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Case Studies

Case studies for discovery services

  1. CYP-X Protein Expression in E.coli for structural analysis
  2. IgM purification
  3. Kinase-Y production using insect cell/baculovirus system
  4. Using multiple platforms for identifying isoform specific antibody
  5. Using WuXi Biologics’ proprietary naive human phage display for antibody discovery
  6. Affinity maturation to achieve Kd value in picomolar range
  7. GPCR Production

Case study: CYP-X Protein Expression in E.coli for structural analysis


Background

Client requested production of CYP-X protein starting from gene sequence and generate crystals from purified protein for structure analysis.

Challenge

  • 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.


Solution/Results

  • 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.

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Case study: IgM purification


Background

Client requested delivery of IgM with 95% purity (as determined by SDS-PAGE and HPLC analysis).

Challenge

  • 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.


Solution/Results

  • 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.

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IgM purif

Case study: Kinase-Y production using insect cell/baculovirus system

Background

Client requested the production of Kinase-Y with high purity.

Challenge

  • 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.


Solution/Results

  • 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%.

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KinaseY2

Case study: Using multiple platforms for identifying isoform specific antibody


Background

  • 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.


Challenge

No functional hits specific to isoform 2 were identified by hybridoma, even after multiple campaigns.


Solution/Results

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.

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multiplat case

Case study: Using WuXi Biologics’ proprietary naive human phage display for antibody discovery

Background

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.

Challenge

  • Two splicing variants (Ag 1a & Ag 1b) differ by >20 different amino acids.
  • Hybridoma approach failed to yield antibodies selective for Ag 1b

Solution/Results

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).

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modified human phage library case study

Case study: Affinity maturation to achieve Kd value in picomolar range

Background

Client requested affinity maturation for their wild-type murine antibody and create a more “human” antibody in the process.

Challenge

Move antibody affinity from moderate binding levels (Kd = 1 nM) to equal or beat benchmark molecule with a double-digit picomolar Kd value.

Solution/Results

  • 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!

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affinity maturation1

Case Study: GPCR Production

Aim

Production of a high quality GPCR protein in insect cells
Reconstitution of GPCR into nanodiscs

Key Challenges

Expression level of GPCR was very low <0.2 mg/L
The protein was unstable and its conformation was very flexible
It was hard to reconstitute GPCR into nanodiscs

Results

  • Expression constructs were optimized
  • ~1.5 mg/L was achieved after optimizing expression and purification protocol
  • Purity >90%
  • GPCR’s conformation was stabilized by suitable ligand
  • GPCR-nanodiscs was successfully assembled

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