Comparative study of recent wide-pore materials of different stationary phase morphology, applied for the reversed-phase analysis of recombinant monoclonal antibodies

Various recent wide-pore reversed-phase stationary phases were studied for the analysis of intact monoclonal antibodies (mAbs) of 150 kDa and their fragments possessing sizes between 25 and 50 kDa. Different types of column technology were evaluated, namely, a prototype silica-based inorganic monolith containing mesopores of ～250 Å and macropores of ～?1.1 μm, a column packed with 3.6 μm wide-pore core-shell particles possessing a wide pore size distribution with an average around 200 Å and a column packed with fully porous 1.7 μm particles having pore size of ～300 Å. The performance of these wide-pore materials was compared with that of a poly(styrene–divinyl benzene) organic monolithic column, with a macropore size of approximately 1 μm but without mesopores (stagnant pores). A systematic investigation was carried out using model IgG1 and IgG2 mAbs, namely rituximab, panitumumab, and bevacizumab. Firstly, the recoveries of intact and reduced mAbs were compared on the two monolithic phases, and it appeared that adsorption was less pronounced on the organic monolith, probably due to the difference in chemistry (C18 versus phenyl) and the absence of mesopores (stagnant zones). Secondly, the kinetic performance was investigated in gradient elution mode for all columns. For this purpose, peak capacities per meter as well as peak capacities per time unit and per pressure unit (PPT) were calculated at various flow rates, to compare performance of columns with different dimensions. In terms of peak capacity per meter, the core-shell 3.6 μm and fully porous 1.7 μm columns outperformed the two monolithic phases, at a temperature of 60 °C. However, when considering the PPT values, the core-shell 3.6 μm column remained the best phase while the prototype silica-based monoliths became very interesting, mostly due to a very high permeability compared with the organic monolith. Therefore, these core-shell and silica-based monolith provided the fastest achievable separation. Finally, at the maximal working temperature of each column, the core-shell 3.6 μm column was far better than the other one, because it is the only one stable up to 90 °C. Lastly, the loading capacity was also measured on these four different phases. It appeared that the organic monolith was the less interesting and rapidly overloaded, due to the absence of mesopores. On the other hand, the loading capacity of prototype silica-based monolith was indeed reasonable.     

Comparison of various silica‐based monoliths for the analysis of large biomolecules†

In the present study, three types of silica‐based monoliths, i.e. the first and second generations of commercial silica monolithic columns and a wide‐pore prototype monolith were compared for the analysis of large biomolecules. These molecules possess molecular weights between 1 and 66 kDa. The gradient kinetic performance of the first‐generation monolith was lower than that of the second generation, for large biomolecules (>14 kDa) but very close with smaller ones (1.3–5.8 kDa). In contrast, the wide‐pore prototype column was particularly attractive with proteins larger than 19 kDa (higher peak capacity). Among these three columns, the selectivity and retention remained quite similar but a possible larger number of accessible and charged residual silanols was noticed on the wide‐pore prototype material, which led to unpredicted small changes in selectivity and slightly broader peaks than expected. The peak shapes attained with the addition of 0.1% formic acid in the mobile phase remained acceptable for MS coupling, particularly for biomolecules of less than 6 kDa. It was found that one of the major issues with all of these silica‐based monoliths is the possible poor recovery of large biomolecules (principally with monoclonal antibody fragments of more than 25 kDa).     

Suppression of radiative losses of surface polaritons on nanostructured thin metal films

The strong electromagnetic coupling between surface plasmon polariton modes on opposite interfaces of a finite thickness periodically nanostructured metal film has been studied. Surface polariton dispersion and associated electromagnetic field distributions have been analyzed. It was shown that at a frequency that corresponds to the crossing of film Bloch modes of different symmetries, the radiative losses of surface polaritons that are related to the polaritons' coupling to light during propagation on the structured surface are suppressed.     

Chlorkalkprüfung

Analytical and Bioanalytical Chemistry -     

Copper-mediated N- and O-arylations with arylboronic acids in a continuous flow microreactor: a new avenue for efficient scalability

A continuous flow procedure has been elaborated for the copper(II)-mediated N- and O-arylation of a range of compounds with arylboronic acids using a commercial microreactor setup. The compounds could be continuously generated in good yields paving the way for efficient scalability.     

