菜单
Service Overviews

Background: Therapeutic recombinant protein drugs expressed in mammalian cells are closer to human natural protein in structure and function. Based on this, Sanyou Bio launched the rapid protein preparation service.


Methods: Rapid protein preparation service integrates Gateway vector construction, mammalian cell expression system, protein purification and physicochemical and biochemical detection technologies, and matches automated high-throughput equipment for rapid and high-quality preparation of various proteins.


Advantages:

1. Fast. Plasmid construction, protein expression and purification could be completed within 14 days. The multi-dimensional quality control system could ensure the delivery of high-quality antigen or antibody proteins. 

2. High-throughput. Using automated high-throughput equipment, the throughput could reach 2000 protein preparations of small amount per week.


Cases: After 6 years of development, Sanyou has accumulated rich experience in protein preparation. It has successfully completed tens of thousands of protein preparations, covering various molecular types such as nanobodies, human or humanized IgG antibodies, cytokines and other types of recombinant proteins.

Service Overviews Background: Therapeutic recombinant protein drugs expressed in mammalian cells are closer to human natural protein in structure and function. Based on this, Sanyou Bio launched the rapid protein preparation service. Methods: Rapid protein preparation service integrates Gateway vector construction, mammalian cell expression system, protein purification and physicochemical and biochemical detection technologies, and matches automated high-throughput equipment for rapid and high-quality preparation of various proteins. Advantages: 1. Fast. Plasmid construction, protein expression and purification could be completed within 14 days. The multi-dimensional quality control system could ensure the delivery of high-quality antigen or antibody proteins. 2. High-throughput. Using automated high-throughput equipment, the throughput could reach 2000 protein preparations of small amount per week. Cases: After 6 years of development, Sanyou has accumulated rich experience in protein preparation. It has successfully completed tens of thousands of protein preparations, covering various molecular types such as nanobodies, human or humanized IgG antibodies, cytokines and other types of recombinant proteins. Service Contents Service No. Service Service Details Client Provides Deliverables and Standards Time SAP-5 Rapid small-scale preparation of antibodies 1. Gene synthesis(optional) 2. Plasmidconstruction 3. Protein expression and purification 4. Quality control tests (SDS-PAGE, SEC, Endotoxin) Antibody gene sequence Deliverables: 1. 0.5-1 mgpurified antibody proteins 2. Analysis certificate Standards: 1. Purities of antibody by SDS-PAGE>95% and by SEC-HPLC 90% 2. Endotoxin<1 EU/mg 3. Affinity assay (optional) ELISA/FACS 4. Affinity kinetics determination (optional):Biacore 2 weeks MAP-300 Rapid medium-scale preparation of antibodies 1.Gene synthesis(optional) 2.Plasmid construction 3.Protein expression and purification 4.Quality control tests(SDS-PAGE, SEC, Endotoxin) Antibody gene sequence Deliverables: 1. 10-30 mgpurified antibody proteins 2. Analysis certificate Standards: 1. Purities of antibody by SDS-PAGE>95% and by SEC-HPLC>90% 2. Endotoxin<1 EU/mg 3. Affinity assay (optional):ELISA/FACS 4. Affinity kinetics determination (optional):Biacore 2-3 weeks SPP-30 Rapid small-scale preparation of antigens (30 mL) 1.Gene synthesis(optional) 2.Plasmid construction 3.Protein expression and purification 4.Quality control tests (SDS-PAGE, SEC, Endotoxin) Antibody gene sequence Deliverables: 1. 0.5-1 mgpurified antibody proteins 2. Analysis certificate Standards: 1. Purities of antibody bySDS-PAGE>85% and by SEC-HPLC>90% 2. Endotoxin<1 EU/mg 3. Affinity assay (optional):ELISA/FACS 4. Affinity kinetics determination (optional):Biacore 2 weeks MPP-300 Rapid medium-scale preparation of antigens (300 mL) 1.Gene synthesis (optional) 2.Plasmid construction 3.Protein expression and purification 4.Quality control tests(SDS-PAGE, SEC, Endotoxin) Antibody gene sequence Deliverables: 1. 1-10 mgpurified antibody proteins 2. Analysis certificate Standards: 1. Purities of antibody bySDS-PAGE>85% and by SEC-HPLC>90% 2. Endotoxin<1 EU/mg 3. Affinity assay (optional):ELISA/FACS 4. Affinity kinetics determination (optional):Biacore 2-3 weeks Service Highlights 1. Mammalian cell expression system, close to natural proteins In the Sanyou rapid protein expression and purification services, mammalian cell expression system (CHO/HEK293) is used, and the recombinant protein obtained by the system has a biological active conformation similar to the natural proteins. 