Adding value
Membrane adsorbers in virus and protein purification

If the search for a new active pharmaceutical ingredient is lengthy in itself, its isolation and preparation are no less daunting. Substances in the pharmaceutical industry need to be of a purer grade now than in the most recent years. This is not even mentioning the increased demand to bring active pharmaceutical ingredients to market faster. Membrane adsorbers now enable fast purification of proteins solutions. Moreover, membrane adsorbers offer additional potential.

No other branch of industry has probably seen 15% annual growth rates like the biopharmaceutical industry is currently reporting. The number of medications developed by this industry is also increasing from year to year, and cell cultured monoclonal antibody concentration is approaching 5–10 g/L, which is discussed as the current “revolution” in this area. However, such booming success usually creates capacity problems. For this reason, new production facilities are being established everywhere for biopharmaceuticals, and producers need a revolutionary purification method as well in the downstream area.
Downstream processing follows production in bioreactors. In this step, the active pharmaceutical ingredient must be isolated from the cells. Membrane adsorbers have already gained a spot in the purification process chain. They are already used as a standard tool in DNA contaminant removal in the production of pharmaceutical proteins and adenovirus purification. Beyond these applications, there is even more potential for membrane adsorbers. Supplied with the appropriate ligands, the membrane adsorbers are ideal policing device for removing proteases, enzymes and toxins. The dynamic binding capacity, which is exceptionally high in comparison to conventional media, allows for quick cycles with the smallest bed volumes. For this reason, protein capturing will gain even greater significance in the future in addition to preparative virus purification.

Large surface areas
There are even applications with adsorbers in high-resolution chromatography; this field is rather atypical though. The preferred area of application for this technology is in fast chromatography using step elution in which very large volumes can be processed. This technology is a consideration whenever the macroporous structure of the membrane (0.45 and 3 µm) and the large surface areas of the unit can be used. The large flow surfaces of up to 1,300 cm2 for a bed volume of 2.1 liters (8 m2 membrane area) result from the design of the adsorber units, such as the Sartobind MultiSep, the cylindrical modules for production. The channel dimensions are designed as a compromise between the smallest possible hold-up volumes and the high flow rate of up to approx. 50 liters at 0.6 MPa (6 bar). Since the membranes are rolled and stacked, the bed is not as densely packed as with a conventional gel in the column. Connections and hold-up volumes are thus dimensioned relatively large for the membrane adsorbers.
Simple handling
A chromatographic column must always be operated with care and the assurance that air bubbles are excluded and that at low flow rates the pressure is monitored on the system. Of course, air bubbles must also be avoided with adsorbers. However, penetration of air bubbles in the adsorber unit does not destroy the bed structure as with conventional column chromatography — you simply ease out air bubbles through the membrane during filling or suction them off, if a syringe filter unit is used, by briefly lifting the injector syringe upward. The larger adsorber capsules and modules have a vent valve through which air is simply bled off. Since the chromatographic bed is attached covalently in membrane adsorbers, there is no cracking or erosion in the chromatographic bed. A pulsing pump does not disrupt a membrane adsorber; a peristaltic pump is completely sufficient for operation as there is almost no back pressure. Membrane adsorbers can be used immediately because you are working in principle with ready-to-use packed “columns”. Gradient elution is possible, but typically step elution is used.
The membrane format may lead lab staff to think that they can entirely dispense with prefiltration. The membrane adsorbers through which the substance flows, however, do not differ in this way at all from conventional columns; prefiltration using 0.45 µm or 0.2 µm filters is thus required.
Membrane adsorbers are more like filters than chromatographic columns and are particularly recommended for users who primarily require simple handling. This requirement has gained such momentum that membrane adsorbers are now even made for single use, such as the Sartobind SingleSep capsules. These types of unit are popular for use in the pharmaceutical production of proteins for removing contaminants via ion exchange chromatography in order to lower costs in an otherwise necessary validation.
The right bed size
The diffusion limitation plays a significant role in conventional chromatography. Due to the low flow rates, it is often not selected with the corresponding binding capacity, but rather according to the flow rate and thus frequently with a bed volume that is too large in order to allow for a sufficiently fast processing time. This, however, increases the adsorptive surface, which, in turn, leads to product losses due to non-specific interaction between the product and the matrix.
At the same time, the validation expense also increases in production, for example, because it is not economical to use large chromatography columns only one time and then not reuse them. The option of “economical single-use chromatography” is thus reserved for membrane adsorbers. By selecting the correct size of chromatographic bed, the yields can be increased, particularly in the production of pharmaceutical proteins.
Membrane ion exchangers (Sartobind Q from Sartorius) were first approved by the FDA in 2001 for pharmaceutical production processes on a ten-thousand liter scale for the quantitative removal of DNA and for reducing viral impurities In this application, an antibody solution that has passed through a protein A-column first is then conducted over 15-layer Sartobind Q MultiSep 15-06 cylinder modules that are 6 cm high or over 15-layer 15-50 cylinder modules that are 50 cm high, and the DNA contamination is reduced to below the detection limit. This quantitative removal of DNA was proven in two fermentation volumes in the production sector. In the first case, eight 2,000 liter batches with an average of 168 pg DNA/mg protein and, in the second case, three 12,000 liter batches with an average of 187 pg DNA/mg protein were cleared of contamination. The successful cleaning of viruses using membrane adsorbers was possible using the macroporous structure of the >3 µm membranes.The binding capacity for very large biomolecules and viruses is inevitably higher with membrane adsorbers than with conventional gels. For example, the purification of adenoviruses using membrane adsorbers has already been established. For parvoviruses, such as densonucleosis viruses, anion and cation exchanger membranes are suitable for purification.
High speed chromatography
A quick screening of a bioreactor harvest for immunoglobulins using a protein A membrane is a typical situation for utilizing an adsorber instead of a column. Instead of an hour, purification takes about ten minutes using a Sartobind Protein A 75 syringe-usable unit. By using the adsorber in the centrifuge units, immunoglobulins can be purified in large numbers at the same time. A protein A membrane adsorber can be operated at an approximately five to ten times higher flow rate than a conventional column. Under these conditions, the back pressure not only increases with gels, but the diffusion limitation also reduces capacity, while with adsorbers, the capacity remains constant at the increased flow rate.
Summary: Membrane adsorbers open a new avenue in preparative chromatography. Instead of the entire amount of the protein to be purified being applied entirely to one large column in one step, the bed volumes of an adsorber can be kept as small as possible and production can be carried out in cycles that take just minutes. This option may be of particular importance in affinity chromatography with expensive ligands.