(176ab) LambdaFabSelect High Throughput Method for Clone Selection in Duetmab Molecules

Dhanesh Gadre, Ellen T O’Connor, Kelcy Newell, Kevin Stewart, Judith Klover

AstraZeneca, One MedImmune Way, Gaithersburg, MD 20878, USA

Duetmabs represent a novel monovalent bispecific antibody format developed at AstraZeneca. They contain two unique light chains of differing subclass (one kappa and one lambda), and two unique heavy chains. Production of Duetmabs is realized by co-expressing the four unique polypeptide chains in a single host cell. While fidelity of polypeptide chain pairing for Duetmabs is improved using protein engineering techniques such as knob-into-hole in the Fc domain, mispaired impurities for individual clones can vary widely, ranging from 10-70%. Higher percentages of mispaired impurities can lead to low overall purification process yields. If clones can be selected with better polypeptide chain pairing fidelity, many of the resulting downstream difficulties can be mitigated. To address the challenge of controlling mispaired impurities, we developed an efficient, high throughput method that can effectively select for clones containing low amounts of mispaired impurities. This method takes advantage of LambdaFabSelect resin’s high selectivity for lambda light chain, thus removing the mispaired byproducts containing only kappa light chains. We used the TECAN EVO200 system to prepare 96 well filter plates coated with LambdaFabSelect resin. The clone samples are applied on the equilibrated plate and are centrifuged after incubation on shaker to obtain the flow through fractions in a collection plate. The ratio of the Protein A titer of the flow through fraction to that of the load determines the percentage of mispaired impurities in the sample. This clone selection technology can potentially result in a large increase in yield of a clinical GMP campaign. In principal this method can also be used using KappaSelect resin in cases where the mispaired impurities contain predominantly lambda light chains.