The value of affinity determination in assay development and throughout drug development
The drive to lower drug dosage, improve healthcare and reduce costs has lead to the search for therapeutic antibodies with high target affinity. As a consequence, optimizing and monitoring the specificity and affinity of drug candidates to the target molecule has become critical in the development of therapeutic antibodies – 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. Characterizing affinity is also important when selecting reagents for quantitative assays or assessing drug product activity in manufacturing release tests. The question is, how can high affinity be measured in the best possible way?
The challenge of accurately measuring high affinity
Therapeutic antibodies are now being developed with affinities down to the femtomolar level, which places particular demands on analytical methods to determine equilibrium dissociation constants (KD) in the low picomolar or sub-picomolar range. 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 can have difficulty in analyzing slow dissociation kinetics. This limits their ability to measure the high affinity KD’s that characterize modern biotherapeutics. Surface-based measurements also require that one of the interactants be attached to the surface, which may affect the determination of KD.
How to determine low picomolar KD in free solution
To meet this need for rapid and accurate determination of high affinity interactions, Gyros Protein Technologies developed Gyrolab Affinity Software for Gyrolab system. This combination enables the determination of affinity down to low picomolar KD, by measuring free unmodified interactants in equilibrated solutions. The flow-through affinity column format in Gyrolab CDs also lowers interaction times for free interactants in equilibrated solutions to a few seconds, avoiding the equilibrium shifts of assays requiring longer incubation times.
Case study: Affinity in the search for therapeutic antibodies with high target coverage
Assays that accurately and precisely measure therapeutic monoclonal antibodies (mAb) are key to successful preclinical and clinical pharmacokinetic (PK) characterization. Dosing studies aimed at delivering high target coverage require measurement of soluble target levels, but this can be challenging due to the complex dynamics of binding equilibriums between bivalent antibodies, corresponding target and antibody-target complexes. Having observed low target coverage of humanized mAbs targeting connective tissue growth factor (CTGF), a team at Boehringer Ingelheim Pharmaceuticals used Gyrolab system combined with Gyrolab Affinity Software to characterize the binding properties of their investigational mAbs and associated reagents. They concluded that this combination workflow would be very valuable in routine assay development.
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