A new UltraScan module for the characterization and quantification of analytical buoyant density equilibrium experiments to determine AAV capsid loading.

Abstract

A method for characterizing and quantifying peaks formed in an analytical buoyant density equilibrium (ABDE) experiment is presented. An algorithm is derived to calculate the concentration of the density forming gradient material at every point in the cell, provided the rotor speed, temperature, meniscus position, bottom of the cell position, and the loading concentration, molar mass, and partial specific volume of the density gradient-forming material are known. In addition, a new peak fitting algorithm has been developed which allows the user to automatically quantify the peaks formed in terms of density, apparent partial specific volume, and relative abundance. The method is suitable for both ionic and non-ionic density forming materials and can be used with data generated from the UV optical system as well as the AVIV fluorescence optical system. These methods have been programmed in a new UltraScan-III module (us_abde). Examples are shown that demonstrate the application of the new module to adeno-associated viral vector preparations and proteins.