Understanding Perfusion Chromatography Media and Its Role in Advancing Biologic Purification

What are perfusion media? Perfusion media are a type of chromatography media that resembles conventional spherical porous microbead media, but they have a distinct pore structure (Figure 1). The term "pore structure" refers to the arrangement and characteristics of the void spaces (pores) within the material. Our discussion will focus on two key factors: pore size and surface area (see the section below for more details).

Perfusion media were developed to address performance challenges associated with conventional media in biologic purification. Conventional spherical microbead media, made of inorganic materials such as silica or organic polymer materials (commonly known as resins), are typically packed in cylindrical columns for the purification of peptides and other small molecules. However, for proteins and other biologics, such as mRNA or virus particles, the pore structure of the conventional media is not optimal for achieving effective separation.

What is pore structure? Porous microbeads contain an internal network of tunnels or channels known as pores. The size of these pores, which open up on the surface of the stationary phase and extend through its interior, determines how accessible an analyte molecule is to the stationary phase's internal surface. Pore size is typically measured in nanometers (nm) or angstroms (Å).

The surface area of the microbeads, often measured in m²/g, is directly related to the number of interaction sites available for analyte molecules.

There is a trade-off between pore size and surface area when dealing with larger molecules. The size of the pores must be optimized to maximize the surface area for interaction with analyte molecules. Smaller pores provide a greater surface area for interaction with smaller molecules, but they limit access to the interior of the stationary phase for larger molecules. Conversely, larger pore sizes generally facilitate better access for larger molecules.

Conventional media typically have a single set of pores with an average pore size of less than or equal to

Figure 1. Depiction of conventional (left) and perfusion (right) media. Image adapted from reference 2.

30 nm (or 300 Å). In contrast, perfusion particles have two sets of pores. The first set consists of larger pores, known as perfusion pores, with sizes greater than or equal to 500 nm (or 5,000 Å). The second set contains smaller pores similar to those found in conventional media. These smaller pore channels originate along the channels of the perfusion pores (see Figure 1) [1].

Relevance of pore structure to chromatography performance. During a chromatography run, the mobile phase (the fluid that transports the analytes) is pumped through the column. The fluid flows through:

1) The interstitial voids—empty spaces between the packed spherical microbeads.

2) The pores are present throughout the spherical microbeads.

The fluid flows freely through the interstitial voids in a type of flow known as convective flow. However, as the fluid permeates the interior of the porous microbeads, it encounters resistance, and the flow within these pore channels is referred to as diffusive flow.

There are dynamic (real-time) differences in the concentration of the fluid molecules between the pores and interstitial voids. These concentration differences, also known as concentration gradients, move fluid’s molecules from areas of higher concentration to areas of low concentration- this type of characteristic of diffusive flow.

Most individual peptides and proteins in solution generally range from 4 to 6 nanometers (40 to 60 Å) in size. Conventional media chromatography, with a pore size of ≤30 nanometers (300 Å), is suitable for most peptide and protein separations. However, larger molecules diffuse slowly within these pores, which can significantly increase band spreading, resulting in a loss of resolution. This occurs because molecules still in the convective stream or bound to the outside of the porous microbeads can elute before those that are diffusing inside the microbeads.

The size of peptide and protein aggregates, as well as some large individual proteins, can vary from tens to hundreds of nanometers. Additionally, other biologics such as mRNA or virus particles often have sizes in the range of tens to hundreds of nanometers. These larger sizes generally prevent them from entering the pores. As a result, the majority of binding for large-sized biologics occurs only on the edges of the beads, with a limited fraction utilizing the voids in the media’s pores. Consequently, this leads to a decrease in binding capacity for these large biologics compared to that for molecules that can enter the pores. Studies have shown that the capacity of a chromatography column can sometimes drop by as much as 30 to 50 times when working with large molecules like DNA.

A high binding capacity can be achieved by increasing the surface area, for example, through the use of highly porous materials or by reducing the size of the particles. However, reducing particle size can dramatically increase flow resistance and back pressure.

To address the diffusion issues for macromolecules, chromatographic media have been designed with larger microbead diameters (ranging from over 50 µm up to 300 µm) and pores that are several micrometers in size. However, simply increasing the pore size does not necessarily enhance binding capacity. Additionally, volumetric capacity — the maximum volume of analyte a chromatography column can hold — may be reduced. Volumetric capacity is crucial because it dictates the maximum amount of sample that can be loaded onto the column without compromising separation quality. Furthermore, increasing particle size may compromise the mechanical strength of the microbeads, limiting their use to low-pressure liquid chromatography setups.

How Does the Pore Structure of Perfusion Media Improve Biologic Chromatography Performance?

The large-sized perfusion pores run throughout the entire interior of the microbeads, facilitating the convective transport of biological molecules. The extent of this flow is determined by the ratio of pore size to particle size. Smaller pores interconnect the perfusion pores, enabling diffusive transport and increasing the surface area, which in turn enhances the volumetric capacity of the column. These smaller pores have very shallow channels, resulting in limited effects from diffusion.

The benefits of using perfusion media for biologic purifications include [2]:

1) Separation times that are 10 to 100 times faster than those achieved with conventional chromatography, while still maintaining the resolution and loading capacity of the column.

2) The rapid transport of materials within the perfusion pores, combined with the short diffusion paths, allows for both resolution and loading capacity to remain independent of the flow rate.

3) Reduced separation times lead to shorter residence times of biologics within the chromatographic column. This decrease may enhance the recovery of biological activity. When biologic molecules are bound to the chromatographic media, they can be exposed to potentially denaturing eluents, proteolytic degradation, and forces that may cause aggregation or damage. The longer these molecules stay on the media, the greater the risk of such damage occurring.

4) As a result of reduced analysis times and the high loading capacity of perfusion supports, the overall cost of large-scale chromatographic processes is lowered.

The advantages of perfusion chromatography are even more pronounced at high mobile-phase velocities (>1000 cm/h), where intraparticle convective transport significantly exceeds the rate of diffusion transport.

Perfusion media allow for low backpressures and improved fluid movement through the interior of the media. However, one challenge with these media can be the entrapment of biologics within their interiors, which may lead to reduced elution concentrations. This issue can often be mitigated by using a very low flow rate.

We have launched a suite of novel NanoPak-C All Carbon perfusion microbeads, designed for the purification of large-sized biologics, including exosomes, virus particles, and mRNA.

NanoPak-C All Carbon perfusion microbead options:

2, 10, 100, or 1000 gram bulk media. Microbead diameter options: 40-300 µm. Diffusive pore size options: 20-40 nm. Perfusion pore options: 0.5-3 µm.

Contact us to discuss how our custom perfusion media can serve your biologic purification challenges. Please email us at inquiry@millennialscientific.com, call us at 855-388-2800, or fill out our online form.

References:

[1] N.B. Afeyan, S.P. Fulton, N.F. Gordon, I. Mazsaroff, L. Várady, F.E. Regnier, Perfusion Chromatography: An Approach to Purifying Biomolecules, Bio/Technology 8(3) (1990) 203-206.

[2] M.C. Garcı́a, M.L. Marina, M. Torre, Perfusion chromatography: an emergent technique for the analysis of food proteins, Journal of Chromatography A 880(1) (2000) 169-187.