A Brief Discussion on the Recombinant IgG Antibodies Expression-Special topic on antibody modification and modification-Tekbiotech-Yeast Display Service,Phage display technology

A Brief Discussion on the Recombinant IgG Antibodies Expression

I. Introduction to IgG Antibody

IgG, as the body's immune guard, mainly plays a protective role in the body's immunity, fighting against most bacteria and viruses. IgG is the only immunoglobulin that can pass through the placenta. IgG can activate the complement system, participate in antibody-mediated cytotoxicity, namely ADCC, and participate in some hypersensitivity reactions.

IgG is a tetrapeptide composed of two heavy chains and two light chains with smaller molecular weight, and presents a Y-shaped structure. The chains are linked by disulfide bonds, with a molecular weight of about 150kDa. IgG molecules are a glycoprotein with a high content in the blood. In normal human blood, almost all IgG Fc parts contain at least two conserved N-glycosylation sites.

Schematic diagram of antibody structure - Tekbiotech.png

Figure 1 Schematic diagram of antibody structure

II. Main Forms of Recombinant Antibodies

Human-mouse chimeric antibody	Humanized antibody	Fully human antibody
The variable region of the antibody comes from mouse monoclonal antibody, while the constant region comes from human antibody. Chimeric antibodies retain the ability of the parent antibody to specifically bind to the antigen, and greatly reduce the immunogenicity of the mouse-derived region in the human body. Their half-life is significantly prolonged, and they are also significantly enhanced in mediating CDC and ADCC.	In order to further reduce the mouse-derived region, CDR grafting technology was developed, which only retains the variable region CDR region of the mouse-derived monoclonal antibody, and the other regions are human-derived, and the proportion of humanized antibodies obtained is as high as 80%-90%.	In order to eliminate allogeneic rejection, phage display technology is used in the development of antibody drugs. Phage antibody library screening uses phage display technology to fuse the gene fragment encoding the variable region of the antibody with the gene encoding the phage surface protein (inserted between the signal peptide and the capsid protein gene), and present it on the surface of the phage in the form of a fusion protein, and use specific proteins/antigens for screening to obtain antibody fragments with high affinity variable regions. When the human antibody variable region gene fragments obtained from human immune cells (or tissues) are used to construct a phage library, and then the display library is screened using a specific antigen, human antibody fragments and corresponding gene sequences against a certain antigen can be obtained. Finally, specific fully human antibodies containing human antibody heavy chains and light chains can be obtained through in vitro protein expression and screening.

III. Expression of Recombinant Antibodies in Mammalian Cells

The traditional way to produce antibodies is to stimulate the animal immune system with antigens. This method requires the preparation of antigens first, and it is difficult to produce antibodies in large quantities using this method (requiring a large amount of experimental consumables and time). The method of producing antibodies in mammalian cells has certain advantages. ① The gene sequence of the known antibody is cloned into an expression vector, introduced into mammalian cells for in vivo culture, and a stable cell line that can stably produce the antibody gene is finally obtained through a series of screening. This process requires a lot of time to screen out a cell line that is sufficiently stable in expression and can be stably produced for a long time in subsequent experiments. ② For the mass production of antibodies with unknown sequences, first stimulate the animal immune system to obtain a small amount of antibodies, obtain the antibody sequence through sequencing, and screen the cell line that stably expresses through recombinant antibody expression in mammalian cells to meet the needs of mass production.

IV. Optimizing Antibody Expression Strategy

4.1 Vector Optimization

The design of expression vectors needs to adapt to the expression system, so as to increase antibody expression, increase cell stability, and improve the efficiency of transcription, secretion, selection, and integration. (1) Various expression vector forms: monocistronic vectors, multi-promoter expression vectors, and multicistronic vectors mediated by IRES or Furin-2A elements. (2) Selection of expression vector elements: promoters and enhancers include CMV promoter, SV40 promoter, EF-1α promoter, CAG promoter, etc.; PolyA includes BGH PolyA, SV40 PolyA, etc.; screening markers include metabolic screening markers MTX/MSX, antibiotic screening markers, etc.

4.2 Gene Site-specific Integration

Use the recombinase system for gene site-specific integration: First, it is necessary to construct a platform cell, use model proteins (such as GFP, etc.) to screen for high expression sites in the host cell genome, and then use the specificity of the recombinase to achieve site-specific integration of the target gene at the site. Such as Flp/FRT recombinase, Cre/loxp recombinase, PhiC31 recombinase, Bxb1 recombinase and other recombinase systems.

V. Advantages of Recombinant Antibodies:

1. Compared with polyclonal antibodies, the performance of polyclonal antibodies prepared by traditional methods is easily affected by the production batch. Recombinant antibodies use molecular cloning and protein expression to greatly reduce the impact of batch production.

2. Compared with monoclonal antibodies, cell lines prepared by traditional monoclonal antibodies have risks such as antibody chromosome loss, cell growth cessation or death after recovery, while recombinant antibodies have the advantages of known sequences, long-term storage of antibody genes, stable antibody properties, and good experimental repeatability. It is a standardized antibody production process that avoids the risk factors in the production of monoclonal and polyclonal antibodies.

3. The development of recombinant antibodies has made monoclonal antibodies no longer limited to mice, and other species can also prepare high-affinity monoclonal antibodies.

TekBiotech's own high-yield series of mammalian expression systems and domesticated 293 series and CHO series cells can achieve efficient secretory expression of full-length antibodies and various forms of recombinant antibody fragments. No immunization is required, and high-yield and reproducible production of monoclonal antibodies and other forms of recombinant antibodies can be achieved.

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