Background: There are large demands for protein materials during R&D and production of antibody drugs, such as in vivo efficacy and PK evaluations, drug formulation process development, and safety evaluation. In addition, the R&D and production of diagnostic reagents also require a large amount of protein materials.
Method: Therefore, Sanyou Bio launches the service of high-quality antibody sample preparation, Based on the GMP cell lines, the stable cell pools are constructed to prepare production of antibody bulk samples. The screened high expression stable cell pools can be used for continuous massive production of proteins, and the whole process only takes for 35 days!
Rich experience, rapid delivery, and high success rate
Accumulation of experience in hundreds of service projects, supported with professional teams having decades of experience, 35-day rapid delivery, over 95% project success delivery guarantee and 90% customer satisfaction experience.
Multi-dimensional quality control, flexible options, and quality guaratee
Advanced antibody production process system with international high-end equipment, production in GMP host cells and all-time C + A class clean environment, comprehensive quality control system, ensure the quality of antibody delivery.
One-time construction, continuous production, and high cost-effictive performance
Large amount production of antibodies by stable cell line construction, whole production process with chemical composition-restricted culture media, ensure the inter-batch difference in cell line expression within a controllable range, and continuous production subsequently.
Provided by customers: target gene sequences or plasmid containing target genes
1. Antibody Yields Analysis
As shown in Fig.1, the average expression titer of cell pools from 39 projects is 2.2 g/L and titers of several cell pools are more than 3.0 g/L.
Fig. 1 Antibody yields analysis
2. Cell Pool Fed-Batch Culture
Fast cell pools are constructed for an antibody of interest. Seven cell pools are selected for fed batch culture in the shake flasks. Growth and metabolism data are collected from the cell pools periodically and the results are shown in Fig. 2. Viable cell density can reach up to 2.5E+7 cells/mL, while the cell viability can remain over 70% after 14 days, and the antibody yield is as high as 3.4 g/L. The lactic acid was always maintained at a relatively low level.
Fig. 2 Cell pool fed-batch culture
3. Antibody Quality Control Analysis Antibody Quality Control Analysis
3.1 SDS-PAGE (SDS polyacrylamide gel electrophoresis)
Purified antibody of interest is tested by SDS-PAGE, and the result is shown in Fig. 3a. The non-reduced and reduced molecular weights are consistent with theoretical molecular weights of the samples and the purities are over 95%, which are compliant with the delivery standards.
Fig. 3a SDS-PAGE
3.2 SEC-HPLC (Size exclusion chromatography)
Purified antibody of interest is tested by SEC to determine the purity, and the result is shown in Fig. 3b. The monomer purity the antibody of interest is 100%, without polymers, which is compliant with the delivery standards.
Fig. 3b SEC-HPLC
4. Antibody Activity Analysis
The activity analysis result of the purified antibody A by FACS is shown in Fig. 4. The EC50 is 0.012 μg/mL, indicating that the sample has good biological activity.
Fig. 4 Binding determination by FACS
5. Cation chromatography purification to increase SEC purity
In one customer's project, after one-step affinity purification of antibody proteins, the SEC purity was less than 90%, which did not meet the delivery standards. After cation or multimode chromatography purification optimization, the SEC purity increased to above 98.0%, and the yield was beyond 85%, which met the delivery standards.
Fig. 5a Purification chromatogram profiles
Fig. 5b SEC-HPLC chromatograms before (left) and after purification (right)
6. DoE design to increase the purity of fusion proteins
In one customer‘s project, the antigen fusion protein purification was carried out. No protein was obtained by one-step ProteinA chromatography, and the purity of non-reducing SDS-Page was low, which did not meet the delivery standards. After optimizations of buffer, loading conditions, elution buffer and elution method for this affinity chromatography, the purity of non-reducing and reducing SDS-Page was respectively increased to above 85% and above 95%, and the yield was beyond 80%, which met the delivery standards and had been recognized by the customer.
Fig. 6 Purification chromatogram (left) and SDS-Page chromatogram (right)
Facilities and Equipment
1000+ m^2 Class C + A clean environment
Cell line construction laboratory
AKTA protein purifier
Agilent 1100 high performance liquid chromatograph