.40 log.sub.10 Pseudorabies virus (PRV) ≥ 6.51 ± 0.34 log.sub.10 ≥6.51 ± 0.29 log.sub.10 ANION EXCHANGE CHROMATOGRAPHY RUN 1 RUN 2 MLV ≥ 6.47 ± 0.38 log.sub.10 ≥ 6.55 ± 0.28 log.sub.10 PRV ≥ 6.28 ± 0.34 log.sub.10 ≥ 6.46 ± 0.38 log.sub.10 Reovirus type-3 (Reo 3) ≥ 8.47 ± 0.29 log.sub.10 ≥ 8.21 ± 0.31 log.sub.10 Mouse minute virus (MMV) ≥ 6.49 ± 0.25 log.sub.10 ≥ 6.49 ± 0.32 log.sub.10 VIRUS REDUCTION FILTRATION RUN 1 RUN 2 MLV ≥ 5.52 ± 0.36 log.sub.10 ≥ 5.44 ± 0.25 log.sub.10 PRV ≥ 5.60 ± 0.25 log.sub.10 ≥ 5.52 ± 0.31 log.sub.10 Reo 3 ≥ 5.60 ± 0.31 log.sub.10 ≥ 5.34 ± 0.30 log.sub.10 MMV ≥ 6.05 ± 0.23 log.sub.10 ≥ 6.40 ± 0.36 log.sub.10

[0175] As can be seen in Table 6, the inactivation and removal ability of MLV, PRV, Reo3, and MMV viruses were confirmed for each step. The results show that there is almost no expression of virus in the population of antibodies according to the process according to the present invention. In particular, in consideration of the virus inactivation and removal ability in each of the steps, it is shown that even though there is unexpected virus expression or contamination in the purification process according to the present invention, it can be removed.

[0176] 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.