Service Overview
Background: Raw material preparation is the first step of R&D of IND antibody drugs. To overcome the drawbacks of long cycle, low throughput, and low rate of success in cell lines preparation, Sanyou Bio proudly launches the construction service of overexpression cell lines for R&D work.
Methods: Overexpression cell lines are constructed with CHO, HEK293 and various of tumor cells by electrotransformation, chemotransformation and lentivirus infection, and are screened by puromycin, hygromycin, G418 and GS high expression screening systems to obtain stable high-expression level cell lines.
Advantages: Starting from transfection, it only takes 4–6 weeks to obtain an overexpression cell pool with high expression and good stability, and only 6–8 weeks to obtain a monoclonal cell line. Moreover, 30 cell lines can be constructed in batches at a time.
Cases: As of Apr. 2022, hundreds of projects has been completed on Sanyou Bio's research cell line construction platform, covering secreted antigens, antibodies, membrane proteins, and transmembrane proteins overexpression cell lines, and more than 3000 overexpression cell lines have been successfully constructed on CHO, HEK293, Jurkat, and other cells, 90% of which can be stably passaged to 20 PDLs.
Service Highlights
Adaptive to multi-type cell line construction
Various targets and species can be selected flexibly.；Overexpression cell lines are constructed with CHO, HEK293, and various of tumor cells by electrotransformation, chemotransformation, and lentiviral infection, and luci reporter system cell lines can be customized.
Stability and quality guarantee
Systematic and comprehensive in vitro efficacy validation performed by an experienced professional team, with 3000+ successful project experience, equipped with Sanyou systematic and comprehensive quality control system, ensured delivery quality.
Rapid one-stop delivery experience
A standardized process-oriented project model with a combination of high-throughput and automation, equipped with Sanyou systematic and comprehensive quality control system, ensuring the successful delivery of hundreds of targets and customer satisfaction. The construction can be completed in 4–6 weeks.
Service Contents
Technical Process
Service Features
1. Ability to construct various types of overexpression cell lines
Complete cell line construction process has been established on Sanyou Bio's research cell line platform for a variety of cell lines, which can be used for immunization, library screening, and functional evaluation. Overexpression cell lines can be mass-produced, and qualified stable overexpression cell lines can be delivered in 4–6 weeks.
Four types of GMP host cells with well-defined origin and rigorous quality control are available, including Thermo-licensed Freedom CHO-S, Horizon-licensed HD-BioP3 Null CHO K1S, Merck-licensed CHOZN and QuaCell-licensed CHO-K1Q, etc. Sanyou Bio has developed a mature cell line construction process for different types of host cells.
2. More than 20 PDL stability assessments to ensure a positive rate > 80%
In the clone screening stage, 12–24 cloned cell lines were evaluated for their early expression levels. After the preferred clones were initially determined, the PDL stability assessmentis were performed on the banked cells to ensure that the stability of the cell bank meets the research needs.
Stability Assesssment Items and Standards
3. Experienced team members
More than 1000 overexpression cell lines have been constructed on Sanyou Bio's research cell line platform, with a success rate of 100%. More than 85% of the overexpression cell lines have a positive rate of more than 80%. The luciferase reporter system can be flexibly customized for targets, and functionally validated by relevant assays.
Number of cell lines
4. Multiple screening to ensure optimal clones
Cell line construction proces：Using multiple screening systems including plate bioreactors, tubular bioreactors, shake flasks, and fermenters, more than 1000 clones are screened through 3 rounds of Batch and 3–4 rounds of Fed- batch to ensure the stable, high-expression cell lines can be obtained.
Overexpression cell lines (membrane protein/transmembrane protein)：Based on multiple screening systems such as 96-well, 24-well, 12-well, and 6-well plates, cell lines are screened by RT-qPCR and FACS to obtain high-expression and stable cell lines.
Tubular bioreactor
3L bioreactor
Shaking table
ABI 7500 Fast QPCR system
Flow cytometer
Case Study
1. Construction of membrane protein overexpression cell lines
1.1 Identification data of CHO cell overexpression cell lines
Objective: To construct an overexpression membrane protein on CHO cells for library screening and functional validation.
