AAV and viral vector purification
Adeno-associated and lentiviral vectors, from clarified harvest to formulated product — and the separation that has no equivalent in any other process: full capsids from empty ones.
Viral vectors are large. An AAV capsid is about 25 nm across, a lentiviral particle roughly four times that — both far larger than a protein, and large enough that the geometry of the chromatography medium starts to matter in a way it does not for an antibody.
They also bring a separation problem that no other modality has. A capsid carrying a genome and an empty one are nearly identical in size and surface chemistry; they differ only in the charge the packaged nucleic acid contributes. Separating them is the hardest step in the process, and the one that defines the route.
The route
| Step | Purpose | Note |
|---|---|---|
| Harvest | Collects vector from cells, supernatant, or both | Serotype-dependent: some release into the medium, some stay cell-associated |
| Nuclease treatment | Digests host DNA and residual plasmid | The nuclease then becomes an impurity to clear |
| Clarification | Removes debris ahead of the column | Depth filtration; every stage costs vector through adsorption |
| Affinity capture | Binds the capsid, clearing most host-cell protein and DNA | Ligand coverage is serotype-specific; elution is usually acidic |
| Full/empty separation | Separates genome-containing from empty capsids | Anion exchange, shallow gradient — the demanding step |
| Polishing and formulation | Final clearance, concentration and buffer exchange | Usually tangential flow filtration |
The full step-by-step version, including where serotype changes the answer, is in AAV purification: the protocol, step by step.
Full/empty separation
This is where method development spends its time. The charge difference between a full and an empty capsid is small, so the gradient has to be shallow, the salt system well controlled and the pH tight. A capsid stressed by earlier steps makes it harder still, because aggregate and damaged particles smear the peak.
A gradient that has to be shallow cannot simply be run faster, which is why this step tends to set the throughput of the whole process. It is also why the flow distribution of the medium matters: anything that broadens the peak further comes directly off the resolution available for the separation.
On full/empty performance we have no data of our own — so we do not claim any. What we can describe is the geometry, and what it does or does not do for a particle of this size — the argument is in which chromatography media are suitable for AAV polishing.
Where MonoCore™ fits
We do not make an AAV affinity ligand. Our part of this process is the anion exchange step: MonoCore™ Q on a modified cellulose matrix with 15 µm channels, in a capsule that connects to the FPLC system you already run. For a ligand of your own, the unfunctionalised matrix is available as custom affinity.
A word on scale. The standard formats are 1.8 and 5.4 mL, with larger on request. That covers process development, screening and clinical batches; if you need a volume beyond the standard sizes, ask us rather than assume.
- How do you separate full and empty AAV capsids?
- On an anion exchanger with a shallow salt or pH gradient. Full and empty capsids differ only in the charge contributed by the packaged genome, so the separation rests on a small difference and is correspondingly sensitive to gradient shape, pH control and the condition of the capsids arriving from earlier steps. It is the most demanding chromatographic step in an AAV process.
- What are the steps in AAV purification?
- Harvest from lysate, supernatant or both; nuclease treatment to digest host DNA; clarification by depth filtration; affinity capture on a capsid-binding ligand; separation of full from empty capsids by anion exchange; then polishing and concentration by tangential flow filtration into the formulation buffer.
- Why does capsid size matter for the chromatography medium?
- Because the particle and the pore are of comparable order: an AAV capsid is about 25 nm, and the pores of a chromatography bead are in the same size range rather than far larger. Once those two dimensions are close, the geometry of the medium becomes something to check against your own material instead of assuming. Our article on media for AAV polishing works through what that means in practice.
- Does AAV survive acidic affinity elution?
- Usually, but not for free. AAV capsids show conformational changes at low pH, so the eluate is normally neutralised immediately and the hold kept short. Where a capsid is particularly sensitive, the alternatives are the same mechanisms used to avoid acid elution of antibodies.
- Can MonoCore™ be used for lentiviral vectors?
- The anion exchange step, yes in principle — but lentiviral particles are larger, enveloped and much less stable than AAV, and we have not characterised the chemistry on them. We would want to run your material before saying anything more definite.
- What scale do you supply?
- 1.8 and 5.4 mL as standard, larger on request. The standard sizes suit process development, screening and clinical batches; for anything beyond them, tell us the volume you need and we will tell you what is possible.