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Method for antibody purification including step where activated carbon material is used

A technology of activated carbon and antibodies, which is applied in the field of high-purity purified antibodies to achieve high virus clearance performance, reduce purification steps and production costs, and reduce the burden of impurities

Pending Publication Date: 2021-08-24
DAIICHI SANKYO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, these reports only illustrate the possibility of removing general impurities (proteins, DNA) originating from the host cell and product-related impurities (e.g. aggregates, variants) originating from the desired product

Method used

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  • Method for antibody purification including step where activated carbon material is used
  • Method for antibody purification including step where activated carbon material is used

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0186] Embodiment 1: common method

[0187] In this example, methods commonly used in Example 2 and subsequent examples will be described.

[0188] 1) Determination of antibody concentration

[0189] Antibody concentration was determined by HPLC using a PA ID Sensor Cartridge (2.1 mm x 3 mm, Applied Biosystems) column.

[0190] 2) HCP determination

[0191] HCP was determined by enzyme-linked immunosorbent assay (ELISA) using the third generation CHO host cell protein kit from Cygnus Technologies or the CHO|360 HCP ELISA kit from BioGENES GmbH. The HCP concentration obtained was converted to a value of ng HCP / mg antibody by dividing by the antibody concentration.

[0192] In Examples 2 to 5 the third generation CHO host cell protein kit from Cygnus Technologies was used, while in Examples 6 to 8 the CHO|360 HCP ELISA kit from BioGENES GmbH was used.

[0193] 3) PLBL2 assay

[0194] PLBL2 was determined by enzyme-linked immunosorbent assay (ELISA) using the Hamster Phospho...

Embodiment 2

[0207] Example 2: Reference example: Purification by CEX chromatography in F / T mode (Mab A)

[0208] In this example, the removal of impurities in the purification procedure of the monoclonal antibody called Mab A will be explained according to the flowchart shown in Table 1 below.

[0209] [Table 1]

[0210]

[0211] 1) Materials and methods

[0212] The cell culture broth containing Mab A produced by CHO cells was filtered through a depth filter to remove cells, and clarified to obtain a cell culture supernatant. Cell culture supernatants were purified by protein A chromatography using KanCapA resin. After equilibrating the column with phosphate buffer (pH 7.5) containing 20 mM NaCl, load the cell culture supernatant. Then, the column was washed with phosphate buffer (pH 7.5) containing 1 M sodium chloride, and then washed with phosphate buffer (pH 7.5) containing 20 mM sodium chloride. Mab A was eluted from the column with acetate buffer (pH 3.6). Absorbance at 280 ...

Embodiment 3

[0217] Example 3: Impurity removal using activated carbon materials (Mab A, Mab B, Mab C, Mab D)

[0218] In this example, the removal of impurities in the purification schemes of various antibodies referred to as Mab A, Mab B, Mab C, and Mab D will be explained according to the purification scheme shown in Table 3 below. It should be noted that the amino acid sequences of Mab A, Mab B, Mab C and MabD and the antigens to be bound are different from each other.

[0219] [table 3]

[0220]

[0221] 1) Impurity removal using activated carbon material (except viruses)

[0222] Millistak+(R) Pod CR40 was used as the activated carbon material. For Mab B and Mab A, antibody recovery and impurity clearance were evaluated. The depth-filtered pools of Mab B and Mab A obtained as described in Example 2 were used as loading solutions for activated carbon filters (activated carbon material loading), respectively. It should be noted that in the case of Mab B, in order to facilitate t...

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Abstract

Provided is a method for antibody purification, in which while production costs are reduced and the purification period is shortened in antibody purification, impurities included in a solution are efficiently eliminated to reliably purify the antibody to a purification level that is sufficient enough to allow the antibody to be used for a human therapeutic purpose. It was found that, by using an activated carbon material, purification can be performed to a higher grade and in a stable manner regardless of the amount or the type of impurities included, and it is possible to reliably achieve a higher virus clearance capability. This finding led to a process in which an activated carbon material is used in a purification process to produce an antibody drug using CHO cells, where the process utilizing an activated carbon material serves as an alternative for an AEX chromatography process having a virus clearance capability. As a result, an antibody can be purified to a purification level that is sufficient enough for use in human therapeutic uses at a reduced cost, yet in a simpler and more efficient manner, compared to conventional purification methods.

Description

technical field [0001] The present invention relates to methods for purifying antibodies to high purity. Background technique [0002] Recently, monoclonal antibodies have attracted great attention and expectations as drugs. Antibodies are expected to have high efficacy and fewer side effects due to their specific binding ability to target antigens, and are attracting attention as anticancer agents in particular. [0003] Many therapeutic antibodies are produced by a production system using CHO cells as hosts (Non-Patent Document 1); however, cell culture supernatants contain not only antibodies as desired products but also impurities derived from host cells (e.g., protein, DNA), product-associated impurities (e.g., aggregates, variants), and virus-like particles. Since proteins derived from host cells (host cell proteins; HCPs) are recognized as heterologous proteins by humans, they are known not only to be antigenic but also to have tumorigenic risks. There is concern t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/16C07K1/18C07K1/22C07K1/36C07K16/00C12P21/08
CPCC07K1/16C07K1/34C07K16/065C07K1/22C07K1/18C07K1/36B01D15/327B01D15/362B01D15/3809B01D15/388C07K1/20
Inventor 增田由美子荻野由香井上航太泉健太武藤智惠
Owner DAIICHI SANKYO CO LTD
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