Plasmids are getting larger. Gene-therapy and vaccine constructs above 10 kbp are now routine, and they have to be produced at scale and at high purity. Anion-exchange chromatography is the standard purification step — and it is exactly where large plasmids cause trouble: yields drop, pressure climbs, and columns clog.
A 2023 study in Electrophoresis looked at where that trouble comes from. Its answer is specific enough to turn into a design rule for choosing media, and it is the reason channel size deserves more attention than it usually gets on a datasheet.
What the study measured
The authors ran plasmids of different sizes on weak anion-exchange monolith columns with channel sizes of 2, 3 and 6 µm. Rather than only reading yield from the elution peak, they followed the mass balance and the isoform composition of the plasmid at the column outlet. That is what makes the result useful: it shows not just that performance drops, but which form of the plasmid goes missing.
The open-circular form is the problem
A plasmid preparation is a mix of isoforms, mainly supercoiled and open-circular. The study identified the open-circular form as an important driver of reduced performance: in supports with smaller channels, open-circular pDNA is trapped as it is carried through by the flow. The same convective entrapment appeared on porous beads and on monoliths.
Porous beads showed a second effect on top. On an 11.6 kbp plasmid they also recovered less of the supercoiled form, because it became trapped by diffusion inside the bead pores. A convective support avoids that second mechanism; it does not avoid the first one unless the channels are wide enough.
The threshold: channels wider than 3.5 µm
The conclusion is a number. Convective anion-exchange monoliths or membranes with a channel diameter above 3.5 µm increased yields and prevented irreversible pressure build-up and column clogging for plasmids at least up to 16 kbp. In narrower channels, the open-circular form can be trapped, and the pressure build-up that follows was not reversible.
Two limits are worth keeping in mind. The work used weak anion-exchange chemistry and a defined set of plasmids, so the exact threshold for another chemistry, a larger construct or a less clean feed can differ. And the threshold describes one cause of clogging. Staying above it does not make a column immune to fouling from other feed components.
Why channel size is a design decision
If wider channels are safer, why not make every channel wide? Because channel size sets two properties in opposite directions. Permeability grows with the square of the channel diameter, so wider channels run at lower pressure and tolerate more of what the feed carries. The internal surface available for binding shrinks as channels widen, so capacity per millilitre of media tends to fall.
Every convective medium sits somewhere on that curve. Channel classes of a few micrometres are common in commercial monoliths — the study itself compared 2, 3 and 6 µm. Choosing a class is a trade between binding surface on one side and hydraulic headroom and clogging tolerance on the other. For large plasmids, the study shows where the floor of that trade is.
Where MonoCore™ sits
MonoCore™ cellulose monoliths ship with a 15 µm standard channel across the line, including MonoCore™ Q for anion exchange and MonoCore™ HIC. That places the standard product well above the published threshold for plasmids up to 16 kbp. Other channel sizes between 10 and 150 µm are available on request; the size is selected per column and is uniform within each monolith.
The same trade-off applies to us. A wider channel buys hydraulic headroom and tolerance to difficult feeds; it does not add binding surface. For pDNA work where clogging and yield loss are the bottleneck, that is the right side of the trade. Where maximum capacity per millilitre is the only thing that matters, a narrower class may still be the better choice — as long as the plasmid is small enough for it.
What to check in your own process
Four measurements tell you whether channel size is limiting a pDNA step. First, the open-circular content of the load, from an agarose gel or an HPLC isoform assay. Second, the isoform mass balance across flow-through, eluate and strip — a missing open-circular fraction points to entrapment rather than to poor binding. Third, the pressure trend over repeated cycles: a baseline that climbs and does not recover after cleaning is the signature of irreversible clogging. Fourth, plasmid size against the channel size of the media you are using.
If the open-circular fraction goes missing and pressure keeps rising, a wider channel is the first thing to try before tuning the gradient.
Source
Kralj Š, Kodermac ŠM, Bergoč I, Kostelec T, Podgornik A, Štrancar A, Černigoj U. Effect of plasmid DNA isoforms on preparative anion exchange chromatography. Electrophoresis 44(24):1953–1966 (2023). doi:10.1002/elps.202300035
Further reading
For why convective transport changes the flow-rate ceiling of packed beds, see our article on convective vs diffusive mass transport in chromatography. For how monoliths and membrane adsorbers compare on hydraulics, resolution and scale-up, see the monolith vs membrane comparison.