Effects of pH, conductivity, host cell protein, and DNA size distribution on DNA clearance in anion exchange chromatography media.

Flowthrough anion exchange chromatography is commonly used as a polishing step in downstream processing of monoclonal antibodies and other therapeutic proteins to remove process-related impurities and contaminants such as host cell DNA, host cell proteins, endotoxin, and viruses. DNA with a wide range of molecular weight distributions derived from Chinese Hamster Ovary cells was used to advance the understanding of DNA binding behavior in selected anion exchange media using the resin (Toyopearl SuperQ-650M) and membranes (Mustang® Q and Sartobind® Q) through DNA spiking studies. The impacts of the process parameters pH (6-8), conductivity (2-15 mS/cm), and the potential binding competition between host cell proteins and host cell DNA were studied.

Protein adsorption and transport in agarose and dextran-grafted agarose media for ion exchange chromatography: Effect of ionic strength and protein characteristics.

This work investigates the effects of ionic strength and protein characteristics on adsorption and transport of lysozyme, BSA, and IgG in agarose-based cation exchangers with short ligand chemistry and with charged dextran grafts. In all cases, the adsorption equilibrium capacity decreased with increasing salt. However, the adsorption kinetics was strongly influenced by the adsorbent structure and protein characteristics.

Theory and applications of refractive index-based optical microscopy to measure protein mass transfer in spherical adsorbent particles.

This work provides the theoretical foundation and a range of practical application examples of a recently developed method to measure protein mass transfer in adsorbent particles using refractive index-based optical microscopy. A ray-theoretic approach is first used to predict the behavior of light traveling through a particle during transient protein adsorption. When the protein concentration gradient in the particle is sharp, resulting in a steep refractive index gradient, the rays bend and intersect, thereby concentrating light in a sharp ring that marks the position of the adsorption front.

Patterns of protein adsorption in chromatographic particles visualized by optical microscopy.

A new method is presented to image transient patterns of protein adsorption in individual spherical chromatographic particles under strong binding conditions. The method takes advantage of the difference in refractive index between the protein-free and protein-saturated adsorbent matrix. When the particles are viewed with an ordinary microscope using white light illumination, the adsorption front appears as a bright ring that moves in time from the surface of the particle to its center.

Protein adsorption and transport in agarose and dextran-grafted agarose media for ion exchange chromatography.

This work examines the relationship between the physical properties of agarose and dextran-grafted agarose cation exchangers and protein adsorption equilibrium and rates. Four different sulfopropyl (SP) matrices were synthesized using a neutral agarose base material--two based on a short ligand chemistry and two obtained by grafting 10 and 40kDa dextran polymers. The pore accessibility, determined by inverse size exclusion chromatography (iSEC) with dextran probes, decreases dramatically as a result of the combined effects of crosslinking, dextran grafting, and the introduction of ionic ligands, with pore radii decreasing from 19nm for the base matrix to 6.