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March 22, 2016

Rapid Determination of High-Affinity KD’s in the Development of Antibody Therapeutics

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    Characterization of protein–protein binding is a critical element in the development of therapeutic antibodies. The desirable characteristics of effective therapeutic antibodies often include a high affinity to the target, which can lower dosage, improve healthcare, and reduce costs. Therefore, determining affinity plays a key role throughout development—from early screening of hybridomas or recombinant antibodies from phage display libraries to affinity maturation and antibody engineering to improve the efficacy, safety, and manufacturability of the final antibody drug product. Affinity characterization is also important when selecting reagents for quantitative analytical assays or assessing activity of the drug product in manufacturing release tests.

    In this article, we present how Gyrolab™ systems simplifies the accurate and rapid analysis of high-affinity antibodies and illustrate with the determination of equilibrium dissociation constants (KD) for three pharmaceutical-grade antagonists of tumor necrosis factor-α (TNF-α) (Figure 1).

    Gyrolab System for Rapid and Accurate Affinity Determination

    Methods commonly used for affinity measurement involve real-time, surface-based measurements, such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and quartz crystal microbalance (QCM) technology. These methods determine kinetic rate constants for the association and dissociation phases from which KD can be calculated, but often exhibit constraints when analyzing slow dissociation kinetics. This limits their ability to measure the high-affinity KD values that characterize modern biotherapeutics. Surface-based measurements also require that one of the interactants is attached to the surface, which may affect the determination of KD.

    The Gyrolab automated, nanoliter-scale immunoassay platform, in contrast, enables rapid and accurate determination of affinity interactions, down to low picomolar KD, by measuring free, unmodified interactants in equilibrated solutions. In addition, the flowthrough affinity column format in Gyrolab CDs is ideal for determination of free interactants in equilibrated solutions because interaction times of only a few seconds between sample and capture column avoid the equilibrium shifts that are inherent in assays requiring longer incubation times.

    Case Study: Determination of KD for Three Leading Therapeutic Antibodies

    Humira® (adalimumab), Remicade® (infliximab), and Enbrel®(etanercept) are all antagonists to TNF-α but differ in structure and mechanism of action: Humira and Remicade are antibodies that bind two TNF-α molecules, whereas Enbrel is a fusion protein consisting of the TNF-α receptor and antibody Fc unit binding one TNF-α molecule. The Gyrolab system was used to determine the affinity of these therapeutic drug products to soluble TNF-α, and the results were compared to those obtained using alternative analytical platforms (KinExA and Biacore).

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    Experimental details

    Binding of soluble recombinant TNF-α to the soluble TNF-α antagonists was performed at room temperature for 24, 48, and 72 hours. Affinity curves were generated with a fixed concentration (10 pM) of TNF-α antagonist and a dilution series of TNF-α ranging from 200 nM to 24 fM. The concentration of the antagonist was set to be in range of the expected KD to ensure an accurate estimate of the KD. The free antagonist in the equilibrated mixtures was measured in the Gyrolab Bioaffy 200 CD in the Gyrolab xP workstation and using a standard method, with biotinylated TNF-α as capture reagent and anti-human immunoglobulin G (IgG) labeled with Alexa Fluor 647 as detection reagent. Each sample was measured in duplicate. The Gyrolab Evaluator Affinity software module was used to fit curves, calculate KD, and compare the antagonists.

    Results of KD determination

    The time course study showed a shift toward complete equilibrium over time (Figure 2). The KD value determined after incubating for 72 hours was closest to the true value. Using this incubation time, the KD values of the three TNF-α antagonists could be determined as being in the range of 1.8–20 pM.

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    Comparison with Other Platforms

    The results from Gyrolab affinity measurements were compared to data published using KinExA and Biacore platforms.The three antibodies were ranked in the same order by all three analytical platforms, but the absolute values for KD differ. The data from all three antagonists correlate well between KinExA and Gyrolab, whereas the affinity improvements, particularly observed for Enbrel, are not easily differentiated by the Biacore platform, presumably due to the limitation of monitoring slow off-rate kinetics. Note that the values for Biacore and KinExA platforms are taken from published literature using different target molecules and experimental conditions, which complicates the comparison of the KD values. Gyrolab and KinExA platforms give comparable values, whereas the variation in affinity between the three antagonists is not easily differentiated by the Biacore platform (Figure 3).

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