Two-dimensional reversed phase-normal phase liquid chromatography for simultaneous achiral-chiral analysis to support high-throughput experimentation.

Typical chromatographic analysis of chiral compounds requires the use of achiral methods to evaluate impurities or related substances along with separate methods to evaluate chiral purity. The use of two-dimensional liquid chromatography (2D-LC) to support simultaneous achiral-chiral analysis has become increasingly advantageous in the field of high-throughput experimentation where low reaction yields or side reactions can lead to challenging direct chiral analysis. Advancements in multi-dimensional chromatography have led to the development of robust 2D-LC instrumentation with reversed phase solvent systems (RPLC-RPLC) enabling this simultaneous analysis, eliminating the need to purify crude reaction mixtures to determine stereoselectivity. However, when chiral RPLC cannot separate a chiral impurity from the desired product, there are few viable commercial options. The coupling of NPLC to RPLC (RPLC-NPLC) continues to remain elusive due to solvent immiscibility between the two solvent systems. This solvent incompatibility leads to lack of retention, band broadening, poor resolution, poor peak shapes, and baseline issues in the second dimension. A study was conducted to understand the effect of various water-containing injections on NPLC and applied to the development of robust RPLC-NPLC methods. Following thoughtful consideration and modifications to the design of a 2D-LC system in regards to mobile phase selection, sample loop sizing, targeted mixing, and solvent compatibility, proof of concept has been demonstrated with the development of reproducible RPLC-NPLC 2D-LC methods to perform simultaneous achiral-chiral analysis. Second dimension NPLC method performance proved comparable to corresponding 1D-NPLC methods with excellent percent difference in enantiomeric excess results ≤ 1.09% and adequate limits of quantitation down to 0.0025 mg/mL for injection volumes of 2 µL, or 5 ng on-column.

Resolving Solvent Incompatibility in Two-Dimensional Liquid Chromatography with In-Line Mixing Modulation.

Two-dimensional liquid chromatography (2D-LC) is a powerful technique used to characterize complex samples such as synthetic polymers, biomacromolecules, and hybrid modalities (conjugates, oligonucleotides, nanoparticles, etc., which fall between traditional small molecules and large molecules). Characterizing such molecules often requires a highly orthogonal 2D-LC workflow to resolve structurally similar impurities. However, it remains a challenge to achieve truly orthogonal 2D-LC coupling due to incompatibility of the chromatographic conditions used in each dimension. In this work, we present a facile strategy of connecting an in-line mixer, in-line mixing modulation (ILMM), to realize challenging 2D-LC workflows: (1) coupling gel permeation chromatography (GPC) with reversed-phase liquid chromatography (RPLC) for hydrophobic oligomer analysis and (2) coupling ion-pair reversed-phase (IPRP) with hydrophilic interaction liquid chromatography (HILIC) for polar antisense oligonucleotide (ASO) analysis. Compared with the state-of-the-art commercially available active solvent modulation (ASM), engaging the ILMM significantly reduces the peak distortion in GPC-RPLC, allowing an at least 67% higher transfer volume from the primary to secondary dimension, and resolves the ASO sample breakthrough in selective comprehensive IPRP×HILIC. Also remarkably, ILMM demonstrated superiority in comprehensive RPLC×RPLC analysis in comparison with ASM, suggesting its potential in broader 2D-LC applications. In addition to chromatography improvement, ILMM offers several advantages over benchmark modulation approaches in regard to alleviating the need of an additional dilution flow and a simple as well as flexible system configuration, opening many opportunities to establish innovative and versatile multidimensional workflows for characterizing compounds with increasing complexity.

Discovery of a dual pathway aggregation mechanism for a therapeutic constrained peptide.

