Chromatographic and Adsorptive Behavior of Bivalent Bispecific Antibodies

Kimerer, Lucas, Chemical Engineering - School of Engineering and Applied Science, University of Virginia
Carta, Giorgio, EN-Chem Engr Dept, University of Virginia

Bivalent bispecific antibodies are structurally comprised of an entire framework immunoglobulin G (IgG) genetically fused to two single chain variable fragments (scFv) via flexible peptide linkers. The flexibility of the linkers allows three extreme configurations where each scFv can be collapsed and interact with the framework IgG, or extended and able to interact with a surface or binding epitope with greater affinity. Slow interconversion between these configurations is found to cause multiple peak elution and complex adsorption kinetics on chromatography media. Surface catalyzed conformational changes greatly decrease separation efficiency and complicate the design of separations. The overall goal of this work is to fundamentally understand the conformational changes of BiSAbs and their major effects on column behavior and adsorption kinetics for cation exchange (CEX) and hydrophobic interaction (HIC) chromatography media.
This work first characterizes the reversible, multiple peak phenomena observed for linear gradient elution. Pure BiSAb samples elute three peaks at room temperature and low residence time (~2 min). All three peaks are monomer-sized, measured by dynamic light scattering. When the residence time is elongated to ~40 min or temperature is elevated to ~55 °C, a single peak elutes with average retention. When the protein is held bound on the resin, the relative size of the strongest binding peak increases. BiSAb fragments with a single linked scFv follow the same trends as the intact molecules, but with reversible two peak elution. BiSAb fragments or the framework IgG, containing zero linked scFvs, elute a single peak regardless of operating conditions. The BiSAb multiple peak elution behavior is qualitatively consistent regardless of: scFv attachment location to the framework IgG, resin structure (pellicular or porous), pH of the mobile phase, and resin functional ligand (weak CEX, strong CEX, or HIC). These effects are therefore driven by the molecular properties of the BiSAbs, rather than resin specific factors. Adsorption kinetics of these complex proteins was studied by viewing transient intraparticle bound profiles using confocal microscopy. The BiSAb intraparticle profiles showed smooth characteristics caused by the slow exchange from the weaker binding, collapsed configuration to the stronger binding, extended configurations. Phenomenological models were developed at the column scale, to predict the multiple peak elution behavior, and at the particle scale, to predict the complex adsorption kinetics.
Finally, the separation between the intact BiSAb molecule and two associated fragments was studied on CEX resins. Both fragments were found to have one scFv missing and elute prior to the intact protein. The fragment with intermediate binding strength contained one additional amino acid, a C-terminal lysine. Linear gradient elution measured fairly small differences in binding strength between the protein species. However, at high load, high selectivity was observed and the intact molecule was found to displace the fragments with relatively fast kinetics.

PHD (Doctor of Philosophy)
Bivalent bispecific antibodies, Conformational changes, Multiple peak elution, Protein chromatography, Mechanistic modeling
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