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Service Overviews

Background: The pilot study is the basis of clinical and commercial production. In order to solve the practical problems faced by pharmaceutical enterprises, such as long process time of fermentation, low antibody expression, substandard antibody quality, and non-compliance with application requirements , Sanyou Bio grandly launched the development service of fermentation process.


Methods: Sanyou Bio’s fermentation development service integrated four technologies of "plate / tube reactor screening, Gator high-throughput detection, matrix bioreactor and DOE experimental design", and established a robust cell culture process combined with systematic and comprehensive process control and product quality control.


Advantages: It only takes 3 months to complete the development of cell culture process, the antibody expression is higher than the industry average level of 3-5 g/L, the antibody quality is up to standard, and the compliance meets the requirements of drug application.


Cases: Since the service was launched in 2020, more than 30 projects of fermentation development have been completed. Sanyou Bio accumulated profound experience and established a stable platform. At present, the average antibody expression in the upstream is 5-6 g/L, up to 12 g/L, which is at the leading level in the industry.

Service Overviews Background: The pilot study is the basis of clinical and commercial production. In order to solve the practical problems faced by pharmaceutical enterprises, such as long process time of fermentation, low antibody expression, substandard antibody quality, and non-compliance with application requirements , Sanyou Bio grandly launched the development service of fermentation process. Methods: Sanyou Bio’s fermentation development service integrated four technologies of "plate / tube reactor screening, Gator high-throughput detection, matrix bioreactor and DOE experimental design", and established a robust cell culture process combined with systematic and comprehensive process control and product quality control. Advantages: It only takes 3 months to complete the development of cell culture process, the antibody expression is higher than the industry average level of 3-5 g/L, the antibody quality is up to standard, and the compliance meets the requirements of drug application. Cases: Since the service was launched in 2020, more than 30 projects of fermentation development have been completed. Sanyou Bio accumulated profound experience and established a stable platform. At present, the average antibody expression in the upstream is 5-6 g/L, up to 12 g/L, which is at the leading level in the industry. Service Contents Service Service Details Client Provides Deliverables and Standards Time R&D of fermentation process 1.Optimization and screening of medium 2. Evaluation of Fermentation tank clone screening 3. Optimization of Fermentation parameters 4. Confirmation of Fermentation preliminary amplification Cell lines 1. Antibody quantity ≥ 3 g/L 2. Qualified antibody ≥ 1 g 3. Upstream process development and confirmation report 4. Antibody quality test report 5. Scanning copies of experimental records 10-14 weeks Service Highlights 1. Surely standard antibody quality Purity: SEC-HPLC monomer ratio ≥ 95%, nrCE-SDS main peak ≥ 92%, CEX main peak ≥ 50%. Activity: The relative binding activity meets the requirements (70%-130%), and the biological activity meets the requirements. 2. Extremely compressed development period Experienced project management team ensures seamless connection between platforms, and completes one round of process optimization within 3-4 weeks. Mature and stable platform technology escorts the rapid process, and the development is completed in 2-3 rounds of tests. 3. International leading antibody production The average expression level of the upstream fermentation is 5-6 g/L, and the highest is 12 g/L. The average recovery rate of the downstream purification is 60%-80%, and the highest is 80%. 4. Compliance with drug declaration requirements The cell lines, culture medium, reagents, consumables and other materials meet the regulatory requirements. The quality of the stock solution of the batch confirmed by the process conforms to the Chinese and US pharmacopoeia standards. 