阅读说明：本技术 一种提高双特异性抗体纯度的分离纯化方法 (Separation and purification method for improving purity of bispecific antibody ) 是由 李智 诸葛鑫 于 2020-03-13 设计创作，主要内容包括：本发明公开了一种提高双特异性抗体纯度的分离纯化方法,包括以下步骤：分别配置澄清过滤平衡缓冲液、EDTA-2Na母液；串联压力表,采用硅胶管将MD0HC过滤器、MX0HC过滤器以及0.22μm除菌过滤器依次连接；分别采用注射用水和澄清过滤缓冲液依次清洗MD0HC过滤器、冲洗MX0HC过滤器；将转染表达的双特异性抗体在生物反应器中进行培养,培养结束后将生物反应器的温度调整为20±5℃,向培养上清液加入EDTA-2Na母液；将上述培养上清液过滤,滤液收集至收集容器；过滤时同时向培养上清液、滤液中通入氧气；最后对滤液进行蛋白纯度进行检测。该方法操作简单,在过滤收集过程中双特异性抗体稳定性好,可有效减少轻链与重链结合的二硫键被破坏,从而获得纯度更高的双特异性抗体。(The invention discloses a separation and purification method for improving purity of a bispecific antibody, which comprises the following steps: respectively preparing a clarifying and filtering balance buffer solution and EDTA-2Na mother liquor; a pressure gauge is connected in series, and an MD0HC filter, an MX0HC filter and a 0.22 mu m sterilizing filter are connected in sequence by adopting a silicone tube; sequentially cleaning an MD0HC filter and washing an MX0HC filter by respectively adopting water for injection and a clarifying and filtering buffer solution; culturing the transfection-expressed bispecific antibody in a bioreactor, adjusting the temperature of the bioreactor to 20 +/-5 ℃ after the culture is finished, and adding EDTA-2Na mother liquor into the culture supernatant; filtering the culture supernatant, and collecting the filtrate into a collection container; introducing oxygen into the culture supernatant and the filtrate during filtration; and finally, detecting the protein purity of the filtrate. The method is simple to operate, the stability of the bispecific antibody is good in the process of filtering and collecting, and the damage of a disulfide bond formed by combining a light chain and a heavy chain can be effectively reduced, so that the bispecific antibody with higher purity can be obtained.)

1. A method for separating and purifying bispecific antibody to improve purity, comprising the following steps:

(1) respectively preparing a clarifying and filtering balance buffer solution and EDTA-2Na mother liquor;

(2) series pressure gauge, sequentially connecting MD0HC filter, MX0HC filter and 0.22 μm sterilizing filter with silicone tube, adjusting peristaltic pump, cleaning MD0HC filter with water for injection for the first time, and washing amount not less than 100L/m²The MX0HC filter is flushed by water for injection, and the flushing amount is more than or equal to 100L/m²(ii) a Adjusting a peristaltic pump, and respectively washing an MD0HC filter and an MX0HC filter by using a clarifying and filtering buffer solution, wherein the using amount of the clarifying and filtering buffer solution is 1-2 times of the volume of the filter;

(3) culturing the transfection-expressed bispecific antibody in a bioreactor, adjusting the temperature of the bioreactor to 20 +/-5 ℃ after the culture is finished, keeping the aeration and stirring parameter settings of the bioreactor unchanged, and adding EDTA-2Na mother liquor into the culture supernatant in the bioreactor;

(4) adjusting the peristaltic pump to adjust the flux to be less than or equal to 100L/m²H, filtering the culture supernatant, wherein the filtering pressure is less than or equal to 1.5 bar; collecting the filtrate into a collection container; keeping introducing oxygen into the culture supernatant of the bioreactor during the filtration process, and simultaneously introducing oxygen into the collection container; and finally, detecting the protein purity of the filtrate.

2. The method of claim 1, wherein in step (1), the clarification filtration equilibration buffer comprises 20 mmol/L Tris, 150 mmol/L NaCl, and the pH is 7.2.

3. The separation and purification method for improving the purity of the bispecific antibody according to claim 1, wherein in step (1), the EDTA-2Na mother liquor comprises 20 mmol/L Tris, 150 mmol/L NaCl, 10.0 g/L EDTA-2Na, and has a pH of 7.2.

4. The method for separating and purifying bispecific antibody according to claim 1, wherein in step (2), the flux of washing the MD0HC filter and MX0HC filter with water for injection is 100-600L/m²·h。

5. The method according to claim 1, wherein the flux of the clarified and filtered buffer solution used to wash the filter in step (2) is 100-600L/m²·h。

6. The method according to claim 1, wherein in step (3), EDTA-2Na mother liquor is added to the culture supernatant in the bioreactor to adjust the concentration of EDTA in the culture supernatant to 0.01-0.3 g/L.

7. The method for separating and purifying a bispecific antibody according to claim 1, wherein in the step (4), the aeration rate of oxygen is 0.2 to 0.4 ml/min-L when oxygen is introduced into the culture supernatant.

8. The method for separating and purifying a bispecific antibody according to claim 1, wherein in the step (4), the amount of oxygen gas is 0.01 to 0.5 ml/min-L when oxygen is introduced into the collection container.

