Chromatography media and methods have evolved continuously since their introduction a half century ago. Traditional methods use columns packed with porous particles. They still dominate chromatography applications in the field of virus purification, but the past 20 years have witnessed the ascendance of alternative supports, namely membranes and monoliths. These newer media exploit the familiar surface chemistries — ion exchange, hydrophobic interaction, and affinity — but they use unique architectures that offer compelling performance features.

The Architecture of Chromatography Media

A monolith can be defined as a continuous stationary phase cast as a homogeneous column in a single piece (1,2,3). Monoliths are further characterized by a highly interconnected network of channels with sizes ranging 1–5 µm. The adsorptive surface is directly accessible to solutes as they pass through the column. The current generation of preparative monoliths have bed heights ranging from a few millimeters to a few centimeters. Strictly speaking, membranes are monoliths. Their channel diameters similarly range from about 0.5 to a few micrometers, but their “bed heights” are fractions of a millimeter. Traditional chromatography media are prepared as porous particles and packed in columns. Most of their adsorptive surface area resides within shallow dead-end pores ranging 50–100 nm in size (4). Figure 1 compares the generalized structures of membranes, monoliths, and porous particles.

Figure 1: (Click to enlarge)

Mass Transport

Membranes and monoliths differ most fundamentally from porous particle media in the mechanisms by which solutes are carried to and from their surfaces. The two primary modes of mass transport are diffusion and convection.

Diffusion can be defined as the migration of solutes from an area of high concentration to an area of low concentration by incremental random thermal movement. Porous particle-based media rely almost exclusively on diffusive mass transport (4). Table 1 lists diffusion constants for representative solutes. Two important points emerge: Diffusion is slow, and it becomes dramatically slower with increasing molecular size. As a direct consequence, dynamic binding capacity on porous particles decreases with increasing flow rates — dramatically so for large solutes such as viral particles (1,2,3,4,5,6,7). Resolution also suffers, also in proportion to flow rate, and also more with large solutes.

Table 1: Diffusion constants for selected solutes (Click to enlarge)

Diffusive limitations on porous particles can be compensated somewhat by reducing flow rate, but the diffusion constants of viral particles may be an order of magnitude slower than proteins, and few industrial users are willing to reduce flow rates to that degree (Table 1). Capacity and fractionation performance are thereby compromised. Pore exclusion is an additional limitation (8). Figure 2 illustrates a scale comparison of MVM and MuLV in relation to 100-nm diffusive pores and 1-µm convective channels. Pore size is roughly five times greater than the diameter of MVM, but MuLV is excluded and has access only to the external particle surface. This severely truncates binding capacity for large viral particles. Table 2 shows that many viral species have diameters above the exclusion limits of most porous particle-based media, but surface access within the monolith is unrestricted.

Figure 2: (Click to enlarge)

Table 2: Approximate diameters of selected viral particles (Click to enlarge)

Convection is movement imposed by an external force, in this case fluid flow delivered by the pumps in a chromatography system. Convective mass transport is not limited by diffusion or by molecular size. A wine cork and a tree trunk both flow down a river at the same rate, which is determined by the velocity of the current. The architecture of membranes and monoliths is designed specifically to take advantage of convective mass transport. Capacity and resolution are largely independent of flow rate, even at velocities 10–20 times faster than are commonly used with diffusive particles (1,2,3, 5,6,7).

High flow rates offer numerous practical benefits. Process development and validation are accelerated, and manufacturing productivity is increased. Short process times may also be beneficial for live and attenuated viruses that are labile under the conditions used to conduct a particular purification step (8, 9). Figure 3 compares the dynamic binding capacity of a common particle-based anion exchanger with that of a monolithic anion exchanger. DNA is used as a surrogate to illustrate the behavior of large solutes. Capacity on the monolith is 30–50 times higher (10), which allows for smaller columns and translates into proportional savings in media, buffer consumption, and use of expensive manufacturing space.

Figure 3: (Click to enlarge)