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Improving separation of empty and full AAV capsids with a new multimodal column chemistry: CIMac EF beta

Separation of empty and full AAV capsids is important analytically as a means of monitoring the effects of different transfection strategies, cell culture conditions, lysis methods, sample preparation and purification methods. It is at least as important on a preparative level because it offers the possibility of removing empty capsids without ultracentrifugation. This poster introduces a new column for performing separation of empty and full capsids.

CIMac EF-beta employs a new ion exchange hydrogen bonding multimodal ligand that provides a new orthogonal option for separation of empty and full capsids. Gross selectivity is similar to strong (QA) anion exchangers eluted with salt gradients, but it generally provides better resolution. Its distinct separation mechanism is documented by the fact that it provides its best results when eluted with increasing pH gradients. This is directly opposite to QA exchangers where increasing pH causes capsids to bind more strongly.

CIMac EF-beta can also be eluted with salt gradients and often provides better resolution than QA, but its best resolution is obtained with pH gradients. Separation of a free capsid protein (CP) occurs only with the pH elution format. CIMac EF-beta can be used instead of classical anion exchange, for example following capture by cation exchange chromatography or affinity. Its distinct separation mechanism also makes it possible to perform orthogonal separations in which it is combined with QA.