Ueber die Reaction auf Alkohol mit einer L?sung von Molybd?ns?ure in concentrirter Schwefels?ure

Ohne Zusammenfassung     

Evaluation of standard and advanced preprocessing methods for the univariate analysis of blood serum 1H-NMR spectra

Proton nuclear magnetic resonance (¹H-NMR)-based metabolomics enables the high-resolution and high-throughput assessment of a broad spectrum of metabolites in biofluids. Despite the straightforward character of the experimental methodology, the analysis of spectral profiles is rather complex, particularly due to the requirement of numerous data preprocessing steps. Here, we evaluate how several of the most common preprocessing procedures affect the subsequent univariate analyses of blood serum spectra, with a particular focus on how the standard methods perform compared to more advanced examples. Carr–Purcell–Meiboom–Gill 1D ¹H spectra were obtained for 240 serum samples from healthy subjects of the Asklepios study. We studied the impact of different preprocessing steps—integral (standard method) and probabilistic quotient normalization; no, equidistant (standard), and adaptive-intelligent binning; mean (standard) and maximum bin intensity data summation—on the resonance intensities of three different types of metabolites: triglycerides, glucose, and creatinine. The effects were evaluated by correlating the differently preprocessed NMR data with the independently measured metabolite concentrations. The analyses revealed that the standard methods performed inferiorly and that a combination of probabilistic quotient normalization after adaptive-intelligent binning and maximum intensity variable definition yielded the best overall results (triglycerides, R = 0.98; glucose, R = 0.76; creatinine, R = 0.70). Therefore, at least in the case of serum metabolomics, these or equivalent methods should be preferred above the standard preprocessing methods, particularly for univariate analyses. Additional optimization of the normalization procedure might further improve the analyses.     

Selection of suitable operating conditions to minimize the gradient equilibration time in the separation of drugs by Ultra-High-Pressure Liquid Chromatography with volatile (mass spectrometry-compatible) buffers

Reversed phase gradient elution is the method of choice for pharmaceuticals analysis since it allows reducing the analysis time while improving both the quality of the separation and the detection limits. The current trends are towards faster separations which can be achieved thanks to equipments withstanding ultra-high pressures and/or high temperatures. Under such conditions, gradient separations can be carried out within a few minutes or even a few tens of seconds. A long equilibration time in addition to the gradient time can be therefore very detrimental. In this work, we investigated the extent to which the gradient equilibration time can be reduced and which parameters mainly affect the retention variability of ionizable compounds when using volatile buffers. We first found out an excellent repeatability between run-to-run experiments whatever the equilibration time and the operating conditions. We then pointed out the key operating parameters which allow achieving reproducible runs when varying the equilibration time between runs. With a view of reducing the equilibration time, the effects of various conditions were examined. The latter include the type of additive for mobile phase pH adjustment, the initial eluent composition, the type of stationary phase, the temperature and the flow-rate. Although much remains to be understood about the equilibration process, our study allows making progress in the knowledge of this phenomenon. Based on the present results, a beneficial effect of both temperature and flow-rate was highlighted and operating conditions leading to faster column equilibration are suggested.     

Aminoglycoside analysis in food of animal origin with a zwitterionic stationary phase and liquid chromatography–tandem mass spectrometry

In this study, fourteen highly polar aminoglycoside (AGs) antibiotics were selected. Various stationary phases were tested, including Obelisc R, ZIC-HILIC, BEH amide and aminopropyl. The nature of the stationary phase, mobile phase (water or buffer solutions and acetonitrile), pH (percentage of formic acid), gradient conditions and injection solvents were systematically studied as relevant parameters for tuning retention selectivity and detectability of AGs in liquid chromatography electrospray tandem mass spectrometry (LC–(ESI)–MS/MS). Only the two zwitterionic phases (Obelisc R and ZIC-HILIC) achieved a proper chromatographic separation considering interferences due to the crosstalk effect in low resolution mass spectrometers. The water/acetonitrile mobile phase containing 1% formic acid used with Obelisc R provided more sensitivity than the highly concentrated buffered mobile phases required for ZIC-HILIC. A solid phase extraction (SPE) clean-up procedure with polymeric weak cation exchange (WCX) cartridges was optimized for honey, milk and liver samples. Different brands of cartridges and elution solvents were tested, and the Taurus WCX offered the best recovery rate with a buffer elution at pH 3. The final optimized method was validated in these matrices according to Decision 2002/657/EC. A monitoring campaign for sixty honey, milk and liver samples was carried out at the Food Authority Control in Geneva. The concentration of dihydrostreptomycin (DSTP) found in one ovine liver exceeded the established maximum residue levels (MRLs) within the European and Swiss legislations but it was compliant taking into account the validation data.     

Robust UHPLC Separation Method Development for Multi-API Product Containing Amlodipine and Bisoprolol: The Impact of Column Selection

This paper describes a new and fast ultra-high pressure liquid chromatographic separation of amlodipine and bisoprolol and all their closely related compounds, for impurity profiling purposes. Computer-assisted method development was applied and the impact of several state-of-the-art stationary phase column chemistries (50 × 2.1 mm, sub-2 μm, and core–shell type materials) on the achievable selectivity and resolution was investigated. The work was performed according to quality by design principles using design of experiment with three experimental factors; namely the gradient time (t _G), temperature (T), and mobile phase pH. Thanks to modeling software, it was proved that the separation of all compounds was feasible on numerous column chemistries within <10 min, by proper adjustments of variables. It was also demonstrated that the reliability of predictions was good, as the predicted retention times and resolutions were in good agreement with the experimental ones. The final, optimized method separates 16 peaks related to amlodipine and bisoprolol within 7 min, ensuring baseline resolution between all peak-pairs.