The technical field is as follows:

the invention relates to the technical field of biology, in particular to a separation and purification method for improving the purity of a bispecific antibody.

Background art:

the bispecific antibody is an artificial antibody containing 2 specific antigen binding sites, can bridge between target cells and functional molecules, can stimulate a guided immune response, is one of genetic engineering antibodies, has become a hotspot in the field of antibody engineering, and has a wide application prospect in the immunotherapy of tumors.

Bispecific antibodies are mostly prepared by expression and large-scale culture of Chinese hamster ovary cells at present, and the harvesting method is basically to use a clarification filtration membrane pack to clarify and harvest at the end of culture and store clarified collection liquid in a closed container. Conventional filtration harvest methods are operated with the bispecific antibody in a suspended aeration state, and the enzymes free in the reactor and in the filtrate readily exert a reducing effect. Furthermore, since the bispecific antibody is unstable in molecular structure and is easily reduced to produce fragments, the method causes reduction of disulfide bonds in the bispecific antibody and drop of light chains on the antibody, thereby producing antibody fragments, resulting in a decrease in purity of the target product.

The invention content is as follows:

the technical problem to be solved by the invention is to provide a separation and purification method for improving the purity of the bispecific antibody aiming at the defects of the prior art, the method is simple to operate, the stability of the bispecific antibody is good in the process of filtering and collecting, and the damage of disulfide bonds combined by a light chain and a heavy chain can be effectively reduced, so that the bispecific antibody with higher purity can be obtained.

In order to better solve the technical problems, the invention adopts the following technical scheme:

a method for separation and purification to improve the purity of a bispecific antibody, comprising the steps of:

(1) respectively preparing a clarifying and filtering balance buffer solution and EDTA-2Na mother liquor;

(2) series pressure gauge, sequentially connecting MD0HC filter, MX0HC filter and 0.22 μm sterilizing filter with silicone tube, adjusting peristaltic pump, cleaning MD0HC filter with water for injection for the first time, and washing amount not less than 100L/m²The MX0HC filter is flushed by water for injection, and the flushing amount is more than or equal to 100L/m²(ii) a Adjusting a peristaltic pump, and respectively washing an MD0HC filter and an MX0HC filter by using a clarifying and filtering buffer solution, wherein the using amount of the clarifying and filtering buffer solution is 1-2 times of the volume of the filter;

(3) culturing the transfection-expressed bispecific antibody in a bioreactor, adjusting the temperature of the bioreactor to 20 +/-5 ℃ after the culture is finished, keeping the aeration and stirring parameter settings of the bioreactor unchanged, and adding EDTA-2Na mother liquor into the culture supernatant in the bioreactor;

(4) adjusting the peristaltic pump to adjust the flux to be less than or equal to 100L/m²H, filtering the culture supernatant, wherein the filtering pressure is less than or equal to 1.5 bar; collecting the filtrate into a collection container; keeping introducing oxygen into the culture supernatant of the bioreactor during the filtration process, and simultaneously introducing oxygen into the collection container; and finally, detecting the protein purity of the filtrate.

As a preferred embodiment of the above technical solution, in step (1), the clarified filtration equilibration buffer comprises 20 mmol/L Tris, 150 mmol/L NaCl, and has a pH of 7.2.

As a preferable mode of the above technical means, in the step (1), the EDTA-2Na mother liquor comprises 20 mmol/L Tris, 150 mmol/L NaCl, 10.0 g/L EDTA-2Na, and the pH value is 7.2.

Preferably, in the step (2), the flux of the water for injection used for washing the MD0HC filter and the MX0HC filter is 100-600L/m²·h。

Preferably, in the step (2), the flux of the clear filter buffer solution used for washing the filter is 100-600L/m²·h。

Preferably, in the step (3), an EDTA-2Na mother liquor is added to the culture supernatant in the bioreactor to adjust the concentration of EDTA in the culture supernatant to 0.01 to 0.3 g/L.

Preferably, in the step (4), when oxygen is introduced into the culture supernatant, the amount of oxygen introduced is 0.2 to 0.4 ml/min-L.

Preferably, in the step (4), the amount of oxygen gas introduced into the collection container is 0.01 to 0.5 ml/min-L.

Due to the adoption of the technical scheme, the invention has the following beneficial effects:

on one hand, the temperature of the bioreactor is reduced after the culture is finished, and a certain amount of EDTA is added into the heavy culture supernatant of the bioreactor; EDTA can chelate metal ions in the solution, thereby reducing the enzyme activity in the solution environment by reducing temperature and reducing metal ions, and reducing the probability of the degradation of the bispecific antibody by the enzyme.

On the other hand, when the culture supernatant is filtered, oxygen is introduced into the culture supernatant and the filtrate and the mixture is stirred at the same time, so that an oxidation environment of a cell culture solution can be provided, a disulfide bond is prevented from being reduced, and the stability of the bispecific antibody is effectively improved; can obviously improve the purity of the bispecific antibody in filtration separation and reduce the generation of antibody protein fragments.

The specific implementation mode is as follows:

the present invention is further illustrated by the following examples, which are provided for the purpose of illustration only and are not intended to be limiting.