David Clicq

Kinetic plot equations for evaluating the real performance of the combined use of high temperature and ultra-high pressure in liquid chromatography. Application to commercial instruments and 2.1 and 1 mm I.D. columns.

The use of ultra-high pressure liquid chromatography (UHPLC) with pressures up to 1000 bar and columns packed with sub-2-microm particles combined with high-temperature mobile phase conditions (up to 90 degrees C) is assessed according to the current available instrumentation via constrained kinetic plot equations. It is shown that the gain in separation speed, theoretically expected from high-temperature UHPLC (HT-UHPLC), is significantly reduced when taking into account the existing instrumental constraints (extra-column band broadening, flow-rate and column length limitations). This study also shows that significant improvements could be expected on the current commercial instruments by increasing the flow-rate limit and/or using packing columns with particle size in the range 2.5-3.5 microm instead of the current sub-2 microm. These particles should obviously withstand very high pressure.

Measurements of diffusion coefficients in 1-D micro- and nanochannels using shear-driven flows

The present paper describes a method for measuring the molecular diffusion coefficient of fluorescent molecules in microfluidic systems. The proposed static shear-driven flow method allows one to perform diffusion measurements in a fast and accurate manner. The method also allows one to work in very thin (i.e. submicron) channels, hence allowing the investigation of diffusion in highly confined spaces. In the deepest investigated channels, the obtained results were comparable to the existing literature values, but when the channel size dropped below the micrometer range, a significant decrease (more than 30%) in molecular diffusivity was observed. The reduction of the diffusivity was most significant for the largest considered molecules (ssDNA oligomers with a size ranging between 25 to 100 bases), but the decrease was also observed for smaller tracer molecules (FITC). This decrease can be attributed to the interactions of the analyte molecules with the channel walls, which can no longer be neglected when the depth of the channel reaches a critical value. The change in diffusivity seems to become more explicit as the molecular weight of the analytes increases.

Enhancement of DNA micro-array analysis using a shear-driven micro-channel flow system

A very simple micro-channel flow system is used to investigate the potential gain in hybridization rate stemming from the induction of a convective flow past the surface of a DNA micro-array. Reporting on a series of preliminary experiments wherein a two-dimensional convective flow is created past the surface of a conventional micro-array slide, the analysis time could be brought down from overnight waiting (16 h) to some 10 to 30 min. The experiments open the road towards the development of novel, convection-driven hybridization systems yielding shorter analysis times, and/or lower detection limits.

On-line near infrared spectroscopy as a Process Analytical Technology (PAT) tool to control an industrial seeded API crystallization

The final step of an active pharmaceutical ingredient (API) manufacturing synthesis process consists of a crystallization during which the API and residual solvent contents have to be quantified precisely in order to reach a predefined seeding point. A feasibility study was conducted to demonstrate the suitability of on-line NIR spectroscopy to control this step in line with new version of the European Medicines Agency (EMA) guideline [1]. A quantitative method was developed at laboratory scale using statistical design of experiments (DOE) and multivariate data analysis such as principal component analysis (PCA) and partial least squares (PLS) regression. NIR models were built to quantify the API in the range of 9-12% (w/w) and to quantify the residual methanol in the range of 0-3% (w/w). To improve the predictive ability of the models, the development procedure encompassed: outliers elimination, optimum model rank definition, spectral range and spectral pre-treatment selection. Conventional criteria such as, number of PLS factors, R(2), root mean square errors of calibration, cross-validation and prediction (RMSEC, RMSECV, RMSEP) enabled the selection of three model candidates. These models were tested in the industrial pilot plant during three technical campaigns. Results of the most suitable models were evaluated against to the chromatographic reference methods. Maximum relative bias of 2.88% was obtained about API target content. Absolute bias of 0.01 and 0.02% (w/w) respectively were achieved at methanol content levels of 0.10 and 0.13% (w/w). The repeatability was assessed as sufficient for the on-line monitoring of the 2 analytes. The present feasibility study confirmed the possibility to use on-line NIR spectroscopy as a PAT tool to monitor in real-time both the API and the residual methanol contents, in order to control the seeding of an API crystallization at industrial scale. Furthermore, the successful scale-up of the method proved its capability to be implemented in the manufacturing plant with the launch of the new API process.

Sub-second liquid chromatographic separations by means of shear-driven chromatography.

