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 0.5-2.0 mol L(-1). An exponential increase of the binding capacity was observed for all proteins. Simple Langmuir one-component isotherm was found suitable for the characterization of the effect of protein concentration in all cases. A combined effect of protein and salt concentrations was expressed via the Langmuir exponential isotherm and fitted the adsorption data for three of the investigated proteins well.

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. Overall, we present a case study on how rational design can streamline downstream process development.

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. Therefore, two screens were undertaken. First, several HIC ligands were screened for different adsorption behavior between virus particles and DNA. Second, elution from pseudo-affinity MA and adsorption of virus particles onto the hydrophobic interaction matrix was explored by a series of buffers using different ammonium sulfate concentrations. Eventually, variations between different cultivation batches and buffer conditions were investigated.The most promising combination, a sulfated cellulose membrane adsorber with subsequent phenyl HIC resulted in overall virus particle recoveries ranging from 76% to 55% depending on the product batch and applied conditions. On average, 61% of the recovered virus particles were infective within all tested purification schemes and conditions. Final DNA content varied from 0.01% to 2.5% of the starting material and the level of contaminating protein was below 0.1%.

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. Given these considerations, a new Sartobind Phenyl membrane adsorber was developed for large-scale purification of biomolecules based on hydrophobic interaction chromatography (HIC) principles. The new hydrophobic membrane adsorber combines the advantages of membrane chromatography-virtually no diffusion limitation and shorter processing time-with high binding capacity for proteins comparable to that of conventional HIC resins as well as excellent resolution. Results from these studies confirmed the capability of HIC membrane adsorber to purify therapeutic proteins with high dynamic binding capacities in the range of 20 mg-MAb/cm(3)-membrane and excellent impurity reduction. In addition the HIC phenyl membrane adsorber can operate at five- to ten-fold lower residence time when compared to column chromatography. A bind/elute purification step using the HIC membrane adsorber was developed for a recombinant monoclonal antibody produced using the PER.C6(R) cell line. Loading and elution conditions were optimized using statistical design of experiments. Scale-up is further discussed, and the performance of the membrane adsorber is compared to a traditional HIC resin used in column chromatography.

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. An additional aspect for the interest in smallpox virus is its potential use as a platform technology for vector vaccines. In particular, the latter requires a high safety level for routine applications. IMVAMUNE, a third generation smallpox vaccine based on the attenuated Modified Vaccinia Ankara (MVA) virus, demonstrates superior safety compared to earlier generations and represents therefore an interesting choice as viral vector. Current downstream production processes of Vaccinia virus and MVA are mainly based on labor-intensive centrifugation and filtration methods, requiring expensive nuclease treatment in order to achieve sufficient low host-cell DNA levels for human vaccines. This study compares different ion exchange and pseudo-affinity membrane adsorbers (MA) to capture chicken embryo fibroblast cell-derived MVA-BN after cell homogenization and clarification. In parallel, the overall performance of classical bead-based resin chromatography (Cellufine sulfate and Toyopearl AF-Heparin) was investigated. The two tested pseudo-affinity MA (i.e., sulfated cellulose and heparin) were superior over the applied ion exchange MA in terms of virus yield and contaminant depletion. Furthermore, studies confirmed an expected increase in productivity resulting from the increased volume throughput of MA compared to classical bead-based column chromatography methods. Overall virus recovery was approximately 60% for both pseudo-affinity MA and the Cellufine sulfate resin. Depletion of total protein ranged between 86% and 102% for all tested matrices. Remaining dsDNA in the product fraction varied between 24% and 7% for the pseudo-affinity chromatography materials. Cellufine sulfate and the reinforced sulfated cellulose MA achieved the lowest dsDNA product contamination. Finally, by a combination of pseudo-affinity with anion exchange MA a further reduction of host-cell DNA was achieved.

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. The trade-off between static binding capacity of the membrane adsorber and its permeability has partially been overcome by adapted architecture of the grafted functional layer achieved via the introduction of uncharged moieties as spacers and via stabilization of the binding layer by chemical cross-linking. The resulting membranes show only negligible effects of flow rate on dynamic binding capacity. There is no considerable size exclusion effect for large proteins due to mesh size of functional cross-linked layers. Investigation of system dispersion based on breakthrough curves confirms that the adapted grafted layer architecture has drastically reduced the contribution of the membrane to total system dispersion. The optimum pore structure of base membranes in combination with the best suited architecture of functional layers was identified in this study.

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. The effect of composition of liquid phase on the pore structure was investigated in redistilled water, phosphate and Tris-HCl buffers containing up to 1M NaCl. Evident differences in the bimodal pore structure were observed here when both the specific volume and size of the hydrogel layer pores significantly decreased with the ionic strength of liquid phase.