Role of configurational flexibility on the adsorption kinetics of bivalent bispecific antibodies on porous cation exchange resins.

The adsorption kinetics of a monoclonal antibody (mAb) used as a reference and of bivalent bispecific antibodies (BiSAb) on a macroporous cation exchanger is studied experimentally by examining the transient patterns of bound protein within the particles using confocal microscopy for a range of protein concentrations, buffer concentrations and pH, and temperatures. The mAb adsorption kinetics is controlled by pore diffusion and conforms to the classical shrinking core model. While the mAb adsorption rate increases with temperature, the ratio of effective and free solution diffusivity, D /D, remains constant and has a value of 0.

Chromatographic and adsorptive behavior of a bivalent bispecific antibody and associated fragments.

The elution and adsorptive behavior of a bivalent bispecific antibody (BiSAb), comprising an IgG1 framework with a scFv domain genetically fused to each heavy chain C-terminus via flexible linkers, and of two associated fragments were studied on two cation exchange chromatography media - ProPac WCX-10, which is pellicular and suitable for analytical use, and Nuvia HR-S, which is macroporous and suitable for preparative and process scale uses. Both fragments were identified by MS as missing one of the two scFv domains and its flexible linker, but one of them also contains an additional C-terminal lysine. The separation of these fragments on both resins occurs as a result of differences in non-specific ligand-protein interactions that are modulated by the salt concentration.

Effects of molecule size and resin structure on protein adsorption on multimodal anion exchange chromatography media.

The effect of bead and ligand structure on protein adsorption was investigated for multimodal anion exchangers combining a quaternary ammonium ion group with hydrophobic moieties: Nuvia aPrime 1 and aPrime 2, based on a 54 μm diameter polymeric bead, and Capto Adhere ImpRes and Capto Adhere, based on agarose beads 51 and 78 μm diameter, respectively. Bovine serum albumin (BSA) monomer, BSA dimer, and thyroglobulin (Tg) were used as model proteins. Based on TEM imaging and iSEC, the Nuvia resins have a microgranular structure and large pores (110 nm radius), while the Capto resins have a fibrous structure and smaller pores (32-36 nm radius).

Structure and functional properties of Capto™ Core 700 core-shell particles.

Capto™ Core 700 is a core-shell chromatographic support with an adsorbing core contained within an inert shell layer designed to purify larger biomolecules and bioparticles in a flow-through mode. The present study aims to characterize the structure and functional properties of this resin using bovine serum albumin (BSA, Mr~65 kDa) and thyroglobulin (Tg, Mr~660 kDa) as model impurity proteins. The functionalized adsorbing core and the inert shell have the same fibrous structure typical of agarose-based beads.

Chromatographic behavior of bivalent bispecific antibodies on hydrophobic interaction chromatography columns.

The elution behavior of bivalent bispecific antibodies (BiSAb) comprising an immunoglobulin G framework genetically fused to a pair of single chain variable fragments (scFvs) was studied on hydrophobic interaction chromatography (HIC) columns using ammonium sulfate gradients. Each of the BiSAb molecules studied exhibited a three-peak elution behavior regardless of the location of scFv attachment to the framework IgG. Collecting and re-injecting each of the isolated peaks and eluting with the same gradient resulted in the same three-peak profile indicating that the behavior is reversible.