Is Your Antibody The Right Affinity?

The importance of affinity determination during drug development and assay development

In order to reduce drug dosage, improve healthcare, and lower costs, therapeutic antibodies that have high target affinity are sought. Optimizing and monitoring the specificity of drug candidates to target molecules has become a critical part of antibody characterization This includes an early screening of hybridomas and recombinant antibodies from phage display library libraries to affinity maturation and engineering antibodies to increase the safety, efficacy and manufacturability. It is important to determine affinity when choosing reagents for quantitative tests or when assessing the activity of drug products in manufacturing release tests. How can high affinity be measured in the best way?

High affinity – This is the challenge

Now, therapeutic antibodies have affinities as low as the femtomolar. This places a special demand on analytical methods for determining equilibrium dissociation constants in the low picomolar and sub-picomolar ranges. The most common methods for affinity measurement are surface-based, real-time measurements like surface plasmon resonance(SPR), biolayer interferometry (BLI) and quartz crystal microbalances (QCM). These methods can calculate KD from the kinetic rate constants of the dissociation and association phases, but it can be difficult to analyze slow dissociation rates. Their ability to measure high-affinity KD’s, which are the hallmark of modern biotherapeutics, is limited. Surface-based measurements require that one interactant be attached to the surface. This may impact the determination of KD.

In a free solution, how to determine low picomolar KD?

Gyros Protein Technologies created Gyrolab Affinity Software for Gyrolab System to meet this need. This combination allows the determination of affinity to low picomolar KD by measuring unmodified interactants free in equilibrated solutions. In Gyrolab CDs, the flow-through affinity column format reduces interaction times for unmodified interactants in equilibrated solution to just a few seconds. This avoids equilibrium shifts that may occur in assays that require longer incubation times.

Case Study: Affinity in Search for Therapeutic Antibodies with High Target Coverage

Preclinical and clinical pharmacokinetic (PK), Pharmacokinetic analysis of antibodies is only possible if assays accurately and precisely measure therapeutic monoclonal antibody (mAb). Dosing studies that aim to achieve high target coverage must measure soluble target levels. However, this can be difficult due to the complex dynamics between binding equilibriums between bivalent antibodies, target complexes, and antibody-target complexes. Boehringer Ingelheim Pharmaceuticals’ team used Gyrolab System and Gyrolab Affinity software to determine the binding properties of their investigational anti-mAbs. This combination workflow proved to be extremely useful in routine assay development.

Pharmacodynamic Mechanisms of Action

There are many antibacterial mAbs that can be used against bacteria, including proteins, polysaccharides, and soluble exotoxins (Tables 1 and 2). The nature and role of the target in bacterial pathogenesis and the mAb structure (i.e. intact IgG mAb, IgG fragments or immunoconjugates) will determine the potential pharmacodynamic mechanism. Anti-exotoxin monoclonal antibodies (mAbs) are known to reduce bacterial pathological activity by neutralizing exotoxins. Monoclonal antibodies that target bacterial surface epitopes can increase bacterial clearance by increasing antibody-dependent and/or complement-mediated bacteriacidal activity or via immune system-independent killing. There has been an increase in interest in immunoconjugates, and immunomodulatory mAbs. These mAbs either have potent antimicrobials to stimulate exhausted immune function functions or both, ugment bactericidal activity. A significant increase in the levels of clinical effectiveness in late-phase clinical trials, Biotechnology industry is witnessing the necessity for greater quantities of mRNA plasmid DNA to be manufactured at greater scales.

Source: https://www.nativesdaily.com/is-your-antibody-the-right-affinity/