Generation of mAb Variants with Less Attractive Self-Interaction but Preserved Target Binding by Well-Directed Mutation.

Strongly attractive self-interaction of therapeutic protein candidates can impose challenges for manufacturing, filling, stability, and administration due to elevated viscosity or aggregation propensity. Suitable formulations can mitigate these issues to a certain extent. Understanding the self-interaction mechanism on a molecular basis and rational protein engineering provides a more fundamental approach, and it can save costs and efforts as well as alleviate risks at later stages of development. In this study, we used computational methods for the identification of aggregation-prone regions in a mAb and generated mutants based on these findings. We applied hydrogen-deuterium exchange mass spectrometry to identify distinct self-interaction hot spots. Ultimately, we generated mAb variants based on a combination of both approaches and identified mutants with low attractive self-interaction propensity, minimal off-target binding, and even improved target binding. Our data show that the introduction of arginine in spatial proximity to hydrophobic patches is highly beneficial on all these levels. For our mAb, variants that contain more than one aspartate residue flanking to the hydrophobic HCDR3 show decreased attractive self-interaction at unaffected off-target and target binding. The combined engineering strategy described here underlines the high potential of understanding self-interaction in the early stages of development to predict and reduce the risk of failure in subsequent development.

Solution Equilibrium Titration for High-Throughput Affinity Estimation of Unpurified Antibodies and Antibody Fragments.

The generation of therapeutic antibodies with extremely high affinities down to the low picomolar range is today feasible with state-of-the art recombinant technologies. However, reliable and efficient identification of lead candidates with the desired affinity from a pool of thousands of antibody clones remains a challenge. Here, we describe a high-throughput procedure that allows reliable affinity screening of unpurified immunoglobulin G or antibody fragments. The method is based on the principle of solution equilibrium titration (SET) using highly sensitive electrochemiluminescence as a readout system. Because the binding partners are not labeled, the resulting KD represents a sound approximation of the real affinity. For screening, diluted bacterial lysates or cell culture supernatants are equilibrated with four different concentrations of a soluble target molecule, and unbound antibodies are subsequently quantified on 384-well Meso Scale Discovery (MSD) plates coated with the respective antigen. For determination of KD values from the resulting titration curves, fit models deduced from the law of mass action for 1:1 and 2:1 binding modes are applied to assess hundreds of interactions simultaneously. The accuracy of the method is demonstrated by comparing results from different screening campaigns from affinity optimization projects with results from detailed affinity characterization.

Simultaneous metal chelate affinity purification and endotoxin clearance of recombinant antibody fragments.

Endotoxins are frequent contaminants of recombinant proteins produced in Escherichia coli. Due to their adverse effects, endotoxins have to be removed from recombinant proteins prior their use in cell-based assays or parenteral application. Reduction of endotoxin to less than 10 EU mg(-1) is, however, one of the most problematic steps during protein purification from E. coli and often associated with substantial loss of biological materials. The present paper describes the use of a single step procedure enabling metal chelate affinity purification and endotoxin clearance from antibody fragments produced in E. coli using a non-ionic detergent. Endotoxin content was as low as 5 to 9 EU mg(-1) with a recovery of antibody fragments of over 90%. Non-ionic detergent treatment did not compromise integrity and functionality of these multimeric molecules. Furthermore, recombinant antibody fragments did not stimulate endotoxin-sensitive cell lines confirming the low endotoxin content. In conclusion, this one-step protocol is a rapid, cost effective and automation-compatible procedure suitable for recombinant antibody fragments.