At-line high throughput site-specific glycan profiling using targeted mass spectrometry

Abstract

Protein post-translational modification (PTM) plays an important role in many biological processes; of which glycosylation is arguably one of the most complex and diverse modifications and is crucial for the safety and efficacy of biotherapeutic proteins. Mass spectrometric characterization of protein glycosylation is well established with clear advantages and disadvantages; on one hand it is precise and information-rich, as well as being relative inexpensive in terms of the reagents and consumables despite the instrumentation cost and, depending on the method, can give site specific information; on the other hand it generally suffers from low throughput, restriction to largely purified samples and is less quantitative, especially for sialylated glycan species. Here, we describe a high throughput, site-specific, targeted mass spectrometric peptide mapping approach to quickly screen/rank candidate production cell lines and culture conditions that give favourable glycosylation profiles directly from conditioned culture media for an Fc-fusion protein. The methodology is fully compatible with automation and combines the speed of 'top-down' mass spectrometry with the site-specific information of 'bottom-up' mass spectrometry. In addition, this strategy can be used for multi-attribute product quality screening/monitoring as an integral part of cell line selection and process development.

Keywords: 2-AB, 2-aminobenzamide labelled UPLC glycan analysis; CM, conditioned media; Cell line selection; DoE, design of experiment; ESI, electrospray ionization; Glycan profiling; Glycosylation; PKPD, pharmacokinetic pharmacodynamic; PTM, post-translation modification; QBD, quality by design; TQ-MS, triple quadrupole mass spectrometry; Targeted mass spectrometry.

Fig. 1

Mass spectral time course showing shifts in charge form to overwhelmingly 3+ state in singly mis-cleaved compare to fully cleaved Fc-glycopeptides and the general dominance of singly mis-cleaved glycopeptide signal over 4 h digestion time. From left to right: fully cleaved glycopeptide at 0.5 h digestion; singly mis-cleaved glycopeptide at 0.5 h digestion; fully cleaved glycopeptide at 1 h digestion; singly mis-cleaved glycopeptide at 1 h digestion; fully cleaved glycopeptide at 2 h digestion; singly mis-cleaved glycopeptide at 2 h digestion; fully cleaved glycopeptide at 4 h digestion; singly mis-cleaved glycopeptide at 4 h digestion.

Fig. 2

Cleavage state and charge-form sorted Fc-glycan profiles against trypsin digestion time showing despite changes in total signal intensities over time the overall glycan profile remains the same. (A) 2+, fully cleaved glycopeptides; (B) 2+, singly mis-cleaved glycopeptides; (C) 3+, fully cleaved glycopeptides; (D) 3+, singly mis-cleaved glycopeptides.

Fig. 3

Cleavage state and charge-form sorted fusion protein-glycan profiles against trypsin digestion time. Despite changes in total signal intensities over time, the overall glycan profile remains the same. In contrast to the Fc-glycopeptide, here glycan profiles between 2+ and 3+ states show a marked difference. (A) 2+, fully cleaved glycopeptides; (B) 2+, singly mis cleaved glycopeptides; (C) 3+, fully cleaved glycopeptides; (D) 3+, singly mis-cleaved glycopeptides.

Fig. 4

Site-specific glycan profiles showing that targeting the subset of 3+ charge state of the singly mis-cleaved glycopeptides in TQ-MS is sufficient to generate results comparable to those obtained by 2-AB methodology despite intrinsic underestimation of the sialylated glycan species. Results obtained by GXII are in contrast, less consistent with the former techniques. Comparison of (A) glycan profiles generated using data from all charge states and cleavage forms, (B) singly mis-cleaved 2+ and 3+ glycopeptides only and (C) 3+ singly mis-cleaved glycopeptide signals only. From left to right: TQ-MS fusion-protein glycan profile; TQ-MS Fc-glycan profile; GXII fusion-protein glycan profile; GXII Fc-glycan profile; 2-AB fusion-protein glycan profile; 2-AB Fc-glycan profile.

Fig. 5

Comparison of purified and CM-spiked sample glycan profiles at 1 h post trypsin digestion. The results are highly similar, confirming the applicability of the analytical approach on crude samples for both (A) Fc-glycans and (B) fusion-protein glycans. From left to right: 2+, fully cleaved glycopeptide signal from purified sample; 2+, fully cleaved glycopeptide signal from CM-spiked sample; 3+, fully cleaved glycopeptide signal from purified sample; 3+, fully cleaved glycopeptide signal from CM-spiked sample; 2+, singly mis-cleaved glycopeptide signal from purified sample; 2+, singly mis-cleaved glycopeptide signal from CM-spiked sample; 3+, singly mis-cleaved glycopeptide signal from purified sample; 3+, singly mis-cleaved glycopeptide signal from CM-spiked sample.