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