Life Sciences: New Innovations in Membrane Chromatography
Biophannaceutical manufacturers face two critical processing issues in liquid chromatography for DNA: plasmid and viral clearance; capacity and flow uniformity. Test data illustrate a number of limitations with traditional, resin- or gel-based column chromatography due to slow diffusion rates, non-uniformity of flow, and inefficient capacity for handling large bio-molecules.lon exchange membrane chromatography is a new technology that can offer significantly faster processing capacity, and dramatic reductions in processing time, both of which help speed up drug production. Recent innovations in membrane technology offer low protein absorption to further enhance performance. In some cases process purification cycles that normally take many hours can be reduced to 30 minutes.
Resin- or gel-based column chromatography is an established and reliable technology utilized by biotechnology and pharmaceutical manufacturers for product capture and purification as well as DNA, viral, and endotoxin clearance and separation. The goal in any liquid chromatographic manufacturing process is to realize a “pure” product in as few purification steps as possible. In chromatography one method of achieving this is using linear isocratic elution, in which different target substances are sequentially eluted in a defined order.
Another method that is more common in manufacturing processes is using step elution, where the salt or pH step is specifically selected to give the maximum separation efficiency. Achieving this “perfect” elution is nearly impossible due to a number of factors that impact on the efficiency of most processes. These limitations include the temperature and viscosity of the solution being eluted, the size of the target molecule, and the type of chemical interaction taking place.
A significant limiting factor of column chromatography is irregular flow during processing. Depending on how a column is packed, flow path and therefore flow rates can vary in different parts of a column, resulting in different absorption and desorption rates, or fluid channeling. In a “worst case” scenario one part of a column may be saturated with the target molecule, while other parts of the column may still have free binding sites, resulting in product binding and breakthrough occurring simultaneously, destroying the resolving power of the column. When poor column packing techniques are employed, fluid channeling is often the result. Channeling prevents proper separation resolution-
a requirement for separating more than one target substance—and this can result in product recovery failure.
Another factor that impacts on flow uniformity is pressure. For gel- or resin-based chromatography high pressures can lead to bed compaction, restricting flow rates and causing channeling, and ultimately poor separation resolution.
Membranes Speed Purification
Ion exchange membrane chromatography is a new technology that offers significant advantages over column chromatography by eliminating slow diffusion times and speeding up purification processes. Not all membranes are alike, and manufacturers need to consider the different membrane chemistries that are utilized, as these impact processing efficiency. A new, proprietary polyether-sulfone membrane has been developed that has low, non-specific protein absorption. This is an important advantage because membranes need to exhibit as low a level of non-specific adsorption as possible in order to perform specific chromatographic processes efficiently.
Membrane chromatography systems are available in two formats. Membranes pleated into cartridges or disposable capsules allow high flow rates, high adsorption capacities, and good resolving capabilities in an easy-to-use disposable format. This allows immediate inclusion into an existing process chain for applications such as DNA and viral clearance, removal of endotoxin, etc. Membrane modules, composed of up to 80 membranes stacked together, are used with stainless steel housings. This format is designed to withstand higher pressures, and the modules offer excellent resolving power, higher capacities compared to capsule or cartridge systems, and are reusable. These modules are specifically designed to give a reliably uniform, parallel flow of sample through the membranes, which prevents channeling.
Surface Binding on Membranes Enables Mass Transfer
The active chemistries are attached to the surface of the membrane structure. Because membranes are three-dimensional structures with open (0.8µm) pores, the active chemistries are immediately available for binding, and diffusional limitations are so low that they have no discernable effect on the efficiency of the system. In chromatography resins and beads, the majority of the active surface area is contained within the pores of the matrix. Target molecules must diffuse into and out of the pores during the binding and elution stages; the diffusional limitations imposed by a porous bead or resin significantly restrict the flow rates that can be used with these systems. Figures 1 and 2 demonstrate the difference in surface areas for chromatographic binding between packed columns and membrane ion exchange. In the packed column (Figure 2, left) the white areas represent resin beads, and blue areas represent flow paths. On the membrane (Figure 2, right) there is much more blue than white, illustrating the greater availability of surface areas for binding, which translates into a much higher flow rate.