2. Multi-dimensional quality control system ensuring high quality The quality of the antigen and antibody proteins could be evaluated by multiple parameters, such as concentration, purity, molecular weight, aggregation, stability, safety, charge heterogeneity, sequence variant, structure analysis and binding or blocking bioactivity analysis. 3. Efficient delivery enabling the completion within 2 weeks It is an integrated service that includes plasmid construction, extraction, transient expression, protein purification and protein quality control, the whole process of which could be completed as fast as 2 weeks. 4. Personalized services providing flexible choices With 3 eukaryotic expression systems, several expression volumes, 2 transient methods, 2 purification tags and 8 quality control indexes provided, customers could obtain a high-precision customization based on the specific needs of the project by combining those five dimensions flexibly. Service Features 1. High-performance Mammalian Cell Expression System Sanyou Bio applies a variety of eukaryotic expression systems such as Expi-CHO transient expression system, Expi-293F transient expression system and 293F transient expression system, which meets the expression needs of most proteins. Table 1 The eukaryotic transient expression systems Cell Lines Applicable Expression Classifications Expi-CHO transient expression system Mainly for antibody preparation Expi-293 Ftransient expression system Mainly for antigen preparation 293 Ftransient expression system Mainly for antigen preparation Fig.2 Expression comparison of 293F and Expi-CHO system 2. Advanced Production Process The international leading vector system integrates the strong promoters, enhancers and other functional elements, benefiting the efficient integration and expression of the target genes; and a high-throughput automated platform is established for plasmid construction, protein expression and protein purification, with large-scale and stable product delivery capabilities. 3. Multi-dimensional Quality Control Analysis 3.1. Commonly used parameters and methods of quality control Table 2 Commonly used parameters and methods of quality control Parameters Tests Analytical Methods Purity and molecular weight Molecular weight and purity identification SDS-PAGE(required) Purity and molecular weight Accurate purity identification SEC(required) Stability Determination of thermal stability DSF Inpurity Endotoxin LAL Concentration Relative concentration identification A280(required) Charge heterogeneity Charge heterogeneity determination CEX Sequence variants Peptide mapping analysis LC-UV-MS/MS Protein N-Glycan analysis N-glycan mapping analysis LC-MS Bioactivity Affinity assay ELISA/FACS(required) Bioactivity Affinity kinetics determination SPR/BLI(required) 3.2. Equipments ・ProteinsimpleMaurice ・Waters ACQUITY UPLC-H-Class PLUS ・BiacoreT200 ・ProbeLifeGator ・Agilent 1260 HPLC ・ABI 7500 Fast RT-PCR Case Stastics 1. Expression Data Of Different Types Of Proteins The expression data based on different expression volumes of 4 different types of proteins (nanobodies, human IgG antibodies, cytokines, and other types of recombinant proteins) are shown in Table 3. It shows that all 4 types of proteins were successfully expressed and purified after 5-7 days of expression, and the expression yields among different types varied greatly. Table 3 Example data for Sanyou recombinant protein preparation Case Protein type Purification Tag Expression System Expression Duration Expression Volume Protein Products Obtained Test results 1 Alpaca-derived nanobodies Fc Expi-CHO 7 days 300 mL 222.3 mg Purity (SDS-PAGE) > 95%, Monomer (SEC) 100% 2 Human/humanized IgG antibodies Fc Expi-CHO 7 days 15 mL 1.9 mg Purity (SDS-PAGE) > 95%, Monomer (SEC) 100% 3 Cytokines his Expi-CHO 7 days 100 mL 7.7 mg Purity (SDS-PAGE) > 85% 4 Other recombinant proteins Fc Expi-293 5 days 50 mL 12.4 mg Purity (SDS-PAGE) > 85% 2. Quality Control Data Case data on concentration, purity, molecular weight, thermal stability, binding or blocking bioactivity analysis and affinity kinetics of protein samples are shown in the following figures: Fig. 2 SDS-PAGE Purity of antibody protein is >95% Fig. 3 DSF The Tm1 (69.3 °C) and Tm2 (81.6 °C) of the marketed antibody drug A are consistent with the Tm from literatures Fig. 4 SEC SEC method Main peak area of protein is >95% Fig. 5 CE SDS Main peak area of protein is >95% Fig. 6 ELISA binding High antibody binding activity Fig. 7 FACS binding High antibody binding activity in vitro Fig. 8 Biacore High antibody affinity with antigen

