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June 15, 2010 (Vol. 30, No. 12)

Rapid Identification of Production Strains

Accelerating Protein Concentration and Functional Analyses to Improve Efficiency

  • How It Works

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    Figure 3. Binding data for granulocyte colony stimulating factor (G-CSF) binding to its receptor on the surface of streptavidin biosensors on an Octet system

    As one example of how the screening process works, Pfenex came up with a high-producing strain for expression of soluble granulocyte colony stimulating factor (G-CSF) for low-cost production of filgrastim as a biosimilar in the therapeutic protein market. The non-glycosylated form of G-CSF is currently produced in E. coli as an inclusion body that requires solubilization and refolding.

    The first stage in developing a production process for G-CSF was to screen Pfenex Expression Technology strains for expression of soluble, correctly folded G-CSF. The Octet system with G-CSF receptor immobilized to Dip and Read streptavidin biosensors was used for titer measurements (Figure 3). The assay demonstrated sufficient range and sensitivity for detecting soluble, functional G-CSF directly in crude cell extracts with minimal sample preparation.

    Expression strains were screened in parallel using the Octet assay following the process outlined in Figure 2. Plasmids were constructed carrying the G-CSF gene fused to 12 P. fluorescens secretion leaders, targeting the protein to the periplasm, and these plasmids were transformed into 20 different host strains with varying phenotypes to create 240 unique expression strains. The soluble fractions of replicate culture lysates were analyzed for G-CSF receptor binding using the BLI method. The resulting screen identified several dozen strains that exhibited soluble expression of G-CSF. Notably, the type of secretion leader had a significant impact on the level of G-CSF expression in each host strain. Six secretion leaders showed low relative G-CSF expression, two showed medium expression, and three secretion leaders showed the highest relative G-CSF expression. One secretion leader did not exhibit any soluble G-CSF expression.  From this initial screen, the highest producing strains were characterized further and selected for fermentation development.

    The entire process to identify a strain took less than five weeks. The Octet Dip and Read assay provided both a means to assess the amount of soluble G-CSF produced by strains as well as an indication of the functional quality of the material by its ability to bind to the G-CSF receptor. The same G-CSF Dip and Read assay was used at Pfenex during fermentation optimization and downstream process development stages of development.

    Several Octet assays have been developed by Pfenex and implemented in expression screens for different classes of proteins including mAbs, antibody derivatives (Fab, Fab´, single-chain antibodies, etc.), growth factors, cytokines, and vaccine antigens. The streptavidin biosensor can be used to load the appropriate capture reagent to allow the analysis of all of the above targets. The Octet platform provides a method for measuring protein titer and verifying functionality in a simple and direct binding assay format that is broadly applicable across a variety of protein types.

    Development of therapeutic proteins requires rigorous measurements of kinetic and thermodynamic binding properties of the protein to its receptor for candidate optimization and clinical dosing strategies. Octet systems monitor binding interactions in real time and measure kinetic constants kon and koff and affinity constant KD.

    The ability to measure concentration as well as kinetic parameters in a single assay makes the Octet platform a useful tool for various stages of the bioprocessing workflow, as shown in this G-CSF example with Pfenex Expression Technology.

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