WO2018053797A1 - Purification method for primary treatment of cho cell culture medium - Google Patents

Abstract

A purification method for a primary treatment of a CHO cell culture medium is provided, comprising the following steps: 1) harvesting a CHO cell culture medium; 2) adding a flocculant, adjusting a pH value and stirring; and 3) clarifying the CHO cell culture medium from step 2). The purification method utilizes a combination method of chitosan, polyaluminum chloride and pH to flocculate cells, cell debris, HCP, DNA and other impurities in the cell culture medium. In addition, the residual chitosan and polyaluminum chloride can be further removed in a downstream process, which can the amount of final chitosan residue to less than 1 ppm.

Description

Purification method for preliminary treatment of CHO cell culture solution Technical field

The present invention relates to the field of monoclonal antibody purification, and in particular to a method for purifying a CHO cell culture solution.

technical background

In the field of modern biopharmaceuticals, monoclonal antibodies produced by animal cell culture technology are playing an increasingly important role in the development of the medical and health industry. The animal cell culture solution is subjected to a step of clarification filtration, purification of the preparation, and the like to obtain a monoclonal antibody. However, impurities such as cell debris, host cell protein (HCP), DNA, and high molecular weight polymer (HMW) contained in the cell culture fluid not only increase the difficulty of clarification filtration, but also increase the cost of downstream purification treatment. In the cost structure of the product, the post-processing cost accounts for 40% to 80% of the total cost.

Currently, methods for separating cells and culture fluids include two layers of deep membrane filtration techniques or centrifugal sedimentation combined with a deep membrane filtration technique. Obtaining a turbidity feedstock by deep membrane filtration requires the use of a deep filtration membrane with a small pore size (possibly below 0.2 um), which is very expensive and has a low filtration flux. Therefore, the membrane filtration cost of separating CHO cell culture fluid is very high. Since the concentration of impurities such as HCP and DNA in the liquid after membrane filtration is high, the service liquid has a great damage to the service life of the filler when passing through the Protein A column, so that the cost of downstream purification is high.

The invention flocculates the cells, cell debris, HCP and other impurities in the CHO cell liquid by flocculation and acid precipitation, increases the particle size of the particles in the liquid solution, reduces the turbidity of the liquid after centrifugation, and can significantly increase the membrane filtration flux. Reduce membrane filtration costs. At the same time, the method of flocculation combined with acid precipitation can adsorb negatively charged impurities such as HCP and DNA, improve the service life of the Protein A column, and reduce the cost of the downstream purification process.

Summary of the invention

The technical problem to be solved by the present invention is to provide a purification method for preliminary treatment of CHO cell culture solution, which can reduce the turbidity of the liquid solution, increase the membrane filtration flux and reduce the membrane filtration cost under the premise of ensuring the yield and quality of the monoclonal antibody. It can reduce the content of impurities such as HCP and DNA, improve the service life of Protein A, save production costs and improve production efficiency.

Specifically, the present invention provides a method for purifying a CHO cell culture solution, which comprises the following steps:

1) harvesting CHO cell culture solution;

2) adding a flocculant, adjusting the pH value, and stirring;

3) Clarify the CHO cell culture solution of step 2).

According to some embodiments of the invention, the flocculating agent is selected from the group consisting of chitosan, polyaluminum chloride, or a combination thereof.

According to some embodiments of the present invention, the final concentration of chitosan is 0.2-0.5 g/L and the final concentration of polyaluminum chloride is 20-70 mg/L in the CHO cell culture solution.

According to some embodiments of the invention, step 2) adjusts the pH to 5.0 to 7.0.

According to some embodiments of the invention, step 2) the agitation time is 30 minutes.

According to some embodiments of the invention, the clarification method is selected from the group consisting of centrifugation, static separation, membrane filtration, or a combination thereof.

According to some embodiments of the invention, the centrifugation, preferably, has a rotational speed of 4000 x g, a time of 15 to 20 minutes, and a temperature of 15 to 25 °C.

The invention utilizes a combination method of chitosan, polyaluminum chloride and pH to flocculate cells, cell debris, HCP and DNA and other impurities in the cell liquid, has obvious effects, simple process, and residual chitosan and polychlorination in the liquid solution. The aluminum can be further removed in the downstream process, and the final chitosan residue can be less than 1 ppm.

detailed description

The invention is further described in detail below with reference to the preferred embodiments thereof, which are set forth to illustrate the invention and not to limit the scope of application of the invention.

The CHO cell (Chinese hamster ovary cell) cell line in the following examples of the present invention was purchased from Invitrogen; the cell line for expressing the recombinant human tumor necrosis factor receptor-Fc recombinant protein (rhTNFR-Fc) antibody was constructed autonomously. The rhTNFR-Fc antibody of the present invention is prepared by fermentation based on the patent US 2005/0069979A.