5. Four sets of authorized host cells Including Freedom CHO-S, HD-BioP3 Null CHO-K1, CHOZN, CHO-K1Q. A robust and efficient platform has been established for four sets of commercial authorized host cells. 6. Experience of more than 30 projects After more than 30 projects of repeated polishing and meticulous crafting, the cell culture process has been dramatically improved. Profound practical experience in increasing antibody expression, improving antibody quality, and reducing metabolic byproducts. Service Features 1. Strict process control and product quality control process control indicators and 7 product quality control indicators. Table 1 Process and Quality Control Standards Category Test Items Platereactor Tubularreactor/shake flask 3 L Bioreactor 15L Bioreactor Acceptance Process control Cell viability before inoculation √ √ √ √ Cell viability ≥ 95% Cell doubling time before seeding √ √ √ √ Doubling time ≤ 26 hours Cell density during culture √ √ √ √ Peak VCD ≥ 10×106 cells /mL Cell viability during culture √ √ √ √ Cell viability at harvest ≥ 60% Culture cycle √ √ √ √ ≥ 12 days Osmotic pressure -- √ -- -- Osmotic pressure of harvest liquid ≤ 450 mOsmol/kg Glucose concentration √ √ √ √ 0.2g /L ≤ Glc ≤ 8 g/L Lactic acid concentration -- √ √ √ Lac ≤ 3 g/L Ammonium ion concentration -- -- -- √ NH4+ ≤ 15 mM Glutamate concentration -- -- -- √ N/A Glutamine concentration -- -- -- √ N/A Carbon dioxide partial pressure -- -- -- √ pCO2 ≤ 120 mmHg Antibody expression (Protein A-HPLC) √ √ √ √ ≥3 g/L Quality Control Molecular size isomer (SEC-HPLC) -- √ √ √ Monomer ≥ 95% Molecular size isomer (nrCE-SDS) -- √ √ √ Main peak ≥ 92% Molecular size isomer (SDS-PAGE) √ -- -- -- purity ≥ 90% charge isomers (CEX-HPLC) -- √ √ √ Main peak ≥ 50% Relative binding activity (ELISA) √ √ -- √ 70%-130% Biological activity -- -- -- √ Meeting the standard Molecular weight (LC-MS) -- -- -- √ Meeting the standard Sugar isomer (UPLC-MS) -- -- -- √ Man5 proportion ≤ 10% Remarks: "√" is a mandatory inspection item; "--" is a non-required inspection item, which will be inspected according to the project situation. 2. Multi-dimensional screening system to improve protein expression and quality The multi-dimensional system is screened and optimized step by step, the antibody expression is gradually increased, and the antibody quality strictly meets the standards. 3. Four sets of officially authorized host cells to choose 4 GMP cell lines with clear sources, including officially authorized host cells from Thermo Fisher, Horizon, Merck, and QuaCell. 4. Fully equipped and well established support system as escort Instruments and equipments: High-throughput instruments and equipments speed up pre-screening; standardized instruments and equipments ensure technology transfer and amplification. Documentation system: All standard operating procedures, management procedures, R&D and production operations are formulated and implemented based on ICH guidelines. Reference regulations: ICH Q8-Q12, NMPA/FDA/EMA guidance for industry; Pharmacopoeia (ChP, USP, EP). Case Stastics 1. Preliminary screening test of the culture Objectives: To screen the medium with high antibody expression by shaking flasks. Methods: The cell lines were inoculated into 9 different kinds of commercial culture medium for Fed-batch in shake flasks, and samples were taken regularly to detect relevant indicators. Results: The viable cell density reached (20-25) × 106 cells /mL, and after 15 days of culture, the cell viability was still higher than 70%, and the antibody expression level was 6-7 g/L. Conclusion: The high expression medium with antibody expression amount of 6-7 g/L was obtained through preliminary screening. Fig. 1 Preliminary screening test of the culture medium of a monoclonal antibody 2. Experiment of improving the expression of antibodies of a bispecific antibody Objectives: To optimize the cell culture process in a 3 L reactor to increase antibody expression and improve cell growth and biochemical metabolism. Methods: The cell lines were inoculated into a 3 L reactor for fed-batch culture, and samples were taken regularly to detect relevant