High throughput detection of antibody self-interaction by bio-layer interferometry

Self-interaction of an antibody may lead to aggregation, low solubility or high viscosity. Rapid identification of highly developable leads remains challenging, even though progress has been made with the introduction of techniques such as self-interaction chromatography (SIC) and cross-interaction chromatography (CIC). Here, we report a high throughput method to detect antibody clone self-interaction (CSI) using bio-layer interferometry (BLI) technology. Antibodies with strong self-interaction responses in the CSI-BLI assay also show delayed retention times in SIC and CIC. This method allows hundreds of candidates to be screened in a matter of hours with minimal material consumption.     

A novel in vitro assay to predict neonatal Fc receptor-mediated human IgG half-life

Immunoglobulin G (IgG) has an unusually long serum half-life in comparison to proteins of a similar size. It is well-known that this phenomenon is due to IgG's ability to bind the neonatal Fc receptor (FcRn) in a pH-dependent manner. FcRn binding properties can vary among IgGs, resulting in altered in vivo half-lives, and therefore it would be beneficial to accurately predict the FcRn binding properties of therapeutic IgG monoclonal antibodies (mAbs). Here we describe the development of an in vitro model capable of predicting the in vivo half-life of human IgG. Using a high-throughput biolayer interferometry (BLI) platform, the human FcRn association rate at acidic pH and subsequent dissociation rate at physiological pH was determined for 5 human IgG1 mAbs. Comparing the combined FcRn association and dissociation rates to the Phase 1 clinical study half-lives of the mAbs resulted in a strong correlation. The correlation was also verified in vivo using mice transgenic for human FcRn. The model was used to characterize various factors that may influence FcRn-mAb binding, including mAb variable region sequence differences and constant region glycosylation patterns. Results indicated that the complementarity-determining regions of the heavy chain significantly influence the mAb's FcRn binding properties, while the absence of glycosylation does not alter mAb-FcRn binding. Development of this high-throughput FcRn binding model could potentially predict the half-life of therapeutic IgGs and aid in selection of lead candidates while also serving as a screening tool for the development of mAbs with desired pharmacokinetic properties.     

Quantifying structural changes and stoichiometry of protein interactions using size and density profiling

Summary The use of Dual Polarisation Interferometry, and emerging analytical biophysical technique, is described for the determination of the optogeometrical properties (thickness and density) at high resolution of adsorbed protein layers at the solid-liquid interface. The technique has been used to quantify, in real time and at subatomic resolution, the structural changes occurring in two well-characterised protein interaction systems, an antibody-antigen interaction and the biotin-streptavidin interaction. The realtime data obtained on structural changes during the interactions is in excellent agreement with previously reported X-ray crystallography and neutron reflection data. The precision of the measurements taken was of the order of 0.01 nm with respect to protein size. The dual-parameter approach also allowed the stoichiometry of both of these interactions to be calculated, giving values that confirm the current understanding of the interactions. This approach provides detailed insights into the inherent and subtle link between structural change and function in proteins, to a degree not previously possible through mass change measurements alone. The technique is expected to find utility in the increasingly important study of protein structure and function.     

Digital holographic microtomography for high‐resolution refractive index mapping of live cells

Quantification of three‐dimensional (3D) refractive index (RI) with sub‐cellular resolution is achieved by digital holographic microtomography (DHμT) using quantitative phase images measured at multiple illumination angles. The DHμT system achieves sensitive and fast phase measurements based on iterative phase extraction algorithm and asynchronous phase shifting interferometry without any phase monitoring or active control mechanism. A reconstruction algorithm, optical diffraction tomography with projection on convex sets and total variation minimization, is implemented to substantially reduce the number of angular scattered fields needed for reconstruction without sacrificing the accuracy and quality of the reconstructed 3D RI distribution. Tomogram of a living CA9‐22 cell is presented to demonstrate the performance of the method. Further, a statistical analysis of the average RI of the nucleoli, the nucleus excluding the nucleoli and the cytoplasm of twenty CA9‐22 cells is performed. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)