In-process control (IPC) is an important task during chemical syntheses in pharmaceutical industry. Despite the fact that each chemical reaction is unique, the most common analytical technique used for IPC analysis is high performance liquid chromatography (HPLC). Today, the so-called “Quality by Design” (QbD) principle is often being applied rather than “Trial and Error” approach for HPLC method development. The QbD approach requires only for a very few experimental measurements to find the appropriate stationary phase and optimal chromatographic conditions such as the composition of mobile phase, gradient steepness or time (tG), temperature (T), and mobile phase pH. In this study, the applicability of a multifactorial liquid chromatographic optimization software was studied in an extended knowledge space. Using state-of-the-art ultra-high performance liquid chromatography (UHPLC), the analysis time can significantly be shortened. By using UHPLC, it is possible to analyse the composition of the reaction mixture within few minutes. In this work, a mixture of route of synthesis of apixaban was analysed on short narrow bore column (50 × 2.1 mm, packed with sub-2 µm particles) resulting in short analysis time. The aim of the study was to cover a relatively narrow range of method parameters (tG
, T, pH) in order to find a robust working point (zone). The results of the virtual (modeled) robustness testing were systematically compared to experimental measurements and Design of Experiments (DoE) based predictions.

Highlights:

• Phorbas amaranthus extract as natural products library
• DryLab® software for Design of Experiments in association with scouting systems
• Sixty-four metabolites chemically characterized by LC-HRMS
• New metabolites inferred based on Global Natural Product Social Molecular Networking (GNPS)

Abstract

The optimization of RP-HPLC separations of plant secondary metabolites can be quickly and easily carried out using DryLab^®, which predicts the chromatographic behavior of the analytes on the basis of a limited number of experiments. Such approach allows the development of robust and reliable RP-HPLC methods and reduces the consumption and waste of harmful and expensive organic solvents and, therefore, is both economic and ecologic.

Highlights:

• Quality-by-design based liquid chromatographic method for new chemical entity
• Exploration of design space using 2D and 3D chromatographic retention modeling
• Establishment of a methodology for achieving analytical method control space
• Demonstration of dependence of method control space on control strategy
• Use of DryLab^® to evaluate method robustness with acceptable accuracy

Highlights

In the present work, we describe the fundamental and practical advantages of a new strategy to improve the resolution of very closely eluting peaks within therapeutic protein samples.

This approach involves the use of multiple isocratic steps, together with the addition of a steep negative gradient segment (with a decrease in mobile phase strength) to "park" a slightly more retained peak somewhere along the column (at a given migration distance), while a slightly less retained compound can be eluted.

First, some model calculations were performed to highlight the potential of this innovative approach. For this purpose, the retention parameters (logk₀ and S) for two case studies were considered, namely the analysis of a mixture of two therapeutic mAbs (simple to resolve sample) and separation of a therapeutic mAb from its main variant (challenging to resolve sample). The results confirm that the insertion of a negative segment into a multi-isocratic elution program can be a good tool to improve selectivity between critical peak pairs. However, it is also important to keep in mind that this approach only works with large solutes, which more or less follow an "on-off" type elution behavior.

Two real applications were successfully developed to illustrate the practical advantage of this new approach, including the separation of a therapeutic mAb from its main variant possessing very close elution behavior, and the separation of a carrier protein from an intact mAb as might be encountered in a quantitative bioanalysis assay. These two examples demonstrate that improved selectivity can be achieved for protein RPLC through the inclusion of a negative gradient slope that selectively bifurcates the elution of two or more peaks of interest.