Example 1

0.2 g of chitosan and 20 mg of polyaluminum chloride were added to 1 L of the harvest liquid, and the pH was adjusted to 5.0 with 0.8 M hydrochloric acid. The above harvested liquid was stirred at 15 ° C for 30 minutes, centrifuged at 4,000 × g for 15 minutes at 15 ° C, and the turbidity was measured to determine the DNA concentration in the preliminary separation liquid and the HCP concentration and the percentage of HMW in the feed liquid after the Protein A step. The results are shown in Table 1.

Detection methods for DNA, HCP and HMW:

DNA detection was performed using real-time fluorescent quantitative PCR. Enzyme activation and pre-denaturation conditions were 95 ° C for 5 min. The PCR reaction conditions were 95 ° C, 15 s; 60 ° C, 60 s; 40 cycles.

The HCP assay uses a sandwich ELISA method. The rotation speed was 180 rpm, the incubation temperature was 37 ° C, and the OD value detection wavelength was 450/630 nm.

HMW uses high performance liquid chromatography. Column TSK-gel G3000SW 7.8*300mm (5μm), mobile phase 150mM phosphate buffer, 100% phase A, flow rate 0.5mL/min, temperature 25°C, pH 7.0, detection time 30min, wavelength 280nm.

Example 2

0.5 g of chitosan and 40 mg of polyaluminum chloride were added to 1 L of the harvest liquid, and the pH was adjusted to 7.0 with 1 M hydrochloric acid. The above harvested liquid was stirred at 15 ° C for 30 minutes, centrifuged at 4,000 × g for 15 minutes at 15 ° C, and the turbidity was measured to determine the DNA concentration in the preliminary separation liquid and the HCP concentration and the percentage of HMW in the feed liquid after the Protein A step. The detection method is the same as that in the first embodiment, and the results are shown in Table 1.

Example 3

0.5 g of chitosan and 70 mg of polyaluminum chloride were added to 1 L of the harvest liquid, and the pH was adjusted to 6.0 with 0.8 M hydrochloric acid. The above harvested liquid was stirred at 15 ° C for 30 minutes, centrifuged at 4,000 × g for 15 minutes at 15 ° C, and the turbidity was measured to determine the DNA concentration in the preliminary separation liquid and the HCP concentration and the percentage of HMW in the feed liquid after the Protein A step. The detection method is the same as that in the first embodiment, and the results are shown in Table 1.

Comparative example 1

The pH of the cell harvested liquid was adjusted to 7.0 with 0.8 M hydrochloric acid, and polyaluminum chloride was added to the cell harvested liquid, and the final concentration of polyaluminum chloride was 40 mg/L. The above harvested liquid was stirred at 15 ° C for 30 minutes, centrifuged at 4,000 × g for 15 minutes at 15 ° C, turbidity was measured, and DNA in the preliminary separation liquid was detected. The concentration and the percentage of HCP and HMW in the feed after the Protein A step were the same as in Example 1. The results are shown in Table 1.

Comparative example 2

0.5 g of chitosan was added to 1 L of the harvest liquid, and the pH was adjusted to 6.0 with 0.8 M hydrochloric acid. The above harvested liquid was stirred in a harvesting liquid at 15 ° C for 30 minutes, centrifuged at 4,000 × g for 15 minutes at 15 ° C, measured for turbidity, and the DNA concentration in the preliminary separation liquid and the HCP concentration and HMW in the feed liquid after the Protein A step were measured. The percentage content and the detection method were the same as those in Example 1, and the results are shown in Table 1.

Table 1 Test results table

	Turbidity after centrifugation (NTU)	DNA concentration (g/L)	HCP concentration (g/L)	HMW percentage (%)
Example 1	22.5	120	820.18	0.83
Example 2	10.9	60	506.08	0.50
Example 3	15.58	81	550.64	0.95
Comparative example 1	98.06	1523	8866.24	0.75
Comparative example 2	84.21	492	2405.37	1.49

It can be seen from the above table that adding chitosan, polyaluminium chloride to the harvesting liquid and adjusting the pH value of the solution can simultaneously reduce the turbidity, DNA, HCP and HMW content of the liquid.

Claims (7)

1. A method for purifying a CHO cell culture solution, which comprises the following steps:

   1) harvesting CHO cell culture solution;

   2) adding a flocculant, adjusting the pH value, and stirring;

   3) Clarify the CHO cell culture solution of step 2).
2. The method of claim 1 wherein the flocculating agent is chitosan, polyaluminum chloride or a combination thereof.
3. The method according to claim 2 or 3, wherein the final concentration of the chitosan is 0.2 to 0.5 g/L, and the final concentration of the polyaluminum chloride is 20 to 70 mg/L.
4. The method of claim 1 wherein said step 2) is adjusted to a pH of from 5.0 to 7.0.
5. The method of claim 1 wherein step 2) is stirred for 30 minutes.
6. The method of claim 1 wherein the clarification is centrifugation, static separation, membrane filtration, or a combination thereof.
7. The method according to claim 7, wherein said centrifugation has a rotational speed of 4000 x g, a time of 15 to 20 minutes, and a temperature of 15 to 25 °C.