Constrained peptides (CPs) have emerged as attractive candidates for drug discovery and development. To fully unlock the therapeutic potential of CPs, it is crucial to understand their physical stability and minimize the formation of aggregates that could induce immune responses. Although amyloid like aggregates have been researched extensively, few studies have focused on aggregates from other peptide scaffolds (e.g., CPs). In this work, a streamlined approach to effectively profile the nature and formation pathway of CP aggregates was demonstrated. Aggregates of various sizes were detected and shown to be amorphous. Though no major changes were found in peptide structure upon aggregation, these aggregates appeared to have mixed natures, consisting of primarily non-covalent aggregates with a low level of covalent species. This co-existence phenomenon was also supported by two kinetic pathways observed in time- and temperature-dependent aggregation studies. Furthermore, a stability study with 8 additional peptide variants exhibited good correlation between aggregation propensity and peptide hydrophobicity. Therefore, a dual aggregation pathway was proposed, with the non-covalent aggregates driven by hydrophobic interactions, whereas the covalent ones formed through disulfide scrambling. Overall, the workflow presented here provides a powerful strategy for comprehensive characterization of peptide aggregates and understanding their mechanisms of formation.

Seeking universal detectors for analytical characterizations.

It is highly desirable to have a universal detector that can detect all types of compounds and give a uniform response regardless of the physiochemical properties of the compounds. With such a universal detector, all components in a sample can be accurately quantified without the need for individual standards. This is especially needed for the characterization of unknowns and for non-targeted analysis, or for samples that have no isolated standards available for each component. Over the years, much effort has been put into seeking a universal detection technology. In this review, we discuss the commonly used detectors for analytical characterization, including UV, RI, ELSD, CAD, CLND, FID, VUV, MS, NMR, and hyphenated detection, with the focuses on the "universal" features of these detectors regarding the types of molecules they can detect and the uniformity of responses.

Variable selection in multivariate modeling of drug product formula and manufacturing process.

Multivariate data analysis methods such as partial least square (PLS) modeling have been increasingly applied to pharmaceutical product development. This study applied the PLS modeling to analyze a product development dataset generated from a design of experiment and historical batch data. Attention was paid in particular to the assessment of the importance of predictor variables, and subsequently the variable selection in the PLS modeling. The assessment indicated that irrelevant and collinear predictors could be extensively present in the initial PLS model. Therefore, variable selection is an important step in the optimization of the pharmaceutical product process model. The variable importance for projections (VIP) and coefficient values can be employed to rank the importance of predictors. On the basis of this ranking, the irrelevant predictors can be removed. To further reduce collinear predictors, multiple rounds of PLS modeling on different combinations of predictors may be necessary. To this end, stepwise reduction of predictors based on their VIP/coefficient ranking was introduced and was proven to be an effective approach to identify and remove redundant collinear predictors. Overall, the study demonstrated that the variable selection procedure implemented herein can effectively evaluate the importance of variables and optimize models of drug product processes.

Simultaneous, sequential quantitative achiral-chiral analysis by two-dimensional liquid chromatography.

In general, chromatographic analysis of chiral compounds involves a minimum of two methods; a primary achiral method for assay and impurity analysis and a secondary chiral method for assessing chiral purity. Achiral method resolves main enantiomeric pairs of component from potential impurities and degradation products and chiral method resolves enantiomeric pairs of the main component and diastereomer pairs. Reversed-phase chromatographic methods are preferred for assay and impurity analysis (high efficiency and selectivity) whereas chiral separation is performed by reverse phase, normal phase, or polar organic mode. In this work, we have demonstrated the use of heart-cutting (LC-LC) and comprehensive two-dimensional liquid chromatography (LC × LC) in simultaneous, sequential achiral and chiral analysis and quantitation of minor, undesired enantiomer in the presence of major, desired enantiomer using phenylalanine as an example. The results were comparable between LC-LC and LC × LC with former offering better sensitivity and accuracy. The quantitation range was over three orders of magnitude with undesired D-phenylalanine detected at approximately 0.3% in the presence of predominant, desired L-phenylalanine (99.7%). The limit of quantitation was comparable to conventional high-performance liquid chromatography. A reversed-phase C18 achiral column in the primary and reversed-phase Chirobiotic Tag chiral column in the secondary dimension were used with a compatible mobile phase.

