Asymmetrical flow field-flow fractionation
(AF4, A4F, AFFFF)
As a leading service provider in biopharmaceutical aggregation and particle characterization Coriolis Pharma offers asymmetrical flow field-flow fractionation (AF4) contract laboratory services for aggregate content and molecular weight distribution as an orthogonal method to size exclusion chromatography or analytical ultracentrifugation.
Asymmetrical flow field-flow fractionation is a well-established method for sizing and quantifying aggregates and particles, since particles spanning from several nanometers to the micrometer range can be separated. Because of the wide separation range, AF4 has also been found appropriate for the analysis of colloidal systems such as liposomes, nanoparticles, polymers or virus-like particles (see relevant publication). At Coriolis, AF4 can be offered in full GMP compliance.
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-link) and analytical ultracentrifugation.
In contrast to HP-SEC, no stationary phase is required, thus reducing the pressure in the system, as well as shear forces and high surface area which might lead to loss of protein due to adsorption. 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. Across the resulting parabolic profile the different components of the sample distribute according to their diffusion coefficients and by that to their hydrodynamic radii. The surface area of the ultrafiltration membrane utilized during field-flow fractionation is lower as compared to a HP-SEC column, which reduces interactions of the analytes with the solid phase. Therefore, the use of AF4 is advantageous for the characterization of biopharmaceuticals that are prone to changes due to experimental conditions in HP SEC, e.g., loss due to adsorption and samples that contain high molecular weight species. Besides, the method is highly flexible with respect to the mobile phase composition (e.g., the ionic strength of the buffer) and might even enable the use of formulation buffer as mobile phase.
Similar to HP-SEC analysis, UV, refractive index and fluorescence detectors are usually used as concentration determining/quantifying detection systems in AF4 analysis. All three detection set-ups, or any combination thereof, are available for R&D as well as GMP analysis at Coriolis. Additionally, multi-angle laser light scattering detection is used to determine the molecular weight and size of biopharmaceuticals and subvisible particles.
Method development for AF4 often requires more effort as compared to HP-SEC because of the higher complexity of the method and the higher number of parameters that can be optimized. Nonetheless good and robust separation results can be achieved by a systematic method development approach.
AF4 analysis can cover a broad variety of biopharmaceutical applications. It is often used in the context of forced degradation studies (see relevant publication) and real-time stability studies, since those often imply the formation of relatively large amounts of aggregates in the size range covered by AF4 (see relevant publication). Furthermore, as an orthogonal method to HP-SEC AF4 is of increasing importance for batch release with respect to the cross-validation of size-exclusion chromatography methods to characterize biopharmaceutical formulations.
At Coriolis, numerous AF4 methods for peptides/proteins, virus-like particles, liposomes and polymers have been successfully developed.