Morphology and separation efficiency of low-aspect-ratio capillary ultrahigh pressure liquid chromatography columns.

We derive a quantitative relationship between the bed morphology and the chromatographic separation efficiency of capillary columns packed with sub-2 µm particles, covering capillary inner diameters from 10 to 75 µm. Our study focuses on wall effects and their impact on band broadening at increasing column-to-particle diameter (aspect) ratios. We approach these complex effects by a morphological analysis of reconstructed column segments composed of several thousand particles that were imaged by confocal laser scanning microscopy. Radial interparticle porosity profiles including wall effects are quantified through an integral porosity deviation, a scalar measure that proves to be a general descriptor of transcolumn porosity heterogeneity. It correlates with the associated transcolumn eddy dispersion, which dominates band broadening in the capillaries and is visualized in the plate height curves by a simple velocity-proportional term. Our comprehensive approach identifies the packing structure features that contribute to decreased efficiency as reflected, e.g., in subtle variations of the wall effect at different aspect ratios, or a particle size-segregation effect in larger-diameter columns as a result of an increased number of packing voids near the wall-bed interface.

State-of-the-art and emerging trends in analytical approaches to pharmaceutical-product commercialization.

The biopharmaceutical landscape continues to evolve rapidly, and associated modality complexity and the need to improve molecular understanding require concomitant advances in analytical approaches used to characterize and release the product. The Product Quality Attribute Assessment (PQAA) and Quality Target Product Profile (QTPP) frameworks help catalog and translate molecular understanding to process and product-design targets, thereby enabling reliable manufacturing of high-quality product. The analytical target profile forms the basis of identifying best-fit analytical methods for attribute measurement and continues to be successfully used to develop robust analytical methods for detailed product characterization as well as release and stability testing. Despite maturity across multiple testing platforms, advances continue to be made, several with the potential to alter testing paradigms. There is an increasing role for mass spectrometry beyond product characterization and into routine release testing as seen by the progress in multi-attribute methods and technologies, applications to aggregate measurement, the development of capillary zone electrophoresis (CZE) coupled with mass spectrometry (MS) and capillary isoelectric focusing (CIEF) with MS for measurement of glycans and charged species, respectively, and increased application to host cell protein measurement. Multitarget engaging multispecific modalities will drive advances in bioassay platforms and recent advances both in 1- and 2-D NMR approaches could make it the method of choice for characterizing higher-order structures. Additionally, rigorous understanding of raw material and container attributes is necessary to complement product understanding, and these collectively can enable robust supply of high-quality product to patients.

Chip-Based Capillary Zone Electrophoresis Mass Spectrometry for Rapid Resolution and Quantitation of Critical Quality Attributes in Protein Biotherapeutics.

The aspiration of the multi-attribute method (MAM) is to utilize a single mass spectrometry-based method that can measure multiple attributes simultaneously, thus enabling data-driven decisions more quickly and efficiently. However, challenges associated with identifying and quantitating critical quality attributes such as asparagine deamidation and isoaspartic acid using conventional ultrahigh-pressure liquid chromatography (UHPLC) coupled to mass spectrometry have necessitated long gradients to ensure sufficient separation for quantitation. Microfluidic chip-based capillary zone electrophoresis mass spectrometry (CZE-MS) shows potential to enable rapid charge-based separation of peptide mixtures, and this approach was evaluated using multipeptide mixtures of synthetic peptides as well as digested protein therapeutics. In these experiments, repeatability, linearity, and peak-to-peak resolution of several peptide families containing asparagine deamidation and/or isoaspartic acid were demonstrated. In addition, a comparison of peptide map results acquired with both UHPLC-MS and CZE-MS for two enzymatically digested biological therapeutics showed comparable sequence coverage and quantitation results between the two approaches. As MAM becomes increasingly utilized for analysis of biological therapeutics, MS instrument demand will rapidly increase, resulting in a bottleneck. A CZE-based separation shows potential to alleviate this bottleneck by drastically increasing MAM throughput while providing results comparable to those acquired using conventional UHPLC separations.

Separation and quantitation of eight isomers in a molecule with three stereogenic centers by normal phase liquid chromatography.

