Application of a micromembrane chromatography module to the examination of protein adsorption equilibrium.

A micromembrane chromatography module based on a 96-well plate design and enabling fast and simple separation of small amounts of proteins was used for the determination of binding capacities of lysozyme, bovine serum albumin, ovalbumin, bovine γ-globulin, and human immunoglobulin G on a hydrophobic membrane Sartobind® Phenyl. Dependence of the binding capacity of the proteins on the ammonium sulfate concentration was examined in the salt concentration range of 0.5-2.

Impact of ligand density on the optimization of ion-exchange membrane chromatography for viral vector purification.

The effect of ligand density on anion-exchange membrane chromatography (AEXmc) for the purification of recombinant baculoviruses (rBVs), potential viral vectors in clinical applications, is studied by surface plasmon resonance on customized AEX surfaces and gradient elution experiments on Sartobind D membrane prototypes with different diethylamine ligand densities, complemented by dynamic light scattering analysis for estimation of the hydrodynamic particle size of the various biologics. A chromatographic-column model based on the steric mass action model of ion exchange is employed to analyze the gradient-elution AEXmc experiments, extrapolate the results to other operating conditions, and provide directions for process improvement. Although counterintuitively, the experimental evidence provided in this study shows that the lowering of ligand density is beneficial for rBV purification by AEXmc in bind-and-elute-mode, because it decreases the residual concentrations of host cell protein, dsDNA, and non-infective rBVs in the eluted product cut, and increases the overall yield by roughly 20% over current standard values.

Purification of cell culture-derived modified vaccinia ankara virus by pseudo-affinity membrane adsorbers and hydrophobic interaction chromatography.

A purification scheme for cell culture-derived smallpox vaccines based on an orthogonal downstream process of pseudo-affinity membrane adsorbers (MA) and hydrophobic interaction chromatography (HIC) was investigated. The applied pseudo-affinity chromatography, based on reinforced sulfated cellulose and heparin-MA, was optimized in terms of dynamic binding capacities, virus yield and process productivity. HIC was introduced as a subsequent method to further reduce the DNA content.

Capturing of cell culture-derived modified Vaccinia Ankara virus by ion exchange and pseudo-affinity membrane adsorbers.

Smallpox is an acute, highly infectious viral disease unique to humans, and responsible for an estimated 300-500 million deaths in the 20th century. Following successful vaccination campaigns through the 19th and 20th centuries, smallpox was declared eradicated by the World Health Organization in 1980. However, the threat of using smallpox as a biological weapon prompted efforts of some governments to produce smallpox vaccines for emergency preparedness.

Development of a polishing step using a hydrophobic interaction membrane adsorber with a PER.C6-derived recombinant antibody.

Membrane chromatography has already proven to be a powerful alternative to polishing columns in flow-through mode for contaminant removal. As flow-through utilization has expanded, membrane chromatography applications have included the capturing of large molecules, including proteins such as IgGs. Such bind-and-elute applications imply the demand for high binding capacity and larger membrane surface areas as compared to flow-through applications.

Influence of pore structure and architecture of photo-grafted functional layers on separation performance of cellulose-based macroporous membrane adsorbers.

New weak cation-exchange membrane adsorbers were prepared via UV-initiated heterogeneous graft copolymerization on Hydrosart macroporous regenerated cellulose membranes. The dynamic performance was investigated in detail with respect to the pore size and pore size distribution of the base membranes, ion-exchange capacity and architecture of the grafted functional layers as well as binding of target proteins. Main characterization methods were pore analysis (BET and permporometry), titration, analysis of protein binding under static conditions including visualization by confocal laser scanning microscopy and chromatographic analysis of dynamic protein binding and system dispersion.

Characterization of pore structure of a strong anion-exchange membrane adsorbent under different buffer and salt concentration conditions.

The quantitative characterization of pore structure of Sartobind Q, a strongly basic membrane anion exchanger that is formed by cross-linked cellulose support and a hydrogel layer on its pore surface, was made combining the results obtained by several experimental techniques: liquid impregnation, batch size-exclusion, inverse size-exclusion chromatography, and permeability. Mercury intrusion and nitrogen sorption porosimetry were carried out for a dry cellulose support membrane in order to get additional information for building a model of the bimodal pore structure. The model incorporated the distribution of the total pore volume between transport and gel-layer pores and the partitioning of solutes of different molecular weights was expressed through the cylindrical pore model for the transport pores and random plane model for the gel layer.