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Therapeutics


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Figure 1: SPR kinetics on a 384-array (a) tile view with each tile representing a discrete mAb-coated spot and (b) close-up view of three mAbs with diverse kinetic profiles (App Note, Carterra)


References 1 Klein, Jan and Nikolaidis, Nikolas. The descent of the antibody-based immune system by gradual evolution. PNAS January 4, 2005. 102 (1) 169-174. 2 Arbabi-Ghahroudi, M. Camelid Single-Domain Antibodies: Historical Perspective and Future Outlook Front Immunol. 2017 Nov 20;8:1589. 3 Feige, Matthias J, Gräwert, Melissa A, Marcinowski, Moritz, Hennig, Janosch, Behnke, Julia, Ausländer, David, Herold, Eva M, Peschek, Jirka, Castro, Caitlin D, Flajnik, Martin, Hendershot, Linda M, Sattler, Michael, Groll, Michael and Buchner, Johannes. The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins. PNAS June 3, 2014. 111 (22) 8155-8160. 4 Hearty, S, Leonard, P, O’Kennedy, R (2012). Measuring Antibody – Antigen Binding Kinetics Using Surface Plasmon Resonance. In: Chames P. (eds) Antibody Engineering. Methods in Molecular Biology (Methods and Protocols), vol 907. Humana Press. Continued on page 57


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residues descended from a limited set of germlines alternate with hypervariable ‘complementary- determining regions’ (CDRs), which are responsi- ble for the enormous sequence and structural diver- sity of an organism’s antibody repertoire. While antibody generation in the pharmaceutical indus- try is highly commoditised, with modern in vivo and in vitro libraries typically producing vast num- bers of clones, the analytical tools used to charac- terise their binding properties in terms of kinetics, affinity and specificity, which are key parameters for assessing their quality and functional activity, lag orders of magnitude behind in throughput. With therapeutic antibodies being the largest class of biotherapeutic proteins that are in clinical trials, there is an ever-increasing demand for higher throughput analytical methods that can match the capacity of antibody production and guide the library-to-leads triage. Since taking an antibody from bench to the market is estimated to cost about $1 billion, there is a need to make antibody screening more efficient and comprehensive to cut costs and timelines.


Using SPR to characterise the binding kinetics and affinities of antibody interactions Label-free biosensors such as those based on opti- cal detection principles such as surface plasmon resonance (SPR) or biolayer interferometry (BLI) are routinely employed in the pharmaceutical industry to characterise antigen/antibody binding


interactions in terms of their kinetic rate constants and affinities4. Knowledge of an interaction’s affin- ity is significant because it dictates the dose or con- centration at which an antibody will be effica- cious5. Oftentimes, selecting an appropriate affini- ty for a given application is an empiric process and can be optimised by engineering. Dissecting a bind- ing affinity into its constituent kinetic rate con- stants provides useful information that can guide the evaluation of an antibody’s performance throughout screening, optimisation and manufac- ture. Label-free methods obviate the need to label or conjugate either of the interacting species and the binding event is monitored in real-time. This makes it ideally suited for screening crude antibody samples that are produced in early-stage research, where numerous clones are available, but in limit- ed quantities. Commonly-used commercially-available biosen-


sor platforms differ in their throughput, sample consumption and ease of use. They are versatile tools and are applied to the study of a broad range of biomolecular interactions, from small molecules to antibodies. Unlike small molecule analysis, where binding interactions are generally transient with weak affinities, as characterised by equilibri-


um dissociation constants (or KD values) in the micromolar range, antigen/antibody binding inter- actions often exhibit affinities that are a million-


fold tighter, with KD values in the picomolar range. Accurately measuring the kinetic rate constants of high affinity interactions requires long binding


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