indicators. Results: The culture time was extended from 12 days to 16 days, and the antibody expression reached 12 g/L. Conclusion: Through the optimization of culture medium, feeding strategy and pH, the expression of antibody was increased by 782%, and the cell growth was significantly improved. Fig. 2 Antibody expression improvement test of a bispecific antibody 3. Purity improvement test of an asymmetric bispecific antibody Objectives: To improve the purity and expression of target double-antibody SEC and nrCE-SDS by optimizing the medium. Methods: The cell lines were inoculated into 14 different commercial culture medium for Fed-batch in shake flasks, and samples were taken regularly to detect relevant indicators. Results: Medium 1 was used before optimization, the antibody expression was 4.3 g/L, the SEC monomer ratio was 67.4%, and the nrCE-SDS main peak ratio was 58.2%. After optimization, medium 12 was used, the antibody expression was 7.4 g/L, the SEC monomer ratio was 83.5%, and the nrCE-SDS main peak ratio was 81.8%. Conclusion: The purity and expression of the target double-antibody SEC and nrCE-SDS were significantly improved by optimizing the medium. Fig. 3 Purity improvement test of an asymmetric bispecific antibody 4. Lactic acid reduction test of a bispecific antibody Objectives: To reduce lactate and increase antibody expression through process optimization. Methods: The cell lines were inoculated into fermentors for Fed-batch culture, and samples were taken regularly to detect relevant indicators. Results: Using the pre-optimized process, the lactic acid concentration reached 5.6 g/L, the cell growth was affected, and the antibody expression was only 1.3 g/L. With the optimized process, the lactate concentration was always lower than 2.5 g/L, the cell growth was improved, and the antibody expression was increased to 3.2 g/L. Conclusion: Through process optimization, lactate was significantly reduced, cell growth was improved, and antibody expression was increased by 146%. Fig. 4 Lactate reduction test of a bispecific antibody 5. Purity Analysis by SEC-HPLC Size Exclusion Chromatography (SEC) separates proteins based on their hydrodynamic size in solution. As shown in Fig. 5, the SEC-HPLC monomer ratio was 97.73%, which met the quality standards (the proportion of SEC-HPLC monomers ≥ 95%). Fig. 5 SEC-HPLC purity analysis of a bispecific antibody 6. Non-reducing CE-SDS Purity Analysis Size Exclusion Chromatography (SEC) separates proteins based on their hydrodynamic size in solution. As shown in Fig. 5, the SEC-HPLC monomer ratio was 97.73%, which met the quality standards (the proportion of SEC-HPLC monomers ≥ 95%). Fig. 6 Non-reducing CE-SDS purity analysis of a bispecific antibody 7. CEX charge heterogeneity analysis Cation Exchange Chromatography (CEX) allows separations based on differences in the electrical charges of proteins in solution. As shown in Fig. 7, the CEX acid peak ratio was 32.8%, the main peak ratio was 62.0%, and the basic peak ratio was 5.2%, which met the quality standards (the proportion of CEX main peaks ≥ 50%). Fig. 7 CEX charge heterogeneity analysis of a double antibody project 8. ELISA Binding Detection Enzyme-linked immunosorbent assay (ELISA) refers to a qualitative and quantitative detection method that binds a soluble antigen or antibody to a solid-phase carrier such as polystyrene, and uses the specific binding of antigen and antibody to carry out an immune reaction. As shown in Fig. 8, the target antibody binding activity was normal, which met the quality standards (70% ≤ relative binding activity ≤ 130%). Fig. 8 ELISA Binding detection of an antibody project 9. Analysis of Glycosylation Modifications Liquid Chromatography-Mass Spectrometry (LC-MS) uses liquid chromatography as the separation system and mass spectrometry as the detection system, combining the high separation ability of chromatography with the high selectivity and sensitivity of mass spectrometry, and the advantages of information to provide relative molecular mass and structure. As shown in Fig. 9, the Man5 contents of the two batches were 3.36% and 3.18%, respectively, which met the quality standard (Man5 ≤ 10%). Fig. 9 ELISA Binding detection of an antibody project

