Utilization of AF4 for characterizing complex nanomaterial drug products: Reexamining sample recovery and its impact...

Asymmetrical flow field-flow fractionation (AF4) with multi-detection has continued to gain wider acceptance for characterizing complex drug products. An important quality attribute for these products is the measurement of the particle size distribution (PSD). Current limitations of established procedures (e.g., dynamic light scattering) for accurately determining PSD can be overcome by AF4. However, while gaining acceptance this technique has not been fully adopted within the pharmaceutical industry. A technical understanding of fundamental operational factors is necessary for the successful application of utilizing any emerging technology. For example, recovery (R% = A_S/A_D*100_, where A_S and A_D are the peak areas from the concentration detector with and without the crossflow field, respectively) is one factor that is used to assess the robustness during AF4 method development, but currently little is known about the interplay between analyte recovery and PSD. This work highlights factors that impact calculated AF4 recovery, and how differences in analyte and absolute recovery ultimately influence the PSD of nanoparticle size standards and complex drug product formulations such as emulsions and liposomes. Factors like ionic strength, buffer composition, and analyte chemistries, which are the most common factors associated with changes to R% in AF4, contributed to changes in A_S. While A_D is not typically examined in detail, the selection of the concentration detector (UV or dRI) along with their instrumental parameters (e.g., wavelength, attenuation value, linear range) and sample preparation was shown to under- or over-estimate A_D thus changing R%. Examining both components of R% and their contributions to analyte and absolute recovery show that decreases in analyte recovery may not be exclusively due to sample loss but could be influenced by changes in analyte-membrane interactions or analyte instability. Because of this, four relationships between recovery and PSD were defined. While R% is used as a tool for assessing AF4 methodology, the factors investigated through this work warrant further considerations when establishing an appropriate R% threshold.