Service overview

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Service details

Service overview

Advanced technology in all key stages

1) Key stage 1: antigen preparation. The methods of preparation of various antigens such as soluble proteins, eukaryotic cells, DNA, and membrane proteins ensure high antigen quality.

2) Key stage 2: antibody production. A variety of antibody screening methods, such as large-capacity phage display fully human antibody library, an immunization antibody library, and hybridoma technology, can be used to obtain dozens of preferred antibody candidate molecules with well-defined sequences and high affinity.

3) Key stage 3: Antibody engineering. The antibody obtained by 3D deep humanization has a humanization more than 90%, also the pharmacological properties. Based on the affinity maturation technology of the large-capacity phage display antibody library, the obtained antibody candidate molecules are extremely optimized, and the affinity can usually be increased by 5-100 times.

4) Key stage 4: Systematic drug screening in vitro. Sanyoubio has established systematic evaluation methods for the biological activity of monoclonal antibody drugs, including ADCC, CDC, cell proliferation inhibition, immune cell co-stimulation, macrophage phagocytosis and other screening methods, which can be used for high efficiency and high throughput screening.

5) Key stage 5: Druggability screening. Combination of the preparation of high expression cell lines, stability study, physical and chemical analysis, in vivo pharmacodynamics screening together could ensure the quality of pre-clinical candidate molecules.

Service highlights

Service highlight 1. Magnetic array immunization antibody library (MIT)

MIT (Magnetic & Immunization & Tandem antibody library) is an antibody preparation method combining animal immunization, array construction and magnetic bead high-throughput screening technology.

Application Case 1: The number of antibodies obtained is extremely large

Through mass selection, primary screening, sequencing analysis and rescreening using MIT immunization antibody library, more than 60 lead antibodies with unique sequences are obtained. The results of the affinity ranking of the obtained antibodies are shown in Figure 1. As can be seen from the figure, the EC50 of the lead antibody binding to the antigen obtained by this project are mostly at the sub-nM level.


Application Case 2: The antibody obtained has higher affinity compared to hybridoma technology

Candidate antibodies against a certain target protein were prepared by MIT immunosorbent library and hybridoma technology, respectively. The results of the affinity comparison test of the antibodies obtained by the two methods are shown in Fig. 2. As can be seen, the preferred antibody clones B1, C4, and B3 derived from the MIT immunization antibody library have significantly higher affinities than the preferred antibodies derived from hybridomas.

Figure 1. Affinity ranking of antibodies obtained from the MIT immune immunization antibody library

Figure 2. Affinity ratio of antibodies from MIT immunization antibody library to antibodies derived from hybridoma technology

Rich experience

Sanyou bio has accumulated a large number of successful cases in the magnetic array immunization antibody library. By January 2018, Sanyou bio has successfully constructed 298 magnetic array immunization antibody libraries.

In addition, Sanyou bio has accumulated a lot of experience in other antibody libraries: including 6 human disease antibody libraries, 50 affinity maturation antibody libraries, 28 naive library sub-libraries and 53 Fab antibody libraries. The accumulated library capacity is more than 1011.

Table 1. Quantity and summary of antibody libraries constructed by Sanyou bio

Till January 2018

Advanced, integrated, and pipelined antibody library construction platform.

Guarantee the quality of MIT antibody library services.

Service Highlight 2: Over 1011 fully human antibody libraries constructed by thousands of samples

At present, the phage display antibody libraries in the world includes whole human naive antibody library, synthetic antibody library, and semi-synthetic antibody library. Most antibody libraries are constructed using only dozens of donors or synthetic antibody genes, and have limitations such as low library capacity, insufficient diversity, low expression ratio, high mismatch ratio, and insufficient druggability, which greatly limit their application. To solve these problems, Sanyou bio uses its dominant platform, and collects four thousand cases of samples, to create a world-leading, fully human naive antibody library with capacity of 1011.

Why choose fully human antibody naive antibody library?

Phage display antibody library technology has many advantages over hybridoma technology.

Phage display fully human antibody library is a well-proven technique. Currently, 8 of the more than 20 fully human antibodies on market are screened from phage display fully human antibody library.

Table 2. Comparison of Sanyou Bio naive antibody library and internationally famous antibody library

Application Case 1: The existing naive antibody library with 1010 capacity has been validated by more than 20 targets, and 5-25 lead antibodies are available for each target.

Through mass selection, primary screening, sequencing analysis and rescreening. A total of more than 20 lead antibodies with unique sequences were obtained from the fully human antibody library.

The results of the affinity ranking of the obtained antibodies are shown in FIG 4. As can be seen from the figure, the EC50 of the lead antibody binding to the antigen obtained by this project are mostly at the sub-nM level.

Figure 3. Affinity ranking of antibodies obtained from the fully human antibody library

Figure 4. Affinity ranking of antibodies obtained from the fully human antibody library

Service Highlight 3: Extreme antibody engineering assures the drugability

3D antibody humanization refers to the humanized design of mouse antibody by computer aid, and the humanized modification of amino acid residues on the surface of heterologous antibody based on crystal structure analysis. The humanized antibody is deeply humanized to the maximum extent, and the degree of humanization is over 90%.

