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Size exclusion chromatography (SEC) is a historical technique, routinely applied for the separation of species possessing different molecular masses (sizes). It is considered as a reference method for the qualitative and quantitative analysis of protein aggregates. In the last few years,several improvements were brought to high performance SEC (HP-SEC or conventional SEC) in terms of particle size, inertness and column dimension. Today, SEC columns of smaller dimensions packed with sub-3 μm particles (also known as ultra-high performance SEC (UHPSEC)) are used, to obtain faster and better separation compared to HP-SEC. This paper discusses the possibilities and limitations of both HP-SEC and UHP-SEC and also describes some possible directions for future applications related to protein biopharmaceuticals analysis.

Reversed phase liquid chromatography (RPLC) is the gold standard analytical strategy in the pharmaceutical industry. In this contribution, we will describe two alternative chromatographic approaches, namely hydrophilic interaction chromatography (HILIC) and supercritical fluid chromatography (SFC), which are more and more commonly used in pharmaceutical analysis. Among their advantages, these two strategies offer better retention of polar substances, alternative selectivities and enhanced MS sensitivity, in comparison with RPLC.

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Relation between the particle size distribution and the kinetic performance of packed columns

Many HPLC analyses could be performed at lower expenditure. This could involve a combination of reducing the analysis time, reducing the resolution between critical peaks, and lowering the consumption of mobile phase. Successfully optimizing the method in such instances - as well as in situations where it is necessary to transfer the method to another laboratory that lacks the same selection of columns - can save the analyst time and money.

In the present study, the possibility of retention modeling in the HILIC mode was investigated, testing several different literature relationships over a wide range of different analytical conditions (column chemistries and mobile phase pH) and using analytes possessing diverse physico-chemical properties. Furthermore, it was investigated how the retention prediction depends on the number of isocratic or gradient trial or initial scouting runs. The most promising set of scouting runs seems to be a combination of three isocratic runs (95,90 and 70%ACN) and one gradient run (95 to 65%ACN in 10 min), as the average prediction errors were lower than using six equally spaced isocratic runs and because it is common in Method development (MD) to perform at least one scouting gradient run in the screening step to find out the best column, temperature and pH conditions. Overall, the retention predictions were much less accurate in HILIC than what is usually experienced in RPLC. This has severe implications for MD, as it restricts the use of commercial software packages that require the simulation of the retention of every peak in the chromatogram. To overcome this problem, the recently proposed predictive elution window shifting and stretching (PEWS2) approach can be used. In this computer-assisted MD strategy, only an (approximate) prediction of the retention of the first and the last peak in the chromatogram is required to conduct a well-targeted trial-and-error search, with suggested search conditions uniformly covering the entire possible search and elution space. This strategy was used to optimize the separation of three representative pharmaceutical mixtures possessing diverse physico-chemical properties (pteridins, saccharides and cocktail of drugs/metabolites). All problems could be successfully handled in less than 2.5 h of instrument time (including equilibration).

Very short columns filled with 1.9 μm particles were evaluated for the ultra-fast analysis of pharmaceutical formulations. Local anaesthetic, mydriatic and anti-hypertensive agents were chosen as analytes and a method was developed and validated for each of these substances, according to ICH guidelines. Excellent quantitative performance was obtained using an optimized Chromatographie system that reduces the importance of extra-column effects and cuts the analysis time to less than 15 s.

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