Towards a comprehensive 2-D-LC-MS separation.

A comprehensive 2-D-LC-MS method has been developed by coupling columns of different selectivity. The primary column eluate is alternately trapped and sampled onto the secondary columns through a guard column interface. When one guard column traps the eluate, the other injects the previously trapped components onto a secondary column. This cycle is repeated throughout the chromatogram. The use of dual secondary columns provides the secondary columns with additional time to generate high-speed chromatograms. Each secondary column generates alternate chromatograms which when combined generate the entire chromatogram. The primary column separation is comparable to conventional HPLC, whereas the secondary column separation is fast. With both the columns operating in reverse phase mode, one would expect strong correlation in the two-dimensional retention and hence inefficiency in separation. However, differences in column operation modes, interaction mechanisms, and vendor silica result in a complementary separation. The system was evaluated by comparing it to one-dimensional counterparts and coupled column chromatography. Although some correlations were observed in 2-D-LC-MS, peaks do show two-dimensional distribution with superior UV and MS data as co-elution is minimized. Also, the ease of converting conventional systems to 2-D-LC-MS is discussed.

Two-dimensional liquid chromatography with mixed mode stationary phases.

Mixed mode stationary phases with ion-pairing reagent (acidic or basic) as integral part of hydrophobic chain offers unique selectivity, and hence, are ideal for multidimensional separations. The retention of hydrophobic components is a function of organic content, whereas that of charged species is a function of organic content, ionogenic modifier and its level in the mobile phase. Hence, by controlling the parameters influencing component retention (stationary phase and mobile phase), the selectivity of chemical components in the two-dimensional plane can be manipulated to improve the separation. A two-dimensional liquid chromatograph has been developed by coupling similar and dissimilar mixed mode stationary phases in the two dimensions. This technique has immense potential in resolving co-eluting components as the retention mechanism in the two-dimensions are complementary. However, with only part of the primary column eluent sampled into the secondary column, the technique is limited to qualitative analysis.

An automated orthogonal two-dimensional liquid chromatograph.

A simple approach to two-dimensional liquid chromatography has been developed by coupling columns of different selectivity using a 12-port, dual-position valve and a standard HPLC system. The valve at the junction of the two columns enables continuous, periodic sampling (injection) of the primary column eluent onto the secondary column. The separation in the primary dimension is comparable to conventional HPLC, whereas the secondary column separation is fast, lasting several seconds. The high-speed separation in the secondary dimension enables the primary column eluent to be sampled with fidelity onto the secondary column throughout the chromatographic run. One might expect a coupled column liquid chromatography system operating in reverse-phase mode to be strongly correlated and, hence, inefficient. However, by applying a solvent gradient in the primary dimension and by progressively incrementing the solvent strength in the secondary dimension (tuning), the inefficiency or cross correlation between the two dimensions is minimized. In a tuned two-dimensional system, the influence of primary column retention (usually hydrophobicity) is minimal on secondary column retention. This enables subtle differences in component interaction with the two stationary phases to dominate the secondary column retention. The peaks are randomly dispersed over a retention plane rather than along a diagonal, resulting in an orthogonal separation. The peak capacity is multiplicative, and each component has a unique pair of retention times, enabling positive identification. In addition, the location of the component provides two independent measures of molecular properties. The 2D-LC system was evaluated by analyzing a test mixture made of some aromatic amines and non-amines on different secondary columns (ODS-AQ/ODS monolith, ODS/amino, ODS/cyano). The relative location of sample components in the two-dimensional plane varied significantly with change in secondary column. Among the secondary columns, the amino and cyano columns offered the most complementary separation, with the retention order of several components reversed in the secondary dimension. The theoretical peak capacity of the 2D-LC system was around 450 for a separation lasting 30 min. A 2D-LC system involving amino and cyano columns resulted in a high-speed separation of the test mixture, with most of the chemical components resolved within a few minutes.