Therapeutics
cycles to enable sufficient information to be gath- ered for both the association and dissociation phases6. Therefore, biosensor platforms that increase the number of interactions monitored in parallel can significantly accelerate the analysis of large panels of antibodies. A recent study benchmarked the Mass-1 system
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from Sierra Sensors against the Biacore 4000 sys- tem from GE Healthcare and found comparable kinetic rate constants for a large panel of antigen/antibody interactions when chip types were matched7. However, with the throughput of these two state-of-the-art instruments limited to monitoring only eight interactions at once via par- allel flow channels, these analyses not only con- sume significant quantities of samples but necessi- tate long run times when addressing hundreds of interactions, making the analysis of more than 100 clones rather tedious. As such, SPR is often used as a secondary tool, after large antibody panels have been whittled down to a subset of clones via pre- liminary screening by higher throughput, but lower information content assays, such as ELISA, which is an end-point analysis lacking real-time kinetic information. ELISA also is prone to a high false hit rate, especially when targets are adsorbed directly to plastic which may expose irrelevant, denatured epitopes or mask native ones. In addition, the use
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of several wash steps may also compromise the ability to identify weak affinity binders, which may have desirable epitopes. Another option is the Octet HTX platform from
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Pall/ForteBio, which is a 96-channel BLI biosensor offering versatility in throughput from 8, 16, 32, 48 to 96-channel modes. Since each interaction necessitates the use of single disposable sensors, running the system in 96-channel mode is costly on sensors and sample consumption scales with the size of the assay. Also, the lack of microfluidics affects the accuracy with which binding kinetics can be measured, because they are often contami- nated by rebinding artifacts, which confound a reliable and detailed analysis8. Indeed, a study by Estep et al in 2013 stated that, “When measuring affinities based on currently available methods, one must compromise either on throughput or accuracy”, which unfortunately has posed a bottle- neck in analytics for decades9. In that paper, they compared the affinities of antigen/antibody inter- actions measured by orthogonal techniques, name- ly the BLI-based 16-channel Octet RED384 plat- form and low and high throughput solution affini- ty methods, respectively the Kinetic Exclusion Assay (KinExA) and Meso Scale Discovery (MSD). The authors reported an excellent correlation between KinExA and MSD affinity determinations
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Continued from page 56
5 Rudnick, Stephen I and Adams, Gregory P. Affinity and Avidity in Antibody-Based Tumor Targeting. Cancer Biotherapy And Radiopharmaceuticals Volume 24, Number 2, 2009. 6 Katsamba, PS, Navratilova, I, Calderon-Cacia, M, Fan, L, Thornton, K, Zhu, M, Bos, TV, Forte, C, Friend, D, Laird- Offringa, I, Tavares, G, Whatley, J, Shi, E, Widom, A, Lindquist, KC, Klakamp, S, Drake, A, Bohmann, D, Roell, M, Rose, L, Dorocke, J, Roth, B, Luginbühl, B, Myszka, DG. Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users. Anal Biochem. 2006 May 15;352(2):208-21. Epub 2006 Feb 23. 7 Kamat, Vishal, Rafique, Ashique. Exploring sensitivity & throughput of a parallel flow SPRi biosensor for characterization of antibody- antigen interaction. Analytical Biochemistry 525 (2017) 8e22. 8 Abdiche, Y, Malashock, D, Pinkerton, A, Pons, J. Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet. Anal Biochem. 2008 Jun 15;377(2):209-17. 9 Patricia Estep, Felicia Reid, Claire Nauman, Yuqi Liu, Tingwan Sun, Joanne Sun and Yingda Xu. High throughput solution-based measurement of antibody-antigen affinity and epitope binning. mAbs 5:2, 270–278; March/April 2013. 10 Salimi-Moosavi, H et al. Rapid affinity measurement by Gyrolab. Anal. Biochem. 426 (2012) 134-141.
Figure 2: Epitope binning a panel of 29 mAbs via BLI into 4 distinct clusters17 Drug Discovery World Summer 2018
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