Utilizing the concept of shear-driven chromatography, we have been able to realize reversed-phase LC separations in flat rectangular nano-channels coated with a C8 monolayer and being as thin as 100 nm. At this scale, the separation kinetics are strongly enhanced, as is witnessed by the extremely short time (< 0.1 s) needed to separate a mixture of coumarin dyes. The observed plate numbers are still relatively small, because the experiments were conducted in ultra-short columns (< or = 1 mm) and under injection band width-limiting conditions.

Experimental demonstration of the possibility to perform shear-driven chromatographic separations in micro-channels

The possibility to perform shear-driven chromatographic separations in micro-channels is demonstrated, using a novel laser-jet printed microfluidic channel system. The obtained theoretical plate numbers are in fair agreement with the theoretical calculations. Theoretical extrapolations of the separation speeds and detection limits which can be achieved when further miniaturizing the current system are presented as well.

Shear-flow-based chromatographic separations as an alternative to pressure-driven liquid chromatography.

It is only by developing specially designed injection and detection systems that shear-driven chromatography can become a viable alternative to HPLC. In the present paper, a dedicated zero dead-volume injection procedure is presented with which sample volumes can be injected reproducibly in the required picoliter range. In addition, a transversal detection groove system is designed which should allow to perform on-line UV-VIS absorption measurements with path lengths in the millimeter range, with an acceptable theoretical plate loss (only 20% in a 5 cm long channel) and acting as a nearly perfect wave guide.

A Process Analytical Technology (PAT) approach to control a new API manufacturing process: Development, validation and implementation

Pharmaceutical companies are progressively adopting and introducing Process Analytical Technology (PAT) and Quality-by-Design (QbD) concepts promoted by the regulatory agencies, aiming the building of the quality directly into the product by combining thorough scientific understanding and quality risk management. An analytical method based on near infrared (NIR) spectroscopy was developed as a PAT tool to control on-line an API (active pharmaceutical ingredient) manufacturing crystallization step during which the API and residual solvent contents need to be precisely determined to reach the predefined seeding point. An original methodology based on the QbD principles was designed to conduct the development and validation of the NIR method and to ensure that it is fitted for its intended use. On this basis, Partial least squares (PLS) models were developed and optimized using chemometrics methods. The method was fully validated according to the ICH Q2(R1) guideline and using the accuracy profile approach. The dosing ranges were evaluated to 9.0-12.0% w/w for the API and 0.18-1.50% w/w for the residual methanol. As by nature the variability of the sampling method and the reference method are included in the variability obtained for the NIR method during the validation phase, a real-time process monitoring exercise was performed to prove its fit for purpose. The implementation of this in-process control (IPC) method on the industrial plant from the launch of the new API synthesis process will enable automatic control of the final crystallization step in order to ensure a predefined quality level of the API. In addition, several valuable benefits are expected including reduction of the process time, suppression of a rather difficult sampling and tedious off-line analyses.

Predictive elution window stretching and shifting as a generic search strategy for automated method development for liquid chromatography.

We report on the possibilities of a new method development (MD) algorithm that searches the chromatographic parameter space by systematically shifting and stretching the elution window over different parts of the time-axis. In this way, the search automatically focuses on the most promising areas of the solution space. Since only the retention properties of the first and last eluting compounds of the sample need to be (approximately) known, the algorithm can be directly applied to samples with unknown composition, and the proposed solutions are not sensitive to any modeling errors. The search efficiency of the algorithm has been evaluated on an extensive set of random-generated in silico samples covering a broad range of different retention properties. Compared to a pure grid-based search, the algorithm could reduce the number of missed components by 50% and more. The algorithm has also been applied to solve three different real-world separation problems from the pharmaceutical industry. All problems could be successfully solved in a very short time (order of 12 h of instrument time).

A variable column length strategy to expedite method development.

A variable length method development (or VL-MD)strategy, exploiting the potential of an automatic column coupling system, is proposed and has been applied to a number of different pharmaceutical and environmental samples with a varying degree of complexity. The proposed strategy consistently produced separation methods that had at least an equally good critical pair resolution and an equally short run time to those of methods produced using commercially available MD assistance software. In some cases, the VL-MD strategy allowed the MD time to be drastically shortened from >30 h to an overnight run of only 12 h. The developed strategy has the potential to become fully automated provided that reliable chromatogram read-out software becomes available. The advantage of combining different stationary phase types to improve the available selectivity and the integration into the general VL-MD strategy was also demonstrated.