Method: The target gene was introduced into cells by electroporation, and the GS screening system was used for two rounds of screening to obtain stable overexpression cell lines.
Results: The expression level of the overexpression cell line was about 100,000 times higher than that of empty cells, and the positive rate was 99.9%.
Conclusions: The overexpression cell line was successfully constructed.
Fig. 2 FACS identification results of CHO-S overexpression cell line
1.2 Identification data of HEK293 cell overexpression cell lines
Objective: To construct an overexpression membrane protein on HEK293 cells for library screening and functional validation.
Methods: The target gene was introduced into cells by electroporation, and the Puromycin screening system was used for two rounds of screening to obtain stable overexpression cell lines.
Results: The expression level of the overexpression cell line was about 100,000 times higher than that of empty cells, and the positive rate was 99.7%.
Conclusions:The overexpression cell line was successfully constructed.
Fig. 3 FACS identification results of HEK293 overexpression cell line
1.3 Identification data of tumor cell overexpression cell lines
Objective: To construct an overexpression membrane protein on tumor cell jurkat for library screening and functional validation.
Methods: The target gene was introduced into cells through lentiviral infection, and the Puromycin screening system was used for two rounds of screening to obtain stable overexpression cell lines.
Results: The expression level of the overexpression cell line was about 10,000 times higher than that of the empty cells, and the positive rate was 100%.
Conclusions: The overexpression cell line was successfully constructed.
Fig. 4 FACS identification results of jurkat overexpression cell line
2. Luciferase reporter gene cell lines
2.1 PD-1/PD-L1 Luciferase reporter system
This reporter gene cell line system covers two cell lines. Jurkat cells are used as host cells for the effector cell line, which overexpress human PD-1 receptor protein and NF-AT-LUC2 response elements, and the monoclonal cells obtained by Puromycin and Hygromycin resistance gene screening systems. CHO-S cells are used as host cells for aAPC cell line, which express αCD3 ScFv antibody and PD-L1 protein on the cell membrane.
Fig. 5 Schematic diagram of PD-L1 luciferase reporter gene detection system
Fig. 6 αPD-1/PD-L1 antagonist antibody activates TCR signaling
2.2 VEGF/VEGFR2 luciferase reporter cell line
HEK293 cells are used as host cells in this reporter gene system, which overexpress human VEGFR2 receptor protein and NF-AT- Luc2 response element, and the monoclonal cells obtained by Puromycin and Hygromycin resistance gene screening systems.
Fig. 7 Schematic diagram
Fig. 8 Recombinant human VEGF protein-activated luciferase reporter gene system
Fig. 9 Neutralization of recombinant human VEGF protein with bevacizumab
3. Construction of secreted stable cell lines
3.1 Expression analysis
Objective: To construct a secreted stable overexpression cell pool by the Bulk pool method for gram-level protein production.
Methods: The target gene was introduced into cells by electroporation, and the GS screening system was used for pressurized screening to obtain a stable overexpression cell pool.
Results: The expression levels of 39 projects in the shake flask phase of cell pools were counted, and the average expression level was 2.2 g/L, with the expression levels of several cell pools higher than 3.0 g/L.
Conclusions: Gram-level protein can be obtained in about 35 days using the overexpression cell pool constructed by the bulk pool method.
Fig. 10 Cell pool expression quantity analysis
3.2 Detection of cell pool fed-batch culture in shake flask
Objective：To detect various parameters in cell expression process, and to comprehensively assess the protein expression level and expression stability.
Methods: The cell pool was inoculated into the culture medium for fed-batch culture in a shake flask, and samples were taken regularly to detect the viable cell density, cell viability, cell expression, and metabolic data such as lactic acid and glucose.
Results: The viable cell density of the cell line was up to 2.5E+7 cells/mL. After 14 days of culture, the cell viability could still be maintained at more than 70%, the antibody expression could reach 3.4 g/L, and the lactic acid is always maintained at a relatively low level.
Conclusions: All the indicators of cells in the process of expression met the standard requirements.
Fig. 11 Cell pool fed-batch culture in shake flask
Achievements
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