Asymmetrical flow field-flow fractionation (AF4)
Asymmetrical flow field-flow fractionation (AF4) is a well-established and highly versatile technique for sizing and quantifying aggregates and particles in protein formulations.
The AF4 separates the various species in a channel that consists of a non-permeable upper wall made of Plexiglas and a semipermeable accumulation wall at the bottom made of an ultrafiltration membrane and a solid frit. In AF4, the underlying force to separate the species, the so called cross-flow, is a physical field directed perpendicular to the laminar flow of the mobile phase. Similar to HP-SEC analysis, in AF4 analysis UV, refractive index and fluorescence detectors are usually used as concentration determining/quantifying detection systems. Additionally, multi-angle laser light scattering (MALLS) detection is used to determine the molecular weight and size of separated species.
Because of its wide separation range from a few nanometers to several micrometers, AF4 has been found appropriate for the analysis of colloidal systems, such as liposomes, nanoparticles, polymers and virus-like particles.
AF4 can be used in all stages of pharmaceutical development and manufacturing – from early research to late stage release testing under GMP, serving as a valuable tool for the orthogonal cross validation of high-performance size-exclusion chromatography [HP-SEC] and analytical ultracentrifugation [AUC]. AF4 is often used in the context of forced degradation studies and real-time stability studies, since those often imply the formation of relatively large amounts of aggregates in the size range covered by AF4.
HP-SEC still represents the current standard in the characterization of most biopharmaceuticals that contain fragments and/or small aggregates and oligomers. However, AF4 can offer benefits for samples that are sensitive towards shear forces, interfaces or extreme buffer condition (such as high salt concentrations typically required by HP-SEC). The lower surface area of an AF4 membrane compared to a column limits interaction between the analytes and solid phase. Also, the lack of a highly packed solid phase enables a lower system pressure, reduces shear forces and increases the upper size limit as compared to HP-SEC. Moreover, it is possible to utilize the formulation buffer as the mobile phase in AF4.
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S. Zölls, R. Tantipolphan, M. Wiggenhorn, G. Winter, W. Jiskoot, W. Friess, A. Hawe, Particles in therapeutic protein formulations, Part 1: overview of analytical methods., J. Pharm. Sci. 101  914–35. doi:10.1002/jps.23001.