菜单
Service Overviews

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 1000 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 Overviews 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 1000 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 Contents Service Service Details Client Provides Deliverables and Standards Time Construction of research cell lines Cell pool construction Gen sequences 1–5 high-expression cell pools 4–6 weeks Construction of research cell lines Monoclonal cell line construction Gen sequences 3–6 monoclonal cell lines 6–8 weeks Construction of research cell lines Cell bank construction (optional) Gen sequences MCB and WCB ≥ 150 vials 4 weeks Construction of research cell lines Cell bank stability study Gen sequences Stability study report 8–10 weeks Service Highlights 1. Extensive varieties: diverse molecular types and abundant host cell bank resources Flexible construction of secreted antigens, antibodies, membrane proteins,transmembrane protein overexpression cell lines and luciferase reporting systems. Overexpression cell lines are constructed with CHO, HEK293, and various of tumor cells by electrotransformation, chemotransformation, and lentiviral infection, which can be screened by puromycin, hygromycin, G418, and GS high-expression screening systems to obtain stable high-expression cell lines. 2. Comprehensive quality assessment: comprehensive evaluation of RT-qPCR, FACS, and PDL The constructed overexpression cell lines are detected by Gator, HPLC, RT-qPCR, and FACS at the protein and gene levels, respectively, the stability of cell lines is evaluated by continuous passage of 20 PDLs, and ensure positive rate > 80%. 3. Fast and efficient: gram-level antibodies can be obtained in 35 days, and stable overexpression cell lines can be obtained in 4–6 weeks From the vector construction, it takes only 35 days at the earliest to obtain gram-level antibodies, and only about 4 weeks at the earliest to obtain a fully qualified overexpression cell line for research that can be ultimately used for the development of therapeutic products. 4. Extensive use: suitable for the whole process of therapeutic product development Stable cell line models constructed can be used for subsequent gene function research and gene therapy research, which can be further used for drug target screening, cell signaling pathway analysis and protein interaction; It is also suitable for in vivo experiments of tumor formation in nude mice. 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. Stability Assessment Items and Standards Item Stability assessment Subject Selected monoclonal cell lines Assessment description Stability assessment on final clones for 20 passages (PDL) Cell line passage stability The duration of cell doubling in the process of passage should be consistent Cell line growth stability The growth curve should overlap with that of seed cells Cell line growth stability There is no significant difference in the highest viable cell density between the cell lines and seed cells Cell line growth stability There is no significant difference in the cellular form between the cell lines and seed cells Cell line expression stability Overexpression should be displayed at both protein and gene levels, and the positive rate should be more than 80% Cell line expression stability The product's physiochemical characteristics are consistent with that of seed cells Genetic stability of cell lines The sequence of target genes is consistent with that of seed cells Genetic stability of cell lines The difference in gene copy number between the product and seed cells should not exceed 30% 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 Assessment Items and Standards Item Stability assessment Subject Selected monoclonal cell lines Assessment description Stability assessment on final clones for 20 passages (PDL) Cell line passage stability The duration of cell doubling in the process of passage should be consistent Cell line growth stability The growth curve should overlap with that of seed cells Cell line growth stability There is no significant difference in the highest viable cell density between the cell lines and seed cells Cell line growth stability There is no significant difference in the cellular form between the cell lines and seed cells Cell line expression stability Overexpression should be displayed at both protein and gene levels, and the positive rate should be more than 80% Cell line expression stability The product's physiochemical characteristics are consistent with that of seed cells Genetic stability of cell lines The sequence of target genes is consistent with that of seed cells Genetic stability of cell lines The difference in gene copy number between the product and seed cells should not exceed 30% 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. Fig. 1 Statistics of different types of cell lines 4. Multiple screening to ensure optimal clones Secreted cell lines: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. Fig. 2 Main instruments and equipments Case Stastics 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. 3 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. 4 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 NCI-N87 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. 5 FACS identification results of NCI-N87 overexpression cell line 2. Luciferase reporter gene cell lines 2.1. PD-1/PD-L1 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. 6 Schematic diagram of PD-L1 luciferase reporter gene detection system Fig. 7 αPD-1/PD-L1 antagonist antibody activates TCR signaling 2.2. VEGF/VEGFR2 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. 8 Schematic diagram Fig. 9 Recombinant human VEGF protein-activated luciferase reporter gene system Fig. 10 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. 11 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 2.5×107 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. 12 Cell pool fed-batch culture in shake flask