Service Contents

Service No.

Service

Service Details

Client Provides

Deliverables and Standards

Time

SAP-5

Rapid small-scale preparation of antibodies

1. Gene synthesis(optional)

2. Plasmidconstruction

3. Protein expression and purification

4. Quality control tests (SDS-PAGE, SEC, Endotoxin)

Antibody gene sequence

Deliverables:

1. 0.5-1 mgpurified antibody proteins

2. Analysis certificate

Standards:

1. Purities of antibody by SDS-PAGE>95% and by SEC-HPLC 90%

2. Endotoxin<1 EU/mg

3. Affinity assay (optional) ELISA/FACS

4. Affinity kinetics determination (optional):Biacore

2 weeks

MAP-300

Rapid medium-scale preparation of antibodies

1.Gene synthesis(optional)

2.Plasmid construction

3.Protein expression and purification

4.Quality control tests(SDS-PAGE, SEC, Endotoxin)

Antibody gene sequence

Deliverables:

1. 10-30 mgpurified antibody proteins

2. Analysis certificate

Standards:

1. Purities of antibody by SDS-PAGE>95% and by SEC-HPLC>90%

2. Endotoxin<1 EU/mg

3. Affinity assay (optional):ELISA/FACS

4. Affinity kinetics determination (optional):Biacore

2-3 weeks

SPP-30

Rapid small-scale preparation of antigens (30 mL)

1.Gene synthesis(optional)

2.Plasmid construction

3.Protein expression and purification

4.Quality control tests (SDS-PAGE, SEC, Endotoxin)

Antibody gene sequence

Deliverables:

1. 0.5-1 mgpurified antibody proteins

2. Analysis certificate

Standards:

1. Purities of antibody bySDS-PAGE>85% and by SEC-HPLC>90%

2. Endotoxin<1 EU/mg

3. Affinity assay (optional):ELISA/FACS

4. Affinity kinetics determination (optional):Biacore

2 weeks

MPP-300

Rapid medium-scale preparation of antigens (300 mL)

1.Gene synthesis (optional)

2.Plasmid construction

3.Protein expression and purification

4.Quality control tests(SDS-PAGE, SEC, Endotoxin)

Antibody gene sequence

Deliverables:

1. 1-10 mgpurified antibody proteins

2. Analysis certificate

Standards:

1. Purities of antibody bySDS-PAGE>85% and by SEC-HPLC>90%

2. Endotoxin<1 EU/mg

3. Affinity assay (optional):ELISA/FACS

4. Affinity kinetics determination (optional):Biacore

2-3 weeks


Service Highlights
  • 1. Mammalian cell expression system, close to natural proteins
    1. In the Sanyou rapid protein expression and purification services, mammalian cell expression system (CHO/HEK293) is used, and the recombinant protein obtained by the system has a biological active conformation similar to the natural proteins.
  • 2. Multi-dimensional quality control system ensuring high quality
    1. The quality of the antigen and antibody proteins could be evaluated by multiple parameters, such as concentration, purity, molecular weight, aggregation, stability, safety, charge heterogeneity, sequence variant, structure analysis and binding or blocking bioactivity analysis.
  • 3. Efficient delivery enabling the completion within 2 weeks
    1. It is an integrated service that includes plasmid construction, extraction, transient expression, protein purification and protein quality control, the whole process of which could be completed as fast as 2 weeks.
  • 4. Personalized services providing flexible choices
    1. With 3 eukaryotic expression systems, several expression volumes, 2 transient methods, 2 purification tags and 8 quality control indexes provided, customers could obtain a high-precision customization based on the specific needs of the project by combining those five dimensions flexibly.

