Solvent extraction is a very powerful purification step in the preparation of VAQTAா, a highly purified, inactivated hepatitis A vaccine. Extraction of an aqueous product-containing protein solution with chloroform through vigorous shaking causes irreversible denaturation of contaminant proteins at the interface. However, the hepatitis A virus (HAV) remains viable and soluble in the aqueous phase. Because three phases (air, aqueous, and organic) are involved, and the mixing is carried out in individual bottles, there is very little theory available to characterize this process, so it must be studied experimentally. This extraction step was characterized by following the removal of a specific impurity from the aqueous phase as a representative marker for the degree of protein precipitation. These experiments led to the identification and optimization of the important variables controlling the extraction step. They were found to be mixing time and size of vessel, with longer mixing times resulting in higher purity and larger bottle size leading to faster kinetics of impurity removal. These parameters are most likely related to solvent/aqueous interfacial area and the resulting shear due to shaking. We conclude that, to scale up this type of mixing, the kinetics of impurity removal need to be determined experimentally for the systems and equipment under consideration.