Search

A simple and versatile continuous air-segmented flow sensor using immobilized luciferase was designed as a general miniaturized platform based on sensitive biochemiluminescence detection. The device uses miniaturized microperistaltic pumps to deliver flows and compact sensitive light imaging detectors based on BI-CMOS (smartphone camera) or CCD technology. The low-cost components and power supply make it suitable as out-lab device at point of need to monitor kinetic-related processes or ex vivo dynamic events. A nylon6 flat spiral carrying immobilized luciferase was placed in front of the detector in lensless mode using a fiber optic tapered faceplate. ATP was measured in samples collected by microdialysis from rat brain with detecting levels as low as 0.4 fmoles. The same immobilized luciferase was also used for the evaluation of bile salt hydrolase (BSH) activity in intestinal microbiota. An aminoluciferin was conjugatated with chenodeoxycholic acid forming the amide derivative aLuc-CDCA. The hydrolysis of the aLuc-CDCA probe by BSH releases free uncaged aminoluciferin which is the active substrate for luciferase leading to light emission. This method can detect as low as 0.5 mM of aLuc-CDCA, so it can be used on real faecal human samples to study BSH activity and its modulation by diseases and drugs.

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. © 2014 Elsevier B.V.