Service Features
1. High-performance Mammalian Cell Expression System

Sanyou Bio applies a variety of eukaryotic expression systems such as Expi-CHO transient expression system, Expi-293F transient expression system and 293F transient expression system, which meets the expression needs of most proteins.


Table 1 The eukaryotic transient expression systems

Cell Lines

Applicable Expression Classifications

Expi-CHO transient expression system

Mainly for antibody preparation

Expi-293 Ftransient expression system

Mainly for antigen preparation

293 Ftransient expression system

Mainly for antigen preparation


Fig.2 Expression comparison of 293F and Expi-CHO system
2. Advanced Production Process

The international leading vector system integrates the strong promoters, enhancers and other functional elements, benefiting the efficient integration and expression of the target genes; and a high-throughput automated platform is established for plasmid construction, protein expression and protein purification, with large-scale and stable product delivery capabilities.


3. Multi-dimensional Quality Control Analysis

3.1. Commonly used parameters and methods of quality control


Table 2 Commonly used parameters and methods of quality control

Parameters

Tests

Analytical Methods

Purity and molecular weight

Molecular weight and purity identification

SDS-PAGE (required) 

Purity and molecular weight

Accurate purity identification

SEC (required)

Stability

Determination of thermal stability

DSF

Inpurity

Endotoxin

LAL

Concentration

Relative concentration identification

A280 (required)

Charge heterogeneity

Charge heterogeneity determination

CEX

Sequence variants

Peptide mapping analysis

LC-UV-MS / MS

Protein N-Glycan analysis

N-glycan mapping analysis

LC-MS

Bioactivity

Affinity assay

ELISA / FACS (required)

Bioactivity

Affinity kinetics determination

SPR / BLI (required)


3.2. Equipments

・ProteinsimpleMaurice

・Waters ACQUITY UPLC-H-Class PLUS

・BiacoreT200

・ProbeLifeGator

・Agilent 1260 HPLC

・ABI 7500 Fast RT-PCR


Case Studies
1. Expression Data Of Different Types Of Proteins

The expression data based on different expression volumes of 4 different types of proteins (nanobodies, human IgG antibodies, cytokines, and other types of recombinant proteins) are shown in Table 3. It shows that all 4 types of proteins were successfully expressed and purified after 5-7 days of expression, and the expression yields among different types varied greatly.


Table 3 Example data for Sanyou recombinant protein preparation

Case 

Protein Type

Purification Tag

Expression System

Expression Duration

Expression Volume

Protein Products Obtained

Test Results

1

Alpaca-derived nanobodies

Fc

Expi-CHO

7 days

300 mL

222.3 mg

Purity (SDS-PAGE) > 95%, Monomer (SEC) 100%

2

Human/humanized IgG antibodies

Fc

Expi-CHO

7 days

15 mL

1.9 mg

Purity (SDS-PAGE) > 95%, Monomer (SEC) 100%

3

Cytokines

his

Expi-CHO

7 days

100 mL

 7.7 mg

Purity (SDS-PAGE) > 85%

4

Other recombinant proteins

Fc

Expi-293

5 days

50 mL

12.4 mg

Purity (SDS-PAGE) > 85%

2. Quality Control Data

Case data on concentration, purity, molecular weight, thermal stability, binding or blocking bioactivity analysis and affinity kinetics of protein samples are shown in the following figures:


Fig. 2 SDS-PAGE

Purity of antibody protein is >95%


Fig. 3 DSF


The Tm1 (69.3 °C) and Tm2 (81.6 °C) of the marketed antibody

drug A are consistent with the Tm from literatures


Fig. 4 SEC

SEC method Main peak area of protein is >95%


Fig. 5 CE SDS

Main peak area of protein is >95%


Fig. 6 ELISA binding

High antibody binding activity


Fig. 7 FACS binding

High antibody binding activity in vitro


Fig. 8 Biacore

High antibody affinity with antigen