Separation and purification method of canine immunoglobulin and application of canine immunoglobulin

A technology for separation and purification of immunoglobulin, applied in the field of blood products, can solve problems such as difficult large-scale preparation, high demand for media, and low production efficiency, and achieve the effects of reducing separation steps, high purity, and improving safety

Pending Publication Date: 2021-03-12
INST OF PROCESS ENG CHINESE ACAD OF SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In addition, combined chromatography is also used to purify plasma immunoglobulins, but the existing combined chromatography requires three or more steps of chromatography, the steps a

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  • Separation and purification method of canine immunoglobulin and application of canine immunoglobulin
  • Separation and purification method of canine immunoglobulin and application of canine immunoglobulin

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Embodiment 1

[0050] This embodiment provides a method for separating and purifying canine immunoglobulin and canine immunoglobulin products, specifically as follows:

[0051] (1) Canine plasma pretreatment: Take fresh frozen canine plasma at -20°C and slowly thaw in a refrigerator at 4°C, then centrifuge (4°C, 10000r / min, 30min), and filter the centrifuged supernatant with gauze (8 layers). Use 0.5mol / L hydrochloric acid to adjust the pH to 5.0, centrifuge again (4°C, 10000r / min, 30min), take the supernatant, and use equilibrium buffer (sodium citrate 50mmol / L, pH=5.0) to dilute to a protein concentration of 5g / L L.

[0052] (2) Cation exchange chromatography: CM QZT 6FF medium is packed in the chromatography column, the ratio of column bed volume to sample volume is 1:5, and the packed chromatography column uses equilibrium buffer (sodium citrate 50mmol / L, pH=5.0) to balance 5 column volumes; the supernatant after centrifugal separation of canine plasma is filtered through a 0.45 μm mic...

Embodiment 2

[0058] This embodiment provides a method for separating and purifying canine immunoglobulin and canine immunoglobulin products, specifically as follows:

[0059] (1) Canine plasma pretreatment: Take fresh frozen canine plasma at -20°C and slowly thaw in a refrigerator at 4°C, then centrifuge (4°C, 10000r / min, 30min), and filter the centrifuged supernatant with gauze (8 layers). Use 0.5mol / L hydrochloric acid to adjust the pH to 6.0, centrifuge again (4°C, 10000r / min, 30min), take the supernatant, and use equilibrium buffer (sodium citrate 50mmol / L, pH=6.0) to dilute to a protein concentration of 10g / L L.

[0060] (2) Cation exchange chromatography: Pack SP QZT 6FF medium in the chromatography column, the ratio of column bed volume to sample volume is 1:5, and the packed chromatography column uses equilibration buffer (sodium citrate 20mmol / L, pH=6.0) to balance 5 column volumes; the supernatant after centrifugation of canine plasma is filtered through a 0.45 μm microfiltrati...

Embodiment 3

[0066] This embodiment provides a method for separating and purifying canine immunoglobulin and canine immunoglobulin products, specifically as follows:

[0067] (1) Canine plasma pretreatment: Take fresh frozen canine plasma at -20°C and slowly thaw in a refrigerator at 4°C, then centrifuge (4°C, 10000r / min, 30min), and filter the centrifuged supernatant with gauze (8 layers). Use 0.5mol / L hydrochloric acid to adjust the pH to 6.5, centrifuge again (4°C, 10000r / min, 30min), take the supernatant, and use equilibrium buffer (sodium citrate 20mmol / L, pH=6.5) to dilute to a protein concentration of 15g / L L.

[0068] (2) Cation exchange chromatography: Pack the Capto S medium in the chromatographic column, the ratio of the column bed volume to the loading volume is 1:15, and the packed chromatographic column uses an equilibrium buffer (sodium citrate 20mmol / L , pH=6.5) to balance 5 column volumes; the supernatant after centrifugation of canine plasma was filtered through a 0.45 μ...

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Abstract

The invention relates to a separation and purification method of canine immunoglobulin and application of the canine immunoglobulin, and the separation and purification method comprises the step of sequentially carrying out cation exchange chromatography and hydrophobic chromatography on a centrifugal supernatant of canine plasma to obtain the canine immunoglobulin. According to the separation andpurification method of the canine immunoglobulin, disclosed by the invention, the purity of the canine immunoglobulin can be improved by creatively carrying out a specific two-step chromatography combination mode, namely cation exchange chromatography and hydrophobic chromatography, on pretreated healthy canine plasma, so that the purity of a product is higher, and the content of impure protein is lower; and the application security is greatly improved. Compared with the traditional low-temperature ethanol precipitation process, the canine immunoglobulin yield loss caused by precipitation, deep filtration and dissolution processes is reduced, and 100-1000L of plasma raw material can be treated on the chromatography scale of 100L of medium, so that the method can be completely amplified toactual production.

Description

technical field [0001] The invention belongs to the technical field of blood products, in particular to a separation and purification method of canine immunoglobulin and its application, in particular to a separation and purification method of canine immunoglobulin with high purity, high yield and simple operation and its application. Background technique [0002] Plasma immunoglobulin is the most abundant plasma protein except albumin, and immunoglobulin G is the most important class of immunoglobulin, which contains a variety of biological activities, and its main function is to participate in the immune defense response. Its indications mainly include preventing bacterial and viral infections, replacing heterogeneous serum products, and inhibiting primary immune responses. [0003] With the increase of pet dogs, the demand for canine plasma products is also increasing. The current methods for purifying plasma immunoglobulins are mainly based on the Cohn-Oncley method or ...

Claims

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Application Information

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IPC IPC(8): C07K16/06C07K1/36C07K1/34C07K1/20C07K1/18
CPCC07K16/065
Inventor 罗坚陈西钊黄永东冯雪刘巧荣马君
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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