Service Contents

Service

Service Details

Client Provides

Deliverables and Standards

Time

R&D of fermentation process

1.Optimization and screening of medium

2. Evaluation of Fermentation tank clone screening

3. Optimization of Fermentation parameters

4. Confirmation of Fermentation preliminary amplification

Cell lines

1. Antibody quantity ≥ 3 g/L

2. Qualified antibody ≥ 1 g

3. Upstream process development and confirmation report

4. Antibody quality test report

5. Scanning copies of experimental records

10-14 weeks


Service Highlights
  • 1. Surely standard antibody quality
    1. Purity: SEC-HPLC monomer ratio ≥ 95%, nrCE-SDS main peak ≥ 92%, CEX main peak ≥ 50%.
    2. Activity: The relative binding activity meets the requirements (70%-130%), and the biological activity meets the requirements.
  • 2. Extremely compressed development period
    1. Experienced project management team ensures seamless connection between platforms, and completes one round of process optimization within 3-4 weeks.
    2. Mature and stable platform technology escorts the rapid process, and the development is completed in 2-3 rounds of tests.
  • 3. International leading antibody production
    1. The average expression level of the upstream fermentation is 5-6 g/L, and the highest is 12 g/L.
    2. The average recovery rate of the downstream purification is 60%-80%, and the highest is 80%.
  • 4. Compliance with drug declaration requirements
    1. The cell lines, culture medium, reagents, consumables and other materials meet the regulatory requirements.
    2. The quality of the stock solution of the batch confirmed by the process conforms to the Chinese and US pharmacopoeia standards.
  • 5. Four sets of authorized host cells
    1. Including Freedom CHO-S, HD-BioP3 Null CHO-K1, CHOZN, CHO-K1Q.
    2. A robust and efficient platform has been established for four sets of commercial authorized host cells.
  • 6. Experience of more than 30 projects
    1. After more than 30 projects of repeated polishing and meticulous crafting, the cell culture process has been dramatically improved.
    2. Profound practical experience in increasing antibody expression, improving antibody quality, and reducing metabolic byproducts.

Service Features
1. Strict process control and product quality control

process control indicators and 7 product quality control indicators.


Table 1 Process and Quality Control Standards

Category

Test Items

Platereactor

Tubularreactor/shake flask

3 L Bioreactor

15L Bioreactor

Acceptance

Process

control

Cell viability before inoculation

Cell viability ≥ 95%

Cell doubling time before seeding

Doubling time ≤ 26 hours

Cell density during culture

Peak VCD ≥ 10×10cells /mL

Cell viability during culture

Cell viability at harvest ≥ 60%

Culture cycle

≥ 12 days

Osmotic pressure

--

--

--

Osmotic pressure of harvest liquid ≤ 450 mOsmol/kg

Glucose concentration

0.2g /L ≤ Glc ≤ 8 g/L

Lactic acid concentration

--

Lac ≤ 3 g/L

Ammonium ion concentration

--

--

--

NH4+ ≤ 15 mM

Glutamate concentration

--

--

--

N/A

Glutamine concentration

--

--

--

N/A

Carbon dioxide partial pressure

--

--

--

pCO2 ≤ 120 mmHg

Antibody expression (Protein A-HPLC)

≥3 g/L

Quality

Control

Molecular size isomer (SEC-HPLC)

--

Monomer ≥ 95%

Molecular size isomer (nrCE-SDS)

--

Main peak ≥ 92%

Molecular size isomer (SDS-PAGE)

--

--

--

purity ≥ 90%

charge isomers (CEX-HPLC)

--

Main peak ≥ 50%

Relative binding activity (ELISA)

--

70%-130%

Biological activity

--

--

--

Meeting the standard

Molecular weight (LC-MS)

--

--

--

Meeting the standard

Sugar isomer (UPLC-MS)

--

--

--

Man5 proportion ≤ 10%



Remarks: "√" is a mandatory inspection item; "--" is a non-required inspection item, which will be inspected according to the project situation.

2. Multi-dimensional screening system to improve protein expression and quality

The multi-dimensional system is screened and optimized step by step, the antibody expression is gradually increased, and the antibody quality strictly meets the standards.

3. Four sets of officially authorized host cells to choose

4 GMP cell lines with clear sources, including officially authorized host cells from Thermo Fisher, Horizon, Merck, and QuaCell.


4. Fully equipped and well established support system as escort

Instruments and equipments: High-throughput instruments and equipments speed up pre-screening; standardized instruments and equipments ensure technology transfer and amplification.

Documentation system: All standard operating procedures, management procedures, R&D and production operations are formulated and implemented based on ICH guidelines.

Reference regulations: ICH Q8-Q12, NMPA/FDA/EMA guidance for industry; Pharmacopoeia (ChP, USP, EP).



Case Studies
1. Preliminary screening test of the culture

Objectives: To screen the medium with high antibody expression by shaking flasks.

Methods: The cell lines were inoculated into 9 different kinds of commercial culture medium for Fed-batch in shake flasks, and samples were taken regularly to detect relevant indicators.

Results: The viable cell density reached (20-25) × 106 cells /mL, and after 15 days of culture, the cell viability was still higher than 70%, and the antibody expression level was 6-7 g/L.

Conclusion: The high expression medium with antibody expression amount of 6-7 g/L was obtained through preliminary screening.


Fig. 1 Preliminary screening test of the culture medium of a monoclonal antibody

2. Experiment of improving the expression of antibodies of a bispecific antibody

Objectives: To optimize the cell culture process in a 3 L reactor to increase antibody expression and improve cell growth and biochemical metabolism.