A normal phase liquid chromatography method was developed for the separation and detection of eight stereoisomers of the key intermediate, CORE + OMe, having three chiral centers. The stereochemistry of this intermediate dictates the stereochemistry of the active pharmaceutical ingredient generated by an additional six synthetic steps. Multiple columns and mobile phases were screened during the development based on a platform approach. The use of dichloromethane as mobile phase additive and adjustment of flow rate and column temperature contributed in achieving resolution of these eight stereoisomers. The separation and detection of these stereoisomers was achieved using a Chiralcel OD-H, 4.6 × 250 mm, 5 µm dp column with heptane: ethanol: dichloromethane in a ratio of 95:3:2 (v:v:v) as mobile phase at a flow rate of 0.7 mL/min. UV detection was carried out at 245 nm and the column temperature was maintained at 15 °C. The analytical method was phase appropriately validated. The limit of detection and limit of quantification were found to be 0.035 and 0.07 µg, respectively. The newly developed method has been implemented for routine utilization to monitor the chiral control during process development and used as the quality control method for chiral purity of the desired compound.

Recent advances in capillary ultrahigh pressure liquid chromatography.

In the twenty years since its initial demonstration, capillary ultrahigh pressure liquid chromatography (UHPLC) has proven to be one of most powerful separation techniques for the analysis of complex mixtures. This review focuses on the most recent advances made since 2010 towards increasing the performance of such separations. Improvements in capillary column preparation techniques that have led to columns with unprecedented performance are described. New stationary phases and phase supports that have been reported over the past decade are detailed, with a focus on their use in capillary formats. A discussion on the instrument developments that have been required to ensure that extra-column effects do not diminish the intrinsic efficiency of these columns during analysis is also included. Finally, the impact of these capillary UHPLC topics on the field of proteomics and ways in which capillary UHPLC may continue to be applied to the separation of complex samples are addressed.

1.1 µm superficially porous particles for liquid chromatography: part II: column packing and chromatographic performance.

The predicted advantages of superficially porous particles over totally porous particles are decreased eddy dispersion, longitudinal diffusion, and resistance to mass transfer contributions to the theoretical plate height. While sub-2 micron superficially porous particles are commercially available, further improvements in performance are predicted by decreasing the particle diameter and decreasing the porous layer thickness. 1.1 µm superficially porous particles with 187Å pores have been synthesized using a layer-by-layer method tuned for production of smaller diameter particles. Following synthesis, these particles were packed into 30 µm i.d. capillary columns and their chromatographic performance evaluated using electrochemical detection. Based on the initial studies, the column efficiency did not meet theory, but was similar to the commercially available products tested. It is believed that the column packing process plays a critical role in the sub-par column performance. To determine if column efficiency could be predicted by solvent-particle interactions, in-solution optical microscopy and sedimentation velocity of particles in various slurry solvents were investigated and compared to column performance. Aggregating slurry solvents, such as methanol were found to produce columns with increased efficiency. The hmin for a column packed with an acetone slurry and a methanol slurry at 3mg/mL were found to be 6.3 and 3.5, respectively. Increasing the slurry concentration to 25mg/mL further improved the efficiency, producing a column with an hmin of 2.6. These efficiency results were accurately predicted by in-solution optical microscopy.

1.1 µm superficially porous particles for liquid chromatography. Part I: synthesis and particle structure characterization.

Superficially porous particles are characterized by a non-porous particle core surrounded by a thin porous layer. Superficially porous particles have been shown to have chromatographic advantages over traditional totally porous particles by reducing the resistance to mass transfer and the eddy diffusion contributions to the theoretical plate height, particularly for biomolecule separations. Currently, 1.7 µm superficially porous particles are commercially available, but a further decrease in the particle diameter and reduction in the porous layer thickness has the potential to further improve the efficiency of the column packing material. In this study, the synthesis of smaller diameter superficially porous particles was investigated. As the particle diameter was decreased, however, synthesis parameters previously reported were rendered unsuitable due to particle agglomeration, non-uniform coating, and porous layer disintegration. Parameters such as colloidal silica size, drying process, and sintering temperature were investigated to improve the structural characteristics of smaller diameter superficially porous particles. Reported is a synthetic route for production of 1.1 µm superficially porous particles having a 0.1 µm porous layer. Based on the revised method, the particles produced have a surface area, pore diameter, and particle size distribution RSD of 52 m(2)/g, 71 Å, and 2.2%, respectively.