Service Contents

Service

Service Details

Client Provides

Deliverables and Standards

Time

Construction of research cell lines

Cell pool construction

Gen sequences

1–5 high-expression cell pools

4–6 weeks

Construction of research cell lines

Monoclonal cell line construction

Gen sequences

3–6 monoclonal cell lines

6–8 weeks

Construction of research cell lines

Cell bank construction (optional)

Gen sequences

MCB and WCB ≥ 150 vials

4 weeks

Construction of research cell lines

Cell bank stability study

Gen sequences

Stability study report

8–10 weeks


Service Highlights
  • 1. Extensive varieties: diverse molecular types and abundant host cell bank resources
    1. Flexible construction of secreted antigens, antibodies, membrane proteins,transmembrane protein overexpression cell lines and luciferase reporting systems. Overexpression cell lines are constructed with CHO, HEK293, and various of tumor cells by electrotransformation, chemotransformation, and lentiviral infection, which can be screened by puromycin, hygromycin, G418, and GS high-expression screening systems to obtain stable high-expression cell lines.
  • 2. Comprehensive quality assessment: comprehensive evaluation of RT-qPCR, FACS, and PDL
    1. The constructed overexpression cell lines are detected by Gator, HPLC, RT-qPCR, and FACS at the protein and gene levels, respectively, the stability of cell lines is evaluated by continuous passage of 20 PDLs, and ensure positive rate > 80%.
  • 3. Fast and efficient: gram-level antibodies can be obtained in 35 days, and stable overexpression cell lines can be obtained in 4–6 weeks
    1. From the vector construction, it takes only 35 days at the earliest to obtain gram-level antibodies, and only about 4 weeks at the earliest to obtain a fully qualified overexpression cell line for research that can be ultimately used for the development of therapeutic products.
  • 4. Extensive use: suitable for the whole process of therapeutic product development
    1. Stable cell line models constructed can be used for subsequent gene function research and gene therapy research, which can be further used for drug target screening, cell signaling pathway analysis and protein interaction; It is also suitable for in vivo experiments of tumor formation in nude mice.

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.


Stability Assessment Items and Standards

Item

Stability assessment

Subject

Selected monoclonal cell lines

Assessment description

Stability assessment on final clones for 20 passages (PDL)

Cell line passage stability

The duration of cell doubling in the process of passage should be consistent

Cell line growth stability

The growth curve should overlap with that of seed cells

Cell line growth stability

There is no significant difference in the highest viable cell density between the cell lines and seed cells

Cell line growth stability

There is no significant difference in the cellular form between the cell lines and seed cells

Cell line expression stability

Overexpression should be displayed at both protein and gene levels, and the positive rate should be more than 80%

Cell line expression stability

The product's physiochemical characteristics are consistent with that of seed cells

Genetic stability of cell lines

The sequence of target genes is consistent with that of seed cells

Genetic stability of cell lines

The difference in gene copy number between the product and seed cells should not exceed 30%

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 Assessment Items and Standards

Item

Stability assessment

Subject

Selected monoclonal cell lines

Assessment description

Stability assessment on final clones for 20 passages (PDL)

Cell line passage stability

The duration of cell doubling in the process of passage should be consistent

Cell line growth stability

The growth curve should overlap with that of seed cells

Cell line growth stability

There is no significant difference in the highest viable cell density between the cell lines and seed cells

Cell line growth stability

There is no significant difference in the cellular form between the cell lines and seed cells

Cell line expression stability

Overexpression should be displayed at both protein and gene levels, and the positive rate should be more than 80%

Cell line expression stability

The product's physiochemical characteristics are consistent with that of seed cells

Genetic stability of cell lines

The sequence of target genes is consistent with that of seed cells

Genetic stability of cell lines

The difference in gene copy number between the product and seed cells should not exceed 30%

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.


Fig. 1 Statistics of different types of cell lines

4. Multiple screening to ensure optimal clones

Secreted cell lines: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.


Fig. 2 Main instruments and equipments


Case Studies
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. 3 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. 4 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 NCI-N87 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. 5 FACS identification results of NCI-N87 overexpression cell line

2. Luciferase reporter gene cell lines

2.1. PD-1/PD-L1

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. 6 Schematic diagram of PD-L1 luciferase reporter gene detection system


Fig. 7 αPD-1/PD-L1 antagonist antibody activates TCR signaling


2.2. VEGF/VEGFR2

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. 8 Schematic diagram


Fig. 9 Recombinant human VEGF protein-activated luciferase reporter gene system


Fig. 10 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. 11 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 2.5×107 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. 12 Cell pool fed-batch culture in shake flask