Resins, Monoliths, or Membranes. Which Chromatographic Method Should You Use?
Today, bioprocessing scientists are spoiled for choice when it comes to chromatography methods. Here, we provide some guidance to help you choose the right matrix for your application.
This article is posted on our Science Snippets Blog
There are a number of important choices you’ll make when it comes to chromatography workflows. Even within one matrix type, such as beaded resins, there are many different ways to separate your precious target from contaminants and impurities. When you add the variety of chromatography matrices available, giving you even more flexibility and choice, it can be difficult to see the best way forward.
However, while the choice can be challenging, there is a lot of data and information that can be used to make your choice based on scientific principles and evidence. In a previous article, we recently discussed how important it is to know the properties of both your target and impurities that need to be removed. We addressed how properties like charge, hydrophobicity, and ligand binding affinity can affect separations. Differences between these properties can be used to choose the most appropriate chromatography mode for your application, but what about different chromatography matrices?
There are three basic types of chromatography matrix to choose from, and it’s likely that you already have knowledge of one of these. Experience with a particular matrix and prior use can count for a lot. Often, this experience comes with a long track record of success and robust processes that have stood the test of time. However, it is always wise to revisit chromatography processes when the opportunity arises and check whether new products might offer enough of an advantage to motivate a review and even a replacement.
In this article, we will explain the different features of three important chromatography matrixes: monoliths, resins, and membranes. Our aim is to equip you with what you need to make well-informed decisions.
Resins could be considered the classical chromatography matrix, acting as a useful benchmark for newer types. Despite their long history, beaded resins are still an essential tool for many separation processes today.
Figure 1 – Resin chromatography uses small particles packed into a column. These particles have chemical properties that provide binding sites (yellow) for target molecules (teal) or impurities (pink).
Because they have been around for such a long time, beaded resins are available from many suppliers and show incredible diversity. There are many materials are used to form the backbone structures, and many separation modes are available - ion exchange (IEX), affinity, hydrophobic interaction (HIC) chromatography, and mixed-mode chromatography are routinely carried out using resins. Resins can be purchased in bulk for you to carefully pack into a column of the right size and specifications, then used to separate biomolecules based on their properties. You can also buy pre-packed, ready-to-use columns for a range of different scales - from a small analytical setup to a medium-scale production run.
One of the key features of the resin beads is that they contain micro-pores where the binding sites are located. This means that solutes must diffuse from the bulk liquid into these micro-pores for binding to occur. This can slow down separations because they will always be diffusion-limited and often require slow flow rates to be effective.
The small size of the pores will also exclude the binding of very large molecules and particles. Anything larger than IgM such as mRNA, plasmid DNA, viral vectors, and vesicles will only be able to reach a fraction of the binding sites in the pores of most resins. However, the maximum capacity for anything smaller than monoclonal antibodies will usually be very high.
Today, resins are the most widely used matrix for purifying biomolecules from lab-scale all the way up to block-buster level production. They are very well established in many processes, such as monoclonal antibody purification.
Monolithic chromatography is relatively new and has very different features from resins. They are hollow cylinders formed from a single block of polymer with a network of interconnected channels. The functional groups for chromatography are distributed over the surface of the walls in these interconnected channels. In contrast to resins, there are no pores in the monolithic matrix, meaning that mass transport is not limited by diffusion. Instead, the bulk eluent flows through the channels carrying the molecules, particles, or vesicles to be separated, and mass transport is achieved via convection alone.
Figure 2 – Monolithic chromatography uses a single-unit structure containing a large network of interconnected channels. These channels contain binding sites (yellow) for target molecules (teal) or impurities (pink).
One of the first signs that you should consider monoliths is if your target molecule is very large, i.e., it will diffuse too slowly to be used with resins, and it may not even enter the pores in resin beads. Using a monolith for large molecules will give very efficient adsorption with steep breakthrough curves and high binding capacities.
Separation using monoliths is independent of the flow rate through the monolithic matrix, meaning that separation can usually be achieved much faster than with resins. This is useful for speeding up processes and improving productivity. However, it might also provide an essential tool for quickly removing aggressive impurities that could damage your target if left in contact for too long.
Another useful feature of monoliths is that the flow through the bed is predominantly laminar. This makes conditions gentler for molecules, such as mRNA, that are particularly sensitive to shear forces. The laminar flow in monoliths contrasts with turbulent flow in packed beds of resin beads. This can lead to strong shear forces, particularly at high flow rates, which can damage shear-sensitive molecules.
An additional benefit is that, due to the low back pressures with monolithic chromatography devices, they are particularly suitable if you have a high viscosity feedstock. This factor is likely to become increasingly valuable with the growing trend towards intensified processing.
Monoliths can be used for various chromatography modes, including IEX, AC (e.g., with an oligo dT ligand), HIC, and MMC. They are delivered as ready-to-use devices in a range of sizes suitable for both the development lab and production scenarios.
Membrane chromatography uses layers of porous membranes with binding sites distributed through the pores. The pores arranged in membrane stacks carry the mobile phase meaning that mass transport is convective.
Figure 3 – Membrane chromatography relies on stacks of porous membranes. Contained within these pores are binding sites (yellow) for target molecules (teal) or impurities (pink).
A hallmark of membrane chromatography is speed. It is possible to tailor the membrane stack height to truly optimize for rapid separations. For example, if you have a shallow bed, it will be possible to pass through large feed volumes at very high flow rates.
Despite the speed, separation is also very efficient owing to the widespread distribution and easily accessible binding sites throughout the membrane structure. As the mass transport is convective, there are also no time limits due to slow diffusion.
Fast separations are particularly useful for molecules that are exposed to enzymic breakdown or other damaging conditions. These need to be separated quickly and transferred to a suitable non-damaging environment. Using membranes can reduce the probability that target molecules are degraded during the separation process.
Membranes used for chromatography can have binding sites for IEX, salt-tolerant IEX, or HIC. They are typically available as plug-and-play, single-use, capsules, and cassettes, making them very flexible and easily adapted to a wide range of different processes.
The large pore sizes make membranes particularly useful for larger particles such as vesicles and viruses. However, it can be challenging to achieve very high capacities in bind-elute mode with small biomolecules such as proteins. Instead, membranes are often used to remove impurities in flow-through mode late in a process. The last impurities are bound, and the target product flows through without interacting with binding sites.
In biomanufacturing, membrane chromatography is widely used to remove trace impurities and contaminants in flow-through mode. It is well established for the removal of endotoxins, host cell proteins, DNA, and viral contaminants. When used in bind-elute mode, membrane matrices can be extremely effective for the purification of viruses and nucleic acids.
Your Final Chromatography Matrix Choice
Choosing chromatography matrices and separation modes is not about picking one winner, even if it sometimes feels as though you select one at the expense of others. Real-life purification processes are often complex and will typically require several rounds of chromatography. In this case, it is possible to mix-and-match chromatography matrices and modalities to give you the best results for each stage in your process.