A multianalytical approach to investigate the effect of nanofiltration on plasma-derived factor IX clinical lots.

Plasma-derived proteins are a subset of relevant biotherapeutics also known as "well-characterized biologicals". They are enriched from plasma through several steps of physical and biochemical methodologies, reaching the regulatory accepted standards of safety, levels of impurities, activity and lot-to-lot consistency. Final products accepted for commercialization are submitted to tight analytical, functional and safety controls by a number of different approaches that fulfill the requirements of sensitivity and reliability. We report here the use of a multianalytical approach for the comparative evaluation of different lots of Factor IX isolated from plasma preparations and submitted or not to a step of nanofiltration. The approach include, among the other, proteomic techniques based on both MALDI-TOF and LC-MS Orbitrap mass spectrometry, circular dichroism for structural characterization, chromatographic and electrophoretic techniques, ELISA and functional assays based on clotting activity and binding to known anticoagulants. Comparative data obtained on two sets of nanofiltered and non-nanofiltered lots with different final activity show that the products have substantially overlapping profiles in terms of activity, contaminants, structural properties and protein content, suggesting that the proposed multianalytical approach is robust enough to be used for the routine validation of clinical lots.