This document summarizes a study that investigated using multimodal metal affinity chromatography to separate empty and full adeno-associated virus (AAV) capsids. The researchers found that empty capsids eluted first from the column, followed by full capsids, and then more empty capsids and debris. They evaluated the fractions using density gradient centrifugation and found that columns charged with different metal ions, including barium, calcium, magnesium, zinc, manganese, and ferric ions, gave similar separation results. Recovery of full serotype 9 capsids was 100% with complete removal of empty capsids. All metal ions also bound contaminants that required sodium hydroxide for removal, with ferric iron and manganese

1. Removal of empty capsids from
adeno-associated virus preparations
by multimodal metal affinity
chromatography
Pete Gagnon, Maja Leskovec, Sara Drmota Prebil, Rok
Žigon, Maja Štokelj, Andrej Raspor, Sebastijan Peljhan,
Ales Štrancar
Cite: Gagnon P., Maja L., Prebil S. D., Žigon R., Štokeli M., Raspor A., Sebastijan P., Štrancar A.(2021)
Removal of empty capsids from adeno-associated virus preparations by multimodal metal affinity
chromatography. Journal of chromatography A, 1649, 462210. https://doi.org/10.1016/j.chroma.2021.462210

2. Abstract
Separation of empty and full adeno-associated virus capsids by multimodal metal affinity
chromatography was investigated using a positively charged metal affinity ligand. A
subpopulation of empty capsids eluted first, followed by full capsids, and later by more empty
capsids and debris. Empty and full capsid composition of chromatography fractions was
evaluated by cesium chloride density gradient centrifugation followed by stratigraphic flow
analysis of the centrifuge tube contents, monitored by intrinsic fluorescence. Columns charged
with barium, calcium, magnesium, zinc, manganese, and ferric ions gave similar results with
respect to capsid separation. Charging with cupric ions maintained resolution between early-
eluting empty capsids and full capsids but caused them to elute at lower conductivity. Empty and
full capsids were fractionated with Tris-borate gradients, sodium chloride gradients, and
magnesium chloride gradients. Recovery of full serotype 9 capsids was 100% with complete
elimination of empty capsids. All metal ions bound contaminant subsets that required sodium
hydroxide for removal. Columns charged with ferric iron and manganese bound more
contaminants than all other metals. Columns charged with calcium, magnesium, barium, and
copper bound the least. Contaminant binding on zinc-charged columns was intermediate
between the two groups.

3. Fig. 8. Multimodal metal affinity chromatography fractionation of AAV9 eluted with a magnesium chloride
gradient at pH 9.0 and density gradient analysis of selected column fractions. A: Elution profile from PrimaT
charged with magnesium, B: density gradient of the sample before metal analysis fractionation, C: density
gradient analysis of the full capsid peak after metal affinity fractionation. IFL refers to intrinsic fluorescence.
Conductivity (mS/cm) is a surrogate indicator for cesium chloride density. Profiles B and C share the same
conductivity scale. HD refers to populations of higher density than the full capsid population. LD refers to the
populations of lower density than the empty capsid population. The dotted vertical lines mark the leading
boundary of the full AAV8 peak.