Research Progress of Fab antibody v1
Authors: Smith Bella 
Published: August 2018

In genetic engineering antibodies, class Fab (Fabs) antibodies have many advantages, such as low molecular weight, specific tissue distribution and low immunogenicity. Class Fab antibodies have become a research hotspot in recent decades. In addition to the widely used enzyme digestion in vitro, various expression systems (E. coli and insects) are used in Fab. The expression of antibodies is the most thorough, and has been applied to the preparation of Fab class of clinical drugs. At present, there are 6 kinds of DDL Class Fab antibody drugs have been approved and marketed by the US FDA, and a variety of Fab drugs are in clinical trials. The structure and characteristics of Fab Natural polyclonal antibodies and biosimilar monoclonal antibodies are all free. IgG molecules, IgG1 molecules from 2 identical 5 x 104. The heavy chain is composed of 2 identical 2.5 * 104 light chains (Fig. 1). The Fab molecule is composed of light chain (VL-CL) and heavy chain (VH-CH1; Fd fragment), about 5 x 104. Interaction of VL-VH and CL-CH1 and the two sulfur bonds between CL and CH1 play a stable role in the double stranded structure of the Fab molecule. The antigen-binding sites of Fab and IgG are composed of six complementary determinant regions, which are located at the light chain (VL; LCDR1, LCDR2, LCDR3) and heavy chains (VH; HCDR1, HCDR2, HCDR3), which constitute the core of the antigen-binding site. In the three-dimensional structure of the variable region, 2 opposite parallel beta - folding peptide chains are formed, the outside is connected by a highly variable CDR ring, and the sandwich structure is formed. The inside is connected by a conservative two sulfur bonds, making the CDR prominent. The development of the Fab antibody fragment is the same as the development of the monoclonal antibody, through 4 stages of mouse derived (murine), chimeric, humanized and humanmonoclonalantibody, in order to reduce the immunogenicity of antibody products. In this process, the recombinant DNA technology has been decided. Qualitative action. Chimeric Fab antibody fragments consist of murine variable region and human constant region, while humanized Fab fragments consist of murine CDR and human frame region. Compared with IgG, Fab has many characteristics: no Fc fragment, relative molecular weight. Fab antibody has become one of the main members of the development of genetic engineering drugs, such as high volume, fluidity and tissue penetration, short cycle half-life, high binding power and no antibody effect, which can be used in the prokaryotic expression system and the simple operation of gene engineering. The strategy of generating, expressing, and improving production of Fab In the Fab fragment drugs that have been marketed in clinic and many preclinical studies, the common method of preparing Fab is enzyme digestion, and many expression systems in Fab. The most widely used expression system is Escherichia coli (E.coli). The two methods of generation and expression are discussed here. Enzymatic production At present, there are mainly 3 kinds of enzymes for enzyme digestion: papaya eggs. White enzyme, pepsin and fig protease. Papain digestion of IgG antibodies is the most traditional way to obtain Fab. Most of the Fab drugs approved and applied to the clinic by FDA are obtained by digesting IgG through papain, such as alcoximumab (abciximab), ASIMO monoclonal antibody (arcitumomab), rattlesnake multivalent immunization Fab and digoxin immune Fab. The enzymolysis method is divided into free enzyme cutting and immobilized enzyme cutting. Free enzymatic hydrolysis reaction is completed in the solution, and the efficiency of enzymatic hydrolysis can be improved by optimizing the enzymolysis conditions. In the study of the preparation of Fab fragments by enzymatic hydrolysis of papain by Tang Siyuan, the effects of enzymatic hydrolysis of pH, enzyme addition, cysteine and enzyme hydrolysis time on the efficiency of IgG enzyme hydrolysis were investigated. Enzymatic hydrolysis efficiency was improved and the conversion rate of IgG was more than 98%. Lei Jiawen and so on [6] improved the preparation efficiency of F (ab ') 2 antibody by optimizing pepsin hydrolysis conditions. It was found that F (ab') 2 fragment could be efficiently prepared by enzymatic hydrolysis of 4.5H in pH4.1 citric acid buffer solution at optimum conditions. Compared with the free enzyme digestion, the new immobilized papain (pepsin and figs) can better control the digestion process and effectively separate the enzyme hydrolysates from the enzymes. In the present market, most of the immobilized enzyme is immobilized enzyme in solid phase carrier, that is, non water-soluble solid phase enzyme, and there is a space hindrance effect and low digestibility when its enzyme is cut into large molecular antibody. Mei Jianfeng and so on [7] found a thermosensitive material and used it as a carrier to immobilize papain, which could eliminate the steric hindrance effect. The N- hydroxy succinimide activated poly (N- isopropyl acrylamide) (NHS-PNIPAM) and papain were covalently combined with papain to prepare a reversible water soluble immobilized enzyme. Expression of Fab in Escherichia coli Over the years, stable antibodies have been obtained for later drug development. It has always been a difficult point to break through. A large number of experiments have compared and evaluated many kinds of expression systems such as Escherichia coli, yeast, plant, insect and mammalian cells. The results show that E.coli and yeast cells have advantages in the expression of small molecular antibody fragments, of which E. Coli expression system is most suitable for expression of small molecule antibody fragments (Fab, ScFv, double chain antibody and Fv fragment), and mammalian cells and plant cells are suitable for the expression of IgG (IgG). E.coli can be used as a unique Fab to generate its own advantages. Such as low cost, high production, fast growth and high conversion efficiency, the target protein can be obtained in a short time and purified and analyzed, and the final protein yield is high. In contrast, the mammalian cell system used to obtain complete antibody molecules is characterized by low expression and high production cost. Fab (fragment of antigen-binding) segment: papain cleaves Ig (immunoglobulin, immunoglobulin) at the N-terminus of the disulfide bond between the heavy chains in the hinge region to form two identical monovalent antigen binding fragments, referred to as Fab segments. The crystallized fragments are referred to as Fc (fragment crystallizable) segments. A Fab fragment is monovalent and binds to the antigen without agglutination or precipitation. The Fc fragment has no antigen-binding activity and is a site where Ig interacts with an effector molecule or cell. Ig is cleaved by pepsin and cleaved at the disulfide bond near the heavy chain of the hinge region to form a bivalent F(ab')2 fragment and some small fragments pFc'. Since the F(ab')2 fragment retains the biological activity of binding to the corresponding antigen and avoids the side effects which may be caused by the antigenicity of the Fc fragment, it is widely used as a biological product. Such as diphtheria antibiotic and tetanus antimycin, after hydrolysis by pepsin, the hypersensitivity reaction is reduced by removing the antigenicity of the Fc fragment. The pFc' fragment is eventually degraded and is not biologically active.