These illustrations show a 20 micron particle, which is typically the size used for analytical chromatography. The typical ligand size for process chromatography is closer to 90 microns, and the diffusional limitation is even more pronounced. The internal surfaces of membranes are coated with affinity chemistries, in this example either “Q” groups (trimethyl ammonium quaternary salts for anionic exchange), or “S” groups (sulfopropyl salts for ionic exchange). These coatings create a high surface area for binding, and as liquid flows through these channels the active affinity molecules are immediately available for reaction.
Removing Flow Rate as a Process Variable
A comparison of flow rates also illustrates the difference between column chromatography and membrane chromatography. Figure 3 demonstrates the results of dynamic binding of Bovine Serum Albumin (BSA) at varying flow rates for a Q-resin and a Q-membrane. The flow rate is shown in column volumes per minute. As flow rate is increased, the ability of the column to absorb BSA is slowed by diffusional limitation. However, as the flow rate increases from one to 100 column volumes per minute, the ability of the Q-membrane to absorb BSA remains virtually the same. This chart demonstrates that under certain conditions, membrane chromatography can remove flow rate as a process variable, since binding capacity will be the same, irrespective of flow rate. This is a major advantage for biopharmaceutical processing, as scale-up for membrane chromatography is linear, and much more predictable.
Membrane Benefits for Large Bio-Molecules
Another limitation with column chromatography is its low binding capacity for large bio-molecules like DNA and viruses. Binding efficiency of these larger particles is restricted because in many instances they are too large to diffuse into the pore structure of the resin. Therefore binding is limited to those active chemistry groups that are on the outer surface of the beads. The large pore size and immediate availability of all active chemistry groups on the surface of a membrane allow high binding capacities of even very large particles such as plasmids, DNA, and viruses.
Due to the limited binding area of beads and resins, manufacturers often oversize chromatography columns and incorporate larger assemblies to allow for high flow rates. By replacing columns with membrane chromatography, systems can be sized much more proportionately to respective processes, with smaller footprints, lower hardware costs, and significantly reduced operating costs. Cycle times can be reduced to as little as 30 minutes.
It is instructive to review the binding capacities for molecules with various molecular weights. Figure 4 shows the binding capacities of several different bio-molecules, comparing column and membrane capacities. The flow rate illustrated in this chart is three column volumes per minute vs. three membrane volumes per minute. The four molecules tested each have varying molecular weights (in parentheses): BSA (67K), Ferritin (440K), Thyroglobulin (669K), and a plasmid (2.88M).
Figure 4 demonstrates that, for each bio-molecule, membrane systems have a significantly higher dynamic binding capacity. Compared to an analytical resin (15um pores), membranes offer between 33 to 50 percent better binding capacity. Compared to a typical resin used in manufacturing (with 90um pores), there is a log difference in rates.
Handling High Loading Concentrations
Manufacturers also face challenges during chromatography cycles when solution loading concentrations are too high, and in this respect as well, membranes offer advantages over columns. As loading concentrations approach 50 percent, the ability of a column to separate product components during elution is limited.
Figures 5 through 8 compare the elution profiles of S-membranes and S-beads at varying loading percentages. At two percent loading there is a very good separation of the two components for both the S-membrane and the S-bead, with well-defined peaks and good baseline resolutions. At 25 percent loading, the membrane still yields a very good resolution, but with the bead, the two peaks start to become much less defined, indicating that the column has been overloaded.
The ability of new membrane chromatography technologies to handle high flow rates and high loading concentrations for DNA, viral, and endotoxin clearance is a major advantage for biopharmaceutical manufacturers. Membranes offer dynamic binding capacities equivalent to column chromatography, but they are not affected by changes in volumetric flow rates, therefore allowing much higher processing throughputs and predictable scale-up. For separation and purification or large particles and bio-molecules, membrane systems are also sized smaller than columns, making them easier to handle than large column assemblies.