Case studies for discovery services
- CYP-X Protein Expression in E.coli for structural analysis
- IgM purification
- Kinase-Y production using insect cell/baculovirus system
- Using multiple platforms for identifying isoform specific antibody
- Using WuXi’s proprietary naive human phage display for antibody discovery
- Affinity maturation to achieve Kd value in picomolar range
Case study: CYP-X Protein Expression in E.coli for structural analysis
Client requested production of CYP-X protein starting from gene sequence and generate crystals from purified protein for structure analysis.
- The expression level was very low (<0.5mg/L).
- The protein had low solubility.
- The protein’s current state had a negative impact on crystallization.
- WuXi tested different combinations of expression vectors and e.coli strains for optimal expression and also added trace elements to the culture media to improve yield.
- The team added a high salt lysis buffer in the purification process and added a weak cation column for process purification optimization.
- These efforts resulted in an improved expression level of 15 mg/L and good diffracting crystals were obtained for structural analysis.
Case study: IgM purification
Client requested delivery of IgM with 95% purity (as determined by SDS-PAGE and HPLC analysis).
- The protein’s large molecular weight created a low binding capacity and low flow rate through origional purification resin.
- The protein’s low solubility and vulnerability to denaturation at extreme pH prevented the usage of traditional affinity columns for purification.
- WuXi team conducted extensive research of relevant literature and applied new technology for convection mass transport.
- Drawn from our experience with large MW proteins, the team established a new three column purification scheme.
- We were able to deliver 200 mg of fully characterized IgM with 97% purity in just 6 weeks.
Case study: Kinase-Y production using insect cell/baculovirus system
Client requested the production of Kinase-Y with high purity.
- The protein is expressed as inclusion bodies in E. coli.
- Low titer and low expression level in insect cells by the client themselves.
- Low purity and degradation due to long purification time with various columns.
- WuXi team generated baculovirus with high titer (108 pfu/ml).
- We optimized the expression with various MOI and time course.
- Purification process was optimized with affinity column and size exclusion column.
- The team finally delivered 25mg Kinase-Y (from 1.6L culture) with the purity >95%.
Case study: Using multiple platforms for identifying isoform specific antibody
- The target protein has two slightly different isoforms.
- We have easily found a handful of functional candidates specific to isoform 1 and enough functional candidates cross-reactive to both isoform 1 and 2 by hybridoma system.
No functional hits specific to isoform 2 were identified by hybridoma, even after multiple campaigns.
By leveraing our in-house human naïve library we were able to identify functional hits by just one campaign. The specific number of campaigns and leads found were shown in the table.
Case study: Using WuXi’s proprietary naive human phage display for antibody discovery
Use WuXi’s proprietary human naive antibody library to identify antibodies specific to each of the two splicing variants of antigen 1 named Ag 1a and Ag 1b.
- Two splicing variants (Ag 1a & Ag 1b) differ by >20 different amino acids.
- Hybridoma approach failed to yield antibodies selective for Ag 1b
Human naïve library was panned on the splicing variant b of the human antigen 1 (Ag 1b) in the presence of splicing variant a (Ag 1a) as a competitor. Following panning, rounds 2 and 3 outputs were screened by monoclonal phage ELISA. Round 3 output shows significant enrichment with Ag 1b specific clones with concurrent reduction of Ag 1a/Ag 1b cross-reactive clones. Sequence analysis of round 3 hits identified 6 unique clones, 2 of which also showed strong binding to cell-surface expressed Ag 1b (data not shown here).
Case study: Affinity maturation to achieve Kd value in picomolar range
Client requested affinity maturation for their wild-type murine antibody and create a more “human” antibody in the process.
Move antibody affinity from moderate binding levels (Kd = 1 nM) to equal or beat benchmark molecule with a double-digit picomolar Kd value.
- WuXi utilized two methodologies to increase affinity binding characteristics of the antibody:
1.Parsimonious mutagenesis of the VHCDR3 region
2.Random mutagenesis of both the VH and VL chains
- Parsimonious mutagenesis resulted in 10-fold increase in affinity
- Subsequent random mutagenesis resulted in generation of 5 clones with enhanced affinity characteristics
- Final selected clone had superior Kd value in picamolar range that was better than the benchmark molecule!