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Dec 1, 2010 (Vol. 30, No. 21)

Adoption of Label-Free Technologies Surges

Methodologies Like SPR and ITC Are Saving Researchers Time and Money

  • High-Specificity Antibodies

    Click Image To Enlarge +
    Inhibitory antibody targeting the serine protease active site: Superposition of the Fab58/HGFA structure (PDB code 2R0K) with the structure of HGFA/peptidic inhibitor (ball and stick) complex (PDB code 2WUC) to illustrate the HGFA protease (surface model–beige) subsite occupancy by Fab58 CDR-loop residues (green). The antibody binding region on HGFA is highlighted in purple. [Genentech]

    Another application where label-free technologies helped to determine structural binding elements of a Fab fragment was presented at DiPIA by Rajkumar Ganesan, Ph.D., a postdoctoral fellow in Daniel Kirchhofer, Ph.D.’s lab at Genentech.

    Dr. Kirchhofer and his colleagues are working on understanding the structural and mechanistic aspects of serine protease inhibition by antibodies targeting the active or allosteric site. “When transmembrane serine proteases such as hepsin are overexpressed in preclinical prostate cancer models they promote tumors; blocking of the active sites of hepsin with an antibody could help inhibit tumor growth,” Dr. Ganesan explained.

    “However, we could not find inhibitory antibodies using classical hybridoma techniques or phage-display libraries. But using an active site blocked version of hepsin and a phage-display library of restricted chemical diversity at the complementarity determining regions, we found a number of Fabs that did bind to hepsin.”

    Dr. Ganesan presented SPR data showing that one of these Fab fragments, Fab25, has high affinity binding for hepsin, and its inhibitory activity was confirmed by enzyme assays using a synthetic substrate. SPR was also used in competition binding experiments with two active site derived inhibitors and demonstrated that Fab25’s binding site has structural elements at or around the hepsin active site region. Using ITC, Dr. Ganesan showed that Fab25’s binding is enthalpically driven, indicating potential hydrogen bond formation and electrostatic interactions between Fab25 and hepsin.

    “Our Fab25 fragment is a potent and specific inhibitor of hepsin and may be a good tool for inhibiting hepsin in prostate cancer.”

    In vaccine design, the use of SPR is spreading because it can be used for specificity testing, epitope mapping, and immune response ranking. “Many of the major pharmas are using SPR to check their viral vaccines because it is now possible to qualify the virus from culture supernatant,” Sundberg explained. “For example, there are vaccine companies using SPR to verify the existence of antigenic sites on virus like particles with specific antibodies.

    “Trends in vaccine development are toward more complex vaccines and lower dose vaccines. These require more flexible, sensitive methods of measuring vaccine concentration, and SPR can achieve this.”

    There is a strong push in viral vaccine production to move from egg- to cell culture-based production, and there is growing interest in using SPR for process development. Additionally, because SPR is being successfully used at the research stage for checking vaccine potency, it could also be used as a manufacturing method for batch-release verification.

    Sundberg presented results of a study where scientists at GE Healthcare compared the current gold standard method for batch release of flu vaccine, the single radial immunodiffusion (SRD) assay, with an SPR method. In the inhibition-based SPR assay, three different virus hemagglutinins (HAs) were immobilized on a Sensor CM5 in a Biacore™ system. Virus from the sample was then mixed with a fixed concentration of anti-serum from the National Institute for Biological Standards and Control and injected over the surface.

    The results showed that using the SRD assay, 5 µg/mL could be measured, whereas the more sensitive SPR assay measured 0.5 µg/m for all three subtypes. “One major advantage of using the SPR assay is that it is less labor intensive,” Sundberg explained.

    “The potency of the three antigens in the flu vaccine can be measured simultaneously, but using the SRD assay the potency of each antigen has to be calculated separately by measuring inhibition zones on three different gels. Since SPR is more sensitive it would be valuable in influenza purification process development, where fractions from optimization experiments tend to have low concentrations of virus. There may also be an opportunity to fill each vaccine vial more accurately and potentially make significant cost savings.”

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