Development of N-Glycan Mass Spectral Library for Therapeutic Glycoproteins


Maria Lorna A. De Leoz, Stephen E. Stein, and Michael J. Tarlov


The number of protein therapeutics has increased considerably in the past two decades with estimated global sales reaching $93 billion in 2009.  The majority of therapeutic proteins are glycosylated, i.e., proteins are modified by the attachment of sugar molecules in complex chains called glycans.  Changes in glycosylation can affect the stability, clearance, activity, and immunogenicity of the therapeutic.  Therefore, the manufacturing process must ensure consistent glycosylation of these products. 


The development of rapid and sensitive techniques to measure glycosylation would be useful for quality control, feedback control, and lot release. Mass spectrometry (MS) has proven to be a powerful tool to analyze glycosylation of therapeutic proteins; however, analysis of MS data remains complex and time consuming.  The availability of comprehensive reference mass spectra of glycans would help to accelerate data analysis, identify glycoforms reliably, and help lessen the occurrence of false positive matches.  Towards this end, we have recently initiated studies to explore the development of a library of reference mass spectra of glycans of therapeutic glycoproteins using high-resolution MS.


Glycans were released from glycoproteins using Peptide-N-glycosidase F.  After desalting and purification by solid phase extraction, the glycoforms were analyzed by nano-electrospray orbitrap mass spectrometry using both ion trap collisionally-induced dissociation and orbitrap higher-energy C-trap dissociation fragmentation over a range of collision energies. 


Using commercial glycoproteins and glycans, we determined the optimal set of conditions, precursor ions, and methods for quality control.  We also refined our mass spectral matching algorithms for glycans.  Tandem mass spectra of a series of glycans commonly found in protein therapeutics have been obtained.  These preliminary data show promise in expanding the scope of the NIST mass spectral library to include glycans.