I. Introduction
With the rapid development of the biopharmaceutical industry, people's demand for new first-class methods and drugs is increasing. As an important biologic in the field of biomedicine, antibody drugs have attracted extensive attention due to their high specificity and immune activity. It is a special class of proteins that can recognize and bind antigens, mainly by binding to target antigens, mediating immune responses, regulating inflammation and promoting apoptosis. It can be used for a variety of diseases, including tumors, infectious diseases, autoimmune diseases, and neurological diseases. However, due to its complex and time-consuming production process, it limits its large-scale production and clinical application.
In addition, in order to ensure the safety and efficacy of antibody drugs, quality control is required, including quality control of the purity, potency, and stability of antibodies. Among them, the purity of the antibody is one of the key indicators, and traditional production methods cannot meet this demand. Therefore, it has become urgent to find an efficient, rapid, and stable method for antibody drug development. As an emerging protein synthesis method, cell-free protein expression (CFPS) technology has shown great potential and advantages in solving the problem of antibody drug production.
2. Antibody discovery and validation
1.Based on traditional methods.
Traditionally, antibody discovery and validation has the following steps:
1) Screening of antigens or candidate gene sequences;
2) amplification of these target genes by PCR;
3) the encoding antibody gene was inserted into the vector plasmid and transfected into a cell line for expression and expansion;
4) purify and separate the expression products;
5) Use ELISA and other techniques to detect whether its specificity and affinity meet the requirements.
However, this method has many limitations, such as the need to culture a large number of cells to obtain a sufficient number of antibodies, the long time required to develop and validate new antibodies, the high cost of human and financial resources, and the inability to quickly develop different types of antibodies for different targets, so it is limited to its application in large-scale production.
Figure 1: Traditional antibody discovery and validation.
2.Based on CFPS technology.
In recent years, cell-free protein expression technology (CFPS), as an emerging protein expression technology, has the following significant advantages:
1) There is no need to cultivate a large number of bacterial animals, which saves manpower, time and money costs, and can also ensure the stability of product quality;
2) Different types of vectors can be flexibly selected, and different vectors can be designed for different targets;
3) cell-free expression systems are typically capable of achieving high levels of protein expression, so protein purification is relatively straightforward;
4) It can be produced in high throughput, and large-scale industrial production can be realized.
This technology can not only improve protein yield, but also reduce costs and simplify the entire process, thus greatly promoting the development and industrialization of antibody drugs.
Figure 2: Antibody discovery and validation based on cell-free protein expression technology.
3. Production of antibody drugs
With the rapid progress and innovation in the field of biotechnology, antibody drugs are an innovative solution that has emerged in many diseases, and its unprecedented potential is profoundly changing the medical landscape. From the early traditional antibody drugs to today's cutting-edge antibody drug conjugates (ADCs) and nuclide conjugate antibody drugs (RAC), researchers have continuously broken through technical barriers and achieved a new level of precise targeting and efficient delivery of drugs. The R&D and application of antibody drugs are gradually moving towards refinement and personalization, which not only broadens the means of first-class diseases, but also indicates a new trend in the future of medical development - formulating precise first-class plans for individual differences.
1.Based on traditional methods.
Early traditional antibody drugs include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, etc., among which monoclonal antibodies and polyclonal antibodies need to be subjected to animal experiments, the preparation process is complex and costly, and for the human body, xenoantibodies are heterologous proteins, which will be recognized by the immune system and trigger an immune response when they enter the human body, with poor stability and batch effect, which is not easy to standardize and scale. However, the cost of chimeric antibody development and production is high, the preparation involves complex gene cloning, recombination and cell culture techniques, and the quality control, purification and large-scale production in the production process are also challenges, and there are potential immunogenicity risks. In addition, technical challenges may arise during preparation, such as the efficiency of gene recombination, the stability of cell transfection, and the yield of antibody expression.
Figure 3: Monoclonal antibody production process.
2.Based on cell-free protein expression technology (CFPS).
CFPS has emerged as a powerful method for the rapid production of multiple antibody drugs, including IgG, single-chain antibodies, FAB, and bispecific antibodies, which enable full-length antibodies to fold and assemble with prokaryotic cell extracts at high titers; Coupled with the relatively simple translation mechanism of E. coli, in vitro protein synthesis reactions based on E. coli have become the main ** of IgG molecules. In addition, unnatural amino acids bind at specific locations and can be used to produce homogeneous antibody-drug conjugates (ADCs). xu, y., lee, j., t.By constructing E. coli with a high-strength constitutive promoter to drive the synthesis of DSBC, FKPA and orthogonal tRNA, the extract of the strain was used for the synthesis of antibody-drug conjugates in the form of continuous fermentation after making a cell-free system, and obtained a higher yield than batch synthesis. This will help unlock the potential of CFPS as a platform for biopharmaceutical production. Cell-free systems have been used to rapidly synthesize a variety of immunoglobulins with rare amino acids that can be used to couple drugs or other functional modules.
Figure 4: Facilitating ADC (antibody-conjugated) drug production through cell-free protein expression technology.
A cell-free protein expression kit from Perotine Biotech provides a method for the preparation of recombinant proteins, which can be applied to the preparation of full-length antibodies, FAB antibodies, high-throughput screening of antibodies, etc., for detection and purification.
The expression system provided with this kit mainly contains:
1. Optimized E. coli extract increases the stability of the structure during DNA transcription and translation, and increases the yield of soluble proteins;
2. Optimized reaction buffer to continuously provide energy for protein synthesis through ATP regeneration system;
3. Optimize the concentration ratio of amino acids to provide sufficient substrate for protein synthesis**;
4. Optimized expression vector containing GFP gene, GFP can be used as a positive control to visually observe the protein expression results.
Fourth, outlook
CFPS can synthesize target proteins directly in vitro using DNA recombinant technology. This approach not only avoids host dependency, but also greatly reduces R&D time and costs; At the same time, it can also ensure the stability of product quality, and can realize large-scale industrial production. It is believed that with the continuous deepening and optimization of related research, there will be more breakthroughs in the research of CFPS in antibody drugs in the future!
Technical exchange: wwwcellfreeprotein.cn
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