Hollow fiber flow field-flow fractionation and size-exclusion chromatography with MALS detection: A complementary approach in biopharmaceutical industry.

Monoclonal antibodies (mAbs) are promising reagents both for the manufacture of drug substances and for their employment as a drug themselves, but to be approved for utilization, according to FDA recommendations and WHO guidelines, they have to undergo verifications regarding their purity, stability and percentage of aggregates. Moreover, stability tests of lots have to be performed in order to verify molecular size distribution over time and lot-to-lot consistency. Recent works in literature have highlighted the need for suitable, sensitive and reliable complementary analytical techniques for the characterization of mAbs and quantification of aggregates. Size-exclusion chromatography (SEC) is the reference technique in the biopharmaceutical industry for its robustness, high performance and simple use; however it presents some limitations especially toward the separation and detection of aggregates with high molecular weight. On the other hand, flow field-flow fractionation (F4) in its miniaturized version (hollow fiber flow field-flow fractionation, HF5) shows comparable performances with interesting additional advantages: a broad size range, gentle separation mechanism with low dilution factor and higher sensitivity. To propose HF5 as a complementary technique for evaluating aggregates' content in mAbs samples, a comparative study of both SEC and HF5 performances has been made. In this work, SEC and HF5 were coupled with UV and multi-angle light scattering detection and employed first in separating standard samples of proteins mixture used as a sample model. Then, a screening of mobile phases and an evaluation of separation performances was performed on a therapeutic mAbs formulation, demonstrating the complementarities between SEC and HF5 and their possible use as a separative platform approach for the characterization and quality control of protein drugs.

Hollow-fiber flow field-flow fractionation with multi-angle laser scattering detection for aggregation studies of therapeutic proteins.

The rapid development of protein-based pharmaceuticals highlights the need for robust analytical methods to ensure their quality and stability. Among proteins used in pharmaceutical applications, an important and ever increasing role is represented by monoclonal antibodies and large proteins, which are often modified to enhance their activity or stability when used as drugs. The bioactivity and the stability of those proteins are closely related to the maintenance of their complex structure, which however are influenced by many external factors that can cause degradation and/or aggregation. The presence of aggregates in these drugs could reduce their bioactivity and bioavailability, and induce immunogenicity. The choice of the proper analytical method for the analysis of aggregates is fundamental to understand their (size) dimensional range, their amount, and if they are present in the sample as generated by an aggregation or as an artifact due to the method itself. Size exclusion chromatography is one of the most important techniques for the quality control of pharmaceutical proteins; however, its application is limited to relatively low molar mass aggregates. Among the techniques for the size characterization of proteins, field-flow fractionation (FFF) represents a competitive choice because of its soft mechanism due to the absence of a stationary phase and application in a broader size range, from nanometer- to micrometer-sized analytes. In this paper, the microcolumn variant of FFF, the hollow-fiber flow FFF, was online coupled with multi-angle light scattering, and a method for the characterization of aggregates with high reproducibility and low limit of detection was demonstrated employing an avidin derivate as sample model.

Gravitational field-flow fractionation integrated with chemiluminescence detection for a self-standing point-of-care compact device in bioanalysis.

A "Point-Of-Care-Testing" (POCT) system relies on portable and simply operated self-standing analytical devices. To fulfill diagnostic requirements, the POCT system should provide highly sensitive simultaneous detection of several biomarkers of the pathology of interest (multiplexing) in a short assay time. One of the main unsolved issues in POCT device development is the integration of pre-analytical sample preparation procedures in the miniaturized device. In this work, an integrated POCT system based on gravitational field-flow fractionation (GrFFF) and chemiluminescence (CL) detection is presented for the on-line sample pre-analytical treatment and/or clean-up and analysis of biological fluids. As a proof of principle for the new GrFFF-CL POCT system, the automatic on-line analysis of plasma alkaline phosphatase activity, a biomarker of obstructive liver diseases and bone disorders, starting from whole blood samples was developed. The GrFFF-CL POCT system was able to give quantitative results on blood samples from control and patients with low sample volume (0.5 µL) and reagent consumption, short analysis time (10 minutes), high reproducibility and with a linear range of 50-1400 IU L(-1). The system can be easily applied to on-line prepare plasma from whole blood for other clinical biomarkers and for other assay formats, based on immunoassay or DNA hybridization.

A new method for immunoassays using field-flow fractionation with on-line, continuous chemiluminescence detection.

Chemiluminescence detection has already been combined with different separation techniques such as HPLC and capillary electrophoresis. In this work, it was applied to gravitational field-flow fractionation, a low-cost, flow-assisted separation technique for micronsized particles suited to further on-line detection of the separated analytes. Horseradish peroxidase was used as model sample, either free in solution or immobilized onto micronsized, polystyrene beads. The chemiluminescent substrates were added directly into the mobile phase, and the continuous, steady-state chemiluminescence generated during elution was detected on-line by either a flow-through luminometer or a CCD camera. Ultra-low detection limits, two orders of magnitude lower than those achievable with spectrophotometric detection, were found. The possibility to fully separate and quantitate free and bead-immobilized enzymes is reported, as a step towards the development of multianalyte, ultra-sensitive, micronsized beads-based flow-assisted immunoassays.