Improvement of Empty|Full Resolution By Implementation of Novel Eluents Using PATfix

In every adeno associated virus (AAV) downstream process the key steps are the enrichment of full capsids and the in-process empty/full analytics. A common approach for enrichment of full capsids is liquid chromatography using ion exchange or multimodal chemistries, which based on particles’ surface charge differences, enables separation of full (F) capsids and product related impurities. Removal of impurities is an important step as they may pose a serious health risk, as well as deteriorate the economics of the production process. A PATfix® triple detector set in combination with an CIMac™ AAVEmpty/Full analytical column by Sartorius BIA Separations was used to determine E/F ratio. The column is a strong anion exchanger which exploits capsid charge differences for separation when employing an ascending salt gradient. Usually, NaCl with the addition of 2 mM magnesium is used, as the presence of magnesium has been shown to improve the peak shape and the removal of empty capsids. According to the Hofmeister series salts have different impact on non-covalent interactions among capsid themselves and between columns and capsids. For example, antichaotrope salts were shown to improve the empty/full capsid ratio, possibly by inducing the hydrophobic interactions between them. Quaternary alkyl ammonium salts (QAAS) are known to have antichaotropic properties and they differ in the substituents. Wang et al. reported that QAASs produced a better empty/full separation than NaCl. This poster presents a screening of different eluents for empty/full analytics. Beside NaCl as a reference salt, two QAAS were chosen: tetramethylammonium chloride- TMAC and a possible nonhazardous alternative choline chloride, which is bifunctional and contains both a quaternary ammonium and hydroxyl groups. The influence of magnesium on E/F separation was also tested.