Methods: The cell lines were inoculated into a 3 L reactor for fed-batch culture, and samples were taken regularly to detect relevant indicators.

Results: The culture time was extended from 12 days to 16 days, and the antibody expression reached 12 g/L.

Conclusion: Through the optimization of culture medium, feeding strategy and pH, the expression of antibody was increased by 782%, and the cell growth was significantly improved.


Fig. 2 Antibody expression improvement test of a bispecific antibody

3. Purity improvement test of an asymmetric bispecific antibody

Objectives: To improve the purity and expression of target double-antibody SEC and nrCE-SDS by optimizing the medium.

Methods: The cell lines were inoculated into 14 different commercial culture medium for Fed-batch in shake flasks, and samples were taken regularly to detect relevant indicators. 

Results: Medium 1 was used before optimization, the antibody expression was 4.3 g/L, the SEC monomer ratio was 67.4%, and the nrCE-SDS main peak ratio was 58.2%. After optimization, medium 12 was used, the antibody expression was 7.4 g/L, the SEC monomer ratio was 83.5%, and the nrCE-SDS main peak ratio was 81.8%.

Conclusion: The purity and expression of the target double-antibody SEC and nrCE-SDS were significantly improved by optimizing the medium.


Fig. 3 Purity improvement test of an asymmetric bispecific antibody

4. Lactic acid reduction test of a bispecific antibody

Objectives: To reduce lactate and increase antibody expression through process optimization.

Methods: The cell lines were inoculated into fermentors for Fed-batch culture, and samples were taken regularly to detect relevant indicators.

Results: Using the pre-optimized process, the lactic acid concentration reached 5.6 g/L, the cell growth was affected, and the antibody expression was only 1.3 g/L. With the optimized process, the lactate concentration was always lower than 2.5 g/L, the cell growth was improved, and the antibody expression was increased to 3.2 g/L.

Conclusion: Through process optimization, lactate was significantly reduced, cell growth was improved, and antibody expression was increased by 146%.


Fig. 4 Lactate reduction test of a bispecific antibody

5. Purity Analysis by SEC-HPLC

Size Exclusion Chromatography (SEC) separates proteins based on their hydrodynamic size in solution. As shown in Fig. 5, the SEC-HPLC monomer ratio was 97.73%, which met the quality standards (the proportion of SEC-HPLC monomers ≥ 95%).


Fig. 5 SEC-HPLC purity analysis of a bispecific antibody

6. Non-reducing CE-SDS Purity Analysis

Size Exclusion Chromatography (SEC) separates proteins based on their hydrodynamic size in solution. As shown in Fig. 5, the SEC-HPLC monomer ratio was 97.73%, which met the quality standards (the proportion of SEC-HPLC monomers ≥ 95%).


Fig. 6 Non-reducing CE-SDS purity analysis of a bispecific antibody

7. CEX charge heterogeneity analysis

Cation Exchange Chromatography (CEX) allows separations based on differences in the electrical charges of proteins in solution. As shown in Fig. 7, the CEX acid peak ratio was 32.8%, the main peak ratio was 62.0%, and the basic peak ratio was 5.2%, which met the quality standards (the proportion of CEX main peaks ≥ 50%).


Fig. 7 CEX charge heterogeneity analysis of a double antibody project

8. ELISA Binding Detection
Enzyme-linked immunosorbent assay (ELISA) refers to a qualitative and quantitative detection method that binds a soluble antigen or antibody to a solid-phase carrier such as polystyrene, and uses the specific binding of antigen and antibody to carry out an immune reaction. As shown in Fig. 8, the target antibody binding activity was normal, which met the quality standards (70% ≤ relative binding activity ≤ 130%).


Fig. 8 ELISA Binding detection of an antibody project

9. Analysis of Glycosylation Modifications
Liquid Chromatography-Mass Spectrometry (LC-MS) uses liquid chromatography as the separation system and mass spectrometry as the detection system, combining the high separation ability of chromatography with the high selectivity and sensitivity of mass spectrometry, and the advantages of information to provide relative molecular mass and structure. As shown in Fig. 9, the Man5 contents of the two batches were 3.36% and 3.18%, respectively, which met the quality standard (Man5 ≤ 10%).


Fig. 9 ELISA Binding detection of an antibody project