Adaptive Modelling of Mutated FMO3 Enzyme Could Unveil Unexplored Scenarios Linking Variant Haplotypes to TMAU Phenotypes

Background: Trimethylaminuria (TMAU) is a rare genetic disease characterized by the accumulation of trimethylamine (TMA) and its subsequent excretion trough main body fluids, determining the characteristic fish odour in affected patients. We realized an experimental study to investigate the role of several coding variants in the causative gene FMO3, that were only considered as polymorphic or benign, even if the available literature on them did not functionally explain their ineffectiveness on the encoded enzyme. Methods: Mutational analysis of 26 TMAU patients was realized by Sanger sequencing. Detected variants were, subsequently, deeply statistically and in silico characterized to determine their possible effects on the enzyme activity. To achieve this goal, a docking prediction for TMA/FMO3 and an unbinding pathway study were performed. Finally, a TMAO/TMA urine quantification by 1H-NMR spectroscopy was performed to support modelling results. Results: The FMO3 screening of all patients highlighted the presence of 17 variants distributed in 26 different haplotypes. Both non-sense and missense considered variants might impair the enzymatic kinetics of FMO3, probably reducing the interaction time between the protein catalytic site and TMA, or losing the wild-type binding site. Conclusions: Even if further functional assays will confirm our predictive results, considering the possible role of FMO3 variants with still uncertain effects, might be a relevant step towards the detection of novel scenarios in TMAU etiopathogenesis.     

High-throughput capillary electrophoresis analysis of biopharmaceuticals utilizing sequential injections

The complexity of biotherapeutic products implies an ever-increasing list of product quality attributes that need to be monitored and characterized. In addition, the growing interest in implementing process analytical technology in biopharmaceutical production has further increased the testing burden, together with the need for rapid testing that can facilitate real-time or near-real-time decision-making. Capillary electrophoresis (CE) has made a place in biopharmaceutical analysis but is regarded as a low-throughput method, with the instrument dead time constituting more than 80% of the total time of analysis. In this study, the dead time of CE was utilized to analyse 3 mAb samples in a single-CE run. This approach resulted in an up to 77% reduction in the total analysis time and increased the productivity by up to 300%, compared to traditional single CE-ultraviolet runs, without compromising resolution or relative peak areas. Additionally, good method reproducibility was observed. The compatibility of the method has been demonstrated with protein A eluate and cation exchange chromatography fractions. We, thus, propose that sequential injections can be applied for fast and robust CE analysis of biopharmaceuticals.     

Rapid multi-attribute characterization of intact bispecific antibodies by a microfluidic chip-based integrated icIEF-MS technology

Rapid, direct identification and quantitation of protein charge variants, and assessment of critical quality attributes with high sensitivity are important drivers required to accelerate the development of biotherapeutics. We describe the use of an enhanced microfluidic chip-based integrated imaged capillary isoelectric focusing-mass spectrometry (icIEF-MS) technology to assess multiple quality attributes of intact antibodies in a single run. Results demonstrate comprehensive detection of multiple charge variants of an aglycosylated knob-into-hole bispecific antibody. Upfront, on-chip separation by icIEF coupled to MS provides the orthogonal separation required to resolve and identify acidic posttranslational modifications including difficult-to-detect deamidation and glycation events at the intact protein level. In addition, on-chip UV detection enables pI determination and relative quantitation of charge isoforms. Six charge variant peaks were resolved by icIEF, mobilized toward the on-chip electrospray tip and directly identified by in-line icIEF-MS using a connected quadrupole time-of-flight mass spectrometer. In addition to acidic charge variants, basic variants were identified as C-terminal lysine, N-terminal cyclization, proline amidation, and the combination of modifications (not typically identified by other intact methods), including lysine and one or two hexose additions. Nonspecific chain cleavages were also resolved, along with their acidic charge variants, demonstrating highly sensitive and comprehensive intact antibody multi-attribute characterization within a 15-min run time.