Application case 1: After humanization, the affinity is basically consistent with the mouse antibody, and the affinity of some antibodies after humanization is higher than that of the mouse antibody.

Through the humanization of antibody obtained, the results of antibody affinity after humanization are shown in FIG 5. As can be seen from the figure, the affinity of the antibody after humanization remains substantially the same as that of the mouse control antibody, and the affinity of some antibodies after humanization is higher than that of the mouse control antibody.

Figure 5. Comparison of the affinity of humanized antibodies to murine antibodies

Figure 6. Crystal structure simulation of humanized antibody and murine antibody

Through computer-aid simulation, selecting the CDR combination to be modified, 109 or even 1010 of mutant antibody library is designed through scientific systematic methods. Preferred antibodies with higher affinity and higher drugability can be screened through mass selection, affinity screening, sequence analysis, affinity kinetic analysis, and expression characteristics analysis and physicochemical properties analysis.

High-affinity guarantee: Optimize 109 or even 1010 amino acid sites in the original sequence. By multi-round screening, the antibody affinity usually increases by 5-100 times.

Advanced technology collection: It integrates advanced technologies such as 3D advanced structure simulation, large-capacity antibody library, and physicochemical analysis.

Application Case 2: Extreme affinity maturation modification improves the affinity by 20 times

Affinity maturation of the obtained antibodies.

The results of the affinity maturation of the obtained antibodies are shown in Fig. 7. As can be seen from the figure, the project increases the affinity by 5-10 times through one round of mutation, and the affinity increases by more than 20 times through two rounds of mutations.


Application Case 3: multiple candidate antibodies are successfully obtained in PH-sensitive project

Affinity maturation of the obtained PH-sensitive antibody.

The results of the affinity maturation of the obtained antibodies are shown in Fig. 8. As can be seen from the figure, by one round of mutation, antibodies having a marked difference in binding characteristics between pH 6.0 and 7.2 are selected as compared with the wild type.

Figure 7. Extreme affinity maturation modification

Figure 8. Extremely affinity maturation modification of PH-sensitive antibody

Service Highlight 4: Systematic in vitro pharmacodynamic evaluation for antibody drug screening for various targets

Cellular functional testing of antibody drugs is critical in drug screening and quality control. At present, the activity analysis methods of antibody drugs are mainly biochemical and cell based functional screening. Sanyou bio has established a systematic evaluation method for the biological activity of monoclonal antibody drugs, which can be used for screening drugs against targets in cytotoxicity, cell proliferation inhibition, cell activation and interaction regulation, human autoimmune system regulation, neutralization antigen, GPCR, etc. It makes the development of antibody drugs close to the clinic stage.

Overview of systematic in vitro pharmacodynamic evaluation

Applications a: isolating human primary cells with high purity suitable for antibody functional screening

After isolating human primary T cells (negative isolation), CD4+ T cells (negative isolation) and monocytes using the German Miltenyi MACS system, isolated cells are labeled using CD3, CD4 and CD14 flowcytometry antibody, respectively and tested with flow cytometry. The result is shown in Figure 9. The figure shows the isolated human primary cells are high purified, is applicable for antibody functional screening.

Figure 9. Human primary cell isolation experiment

Application Case 2: FACS affinity screening of some candidate antibody molecules has the same affinity as the positive control antibody.

The cells to be tested are sequentially incubated with candidate antibody of different concentration gradients and fluorescent secondary antibody, and then the affinity of candidate molecules is detected using flow cytometer. The result is shown in Figure 10. As can be seen from the figure, some of the candidate antibodies have the same affinity as the positive control antibody.

Figure 10. FACS affinity screening experiment

Application Case 3: The biological activity of antibody clones obtained by co-stimulation culture experiments is significantly better than that of control antibodies.

Human primary T cells are co-stimulated with control antibody and different concentrations of candidate antibodies, and ELISA assay is used to detect the amount of cytokine secretion in the supernatant of the cultured cells. The result is shown in Figure 11. As can be seen from the figure, the candidate antibodies can effectively activate the T cell response.

Figure 11. Costimulatory culture experiment

Application Case 4: Flowcytometry detecting the efficiency of macrophages phagocytic target cells

By differentiating human monocytes to macrophages, the target cells are incubated with macrophages and antibody A or candidate antibodies of different concentration gradients, and the efficiency of macrophage phagocytosis of target cells is detected by flow cytometry. The result is shown in Figure 11. As can be seen from the figure, both candidate antibody and antibody A can effectively promote macrophage phagocytosis of target cells, and two of the candidate antibodies are better than positive control antibody.

Application Case 5: the screened antibodies effectively enhance the immune response

The isolated monocytes are differentiated into macrophages in vitro, and plated with CD4+ T cells from different human in a certain ratio and different concentrations of the target antibodies are added to the plate. The result is shown in FIG 13. As can be seen from the figure, antibodies can effectively enhance the immune effect.

Figure 12. Flow cytometry detection of macrophage phagocytosis of target cells

Figure 13. Mixed lymphocyte reaction experiment

Service details

Service details

Candidate molecules with good drugability can be obtained through four milestones

Service mode

Sanyou bio adopt a variety of flexible service modes such as overall outsourcing, milestone cooperation development, and personalized service which halve the risk of the customer and double the success.

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