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Biomagnetic microspheres as well as preparation method and application thereof

A magnetic microsphere, biological technology, applied in the field of biochemistry, can solve the problems of reduced binding performance of purification media, reduced purification effect, large labor and time, etc., to increase the capture rate and binding amount, reduce retention time, high-pass The effect of volume separation

Inactive Publication Date: 2021-06-01
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the covalent coupling method can ensure that the purification medium (such as protein A) is firmly immobilized on the carrier, it will reduce the binding performance of the purification medium (such as protein A) after the purification column (such as protein A column) is used many times , the purification effect is reduced
Therefore, in order to ensure high purification efficiency and quality, operators need to replace all the fillers in the affinity chromatography column in time. This process not only consumes a lot of consumables, but also consumes a lot of labor and time, resulting in high purification costs.

Method used

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  • Biomagnetic microspheres as well as preparation method and application thereof
  • Biomagnetic microspheres as well as preparation method and application thereof
  • Biomagnetic microspheres as well as preparation method and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0401] A typical preparation method of the biomagnetic microspheres (reference image 3 ), including the following steps:

[0402] Step (1): providing a magnetic microsphere body, chemically modifying the magnetic microsphere body, introducing an amino group onto the outer surface of the magnetic microsphere body, and forming an amino group-modified magnetic microsphere A.

[0403] In some preferred modes, the body of the magnetic microspheres is chemically modified with a coupling agent.

[0404] In some preferred modes, the coupling agent is an aminated silane coupling agent.

[0405] In some preferred ways, the magnetic microsphere body is SiO 2 The encapsulated magnetic material uses a silane coupling agent to chemically modify the body of the magnetic microsphere; the silane coupling agent is an aminated silane coupling agent in some preferred modes.

[0406] Step (2): using the covalent reaction between the carboxyl group and the amino group to covalently couple the acry...

Embodiment 1

[0503] The preparation of embodiment 1 biomagnetic microsphere D (in conjunction with biotin)

[0504] Preparation of silica-coated magnetic microspheres (also known as magnetic microsphere bodies, magnetic beads, glass beads)

[0505] 20g Fe 3 o 4 The microspheres were placed in a mixed solvent of 310mL ethanol and 125mL water, 45mL 28% (wt) ammonia water was added, 22.5mL tetraethyl orthosilicate was added dropwise, and the reaction was stirred at room temperature for 24h, and washed with ethanol and water after the reaction. Ferric oxide microspheres with different particle sizes (about 1 μm, 10 μm, 100 μm) are used as raw materials to control the particle size of the obtained glass beads. The ferric oxide microspheres with different particle sizes can be prepared by conventional technical means.

[0506] The prepared magnetic microspheres are used as the basic raw material for modifying the purification medium or linking elements-purification medium, so they are also ca...

Embodiment 2

[0515] Example 2 The preparation of biomagnetic microspheres F (a kind of biomagnetic microspheres F binding affinity proteins) bound to avidin-protein A (with protein A as the purification medium, with the affinity of biotin-avidin complexes as connecting elements)

[0516] 2.1 Synthesis of ProteinA-eGFP-avidin fusion protein

[0517] The DNA sequences consisting of three genes of the fusion proteins ProteinA-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2 were respectively constructed.

[0518] Wherein, the sequence of Protein A (Protein A) comes from Staphylococcus Aureus, referred to as SPA. The total length of the amino acid sequence of SPA is 516 amino acid residues, and its 37-327 amino acids are selected as the gene sequence used in the construction of the fusion protein, that is, the antibody binding domain of SPA. After optimizing the sequence with an optimization program, the nucleotide sequence is obtained, see SEQ ID NO.:1.

[0519] Tamvavidin2, an avidin anal...

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Abstract

The invention relates to biomagnetic microspheres, which comprises a magnetic microsphere body, at least one polymer with a linear main chain and a branched chain is arranged on the outer surface of the magnetic microsphere body, one end of the linear main chain is fixed to the outer surface of the magnetic microsphere body, the other end of the polymer is dissociated on the outer surface of the magnetic microsphere body, and biotin is connected to the tail end of a branch chain of the polymer of the biological magnetic microspheres. The invention also provides an improvement based on the biomagnetic microsphere provided in the first aspect, and a preparation method and an application thereof. The biomagnetic microspheres are convenient to operate and use and can be rapidly dispersed and rapidly settled in a solution, and large-scale experimental equipment such as a high-speed centrifugal machine does not need to be used; the product has the advantages of simple operation and wide application, can be connected with selective purification media (such as avidin, affinity protein, polypeptide / protein tag and the like) through biotin, and can be universally applied to separation and purification of proteins including but not limited to target objects such as antibody substances and the like on a large scale.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a biomagnetic microsphere and a preparation method and application thereof. Background technique [0002] The separation and purification of proteins, including but not limited to antibodies, antibody fragments or their fusion proteins, is an important downstream link in the production process of biopharmaceuticals. The effect and efficiency of separation and purification directly affect the quality and production cost of protein drugs. For protein purification, materials such as agarose gel are commonly used as purification columns or purification microsphere carriers at this stage. In the prior art, for the separation and purification of antibody substances (such as antibody molecules, antibody fragments or their fusion proteins), production technicians mainly use protein A (referred to as: Protein A) affinity adsorption column to separate and purify the substa...

Claims

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Application Information

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IPC IPC(8): C12P21/00C07K1/14
CPCC12P21/00C07K1/14C07K1/22B01J13/14B01J13/22C12P21/02B01D15/3809B01D15/3823B01J20/28009B01J20/28019B01J20/06B01J20/103B01J20/261B01J20/265B01J20/3204B01J20/3227B01J20/3242B01J20/3219B01J20/3234B01J20/3293B01J20/328C07K17/14B01J13/02G01N33/532G01N33/5434
Inventor 郭敏吴亮徐丽琼于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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