HPLC- and UHPLC-MS Analysis of Pharmaceutically Relevant Biomacromolecules on the Analytical and Capillary Scale

I would like to thank Hayley Herderschee, Ph.D. (c) for sharing her poster from HPLC 2022. Although we are not related to this poster, other than receiving it, I found the chromatography interesting. The poster is entitled “HPLC- and UHPLC-MS Analysis of Pharmaceutically Relevant Biomacromolecules on the Analytical and Capillary Scale” by Hayley Hederschee et al. They are also writing a manuscript for this poster.

The following is taken from the poster.

They successfully developed a repeatable, quantitative, and structurally informative method for pneumococcal polysaccharide analysis with implications of monitoring drug product stability over time. To effectively analyze their stability, they employed skimmer-CID mass spectrometry (sCID MS), a powerful technique for complex serotype mixtures. The large size and lack of chromophore in these serotypes make sCID an attractive detector [1,2].

Biomacromolecules, such as monoclonal antibodies, proteins, and polysaccharides, are key components of many top-grossing drugs worldwide [3]. However, their chromatographic analysis poses challenges due to complexity, size, and slow diffusion [4].

The objective includes:

1. HPLC-MS of Pneumococcal PS:
They successfully developed a quantitative and repeatable method to assess the stability of pneumococcal PS in vaccines over time, ensuring their safety and efficacy. Through reversed-phase liquid chromatography (RPLC), they achieved effective separation of different intact pneumococcal serotypes. Additionally, intact mass spectra produced distinct profiles for each serotype, facilitating their quantitation and differentiation.

2. UHPLC of Intact Biomacromolecules:
By leveraging pressures above 15,000 psi, they achieved good results in the separation of intact proteins. Using smaller particle sizes and longer column lengths, they attained nc ≈ 2400 in a 10-hour gradient experiment at 35 kpsi, employing a 100 cm capillary column with 1.1 µm particles. This advancement enhances the ability to analyze intact proteins, enabling novel discoveries of post-translational modifications and valuable structural information for top-down proteomics.

Other implications include:

• Monitoring drug safety and efficacy:
The development of a reliable and quantitative method to characterize pneumococcal PS over time structurally has significant implications for monitoring the drug product stability, safety, and effectiveness of various drugs. This method can be applied not only to pneumococcal PS but also to other polysaccharides, broadening its utility.

• Advancing top-down proteomics:
Breakthroughs in UHPLC of proteins enable more efficient and accurate analysis of intact proteins, providing crucial insights into post-translational modifications and other structural information. This advancement contributes to the field of top-down proteomics.

Please see the details here.