Hollow fiber flow-field-flow fractionation (HF5)
Hollow fiber flow field-flow fractionation (HF5) is a valuable variation of the more frequently used AF4 technique for sizing and quantifying aggregates and particles in protein formulations.
HF5 separates the various species in a hollow fiber made of an ultrafiltration membrane. Just as for AF4, the underlying force to separate the species is a cross-flow. In HF5 however, the cross-flow is applied radially inside the hollow fiber. Similar to HP-SEC analysis, in HF5 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 fractionated species.
Advantages of the HF5 system over the classical AF4 set-up are a better resolution, less dilution effects due to low volume in the hollow fiber, and in some cases, a higher sensitivity. Currently, however, HF5 systems do not offer the same flexibility regarding channel dimensions, membrane material and membrane pore-size as AF4.
Because of its wide separation range from a few nanometers to micrometers, HF5 has been found appropriate for the analysis of colloidal systems, such as liposomes, nanoparticles, polymers and virus-like particles (see relevant publication). HF5 can be used in all stages of pharmaceutical development and manufacturing – from early research to late stage release testing.
HF5 offers some benefits over HP-SEC
HP-SEC still represents the current standard in the characterization of most biopharmaceuticals that contain fragments and/or small aggregates and oligomers. However, HF5 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 HF5 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. Further, it is possible to utilize formulation buffer as the mobile phase in HF5.
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D.J. Houde, A.S. Berkowitz, eds., Biophysical Characterization of Proteins in Developing Biopharmaceuticals, 1st ed., Newnes, 2014
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.
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