4 research outputs found
Development of a PNGase Rc column for online deglycosylation of complex glycoproteins during HDX-MS
Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins
Mass spectrometry-based multi-attribute method in protein therapeutics product quality monitoring and quality control
ABSTRACTThe multi-attribute method (MAM), a liquid chromatography-mass spectrometry (LC-MS)-based peptide mapping method, has gained increased interest and applications in the biopharmaceutical industry. MAM can, in one method, provide targeted quantitation of multiple site-specific product quality attributes, as well as new peak detection. In this review, we focus on the scientific and regulatory considerations of using MAM in product quality attribute monitoring and quality control (QC) of therapeutic proteins. We highlight MAM implementation challenges and solutions with several case studies, and provide our perspective on the opportunities to use MS in QC for applications other than standard peptide mapping-based MAM
Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS
Protein
glycosylation is one of the most common PTMs and many cell
surface receptors, extracellular proteins, and biopharmaceuticals
are glycosylated. However, HDX-MS analysis of such important glycoproteins
has so far been limited by difficulties in determining the HDX of
the protein segments that contain glycans. We have developed a column
containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup
to allow improved analysis of glycoproteins. We show that HDX-MS with
the PNGase Rc column enables efficient online removal of N-linked
glycans and the determination of the HDX of glycosylated regions in
several complex glycoproteins. Additionally, we use the PNGase Rc
column to perform a comprehensive HDX-MS mapping of the binding epitope
of a mAb to c-Met, a complex glycoprotein drug target. Importantly,
the column retains high activity in the presence of common quench-buffer
additives like TCEP and urea and performed consistent across 114 days
of extensive use. Overall, our work shows that HDX-MS with the integrated
PNGase Rc column can enable fast and efficient online deglycosylation
at harsh quench conditions to provide comprehensive analysis of complex
glycoproteins
Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS
Protein
glycosylation is one of the most common PTMs and many cell
surface receptors, extracellular proteins, and biopharmaceuticals
are glycosylated. However, HDX-MS analysis of such important glycoproteins
has so far been limited by difficulties in determining the HDX of
the protein segments that contain glycans. We have developed a column
containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup
to allow improved analysis of glycoproteins. We show that HDX-MS with
the PNGase Rc column enables efficient online removal of N-linked
glycans and the determination of the HDX of glycosylated regions in
several complex glycoproteins. Additionally, we use the PNGase Rc
column to perform a comprehensive HDX-MS mapping of the binding epitope
of a mAb to c-Met, a complex glycoprotein drug target. Importantly,
the column retains high activity in the presence of common quench-buffer
additives like TCEP and urea and performed consistent across 114 days
of extensive use. Overall, our work shows that HDX-MS with the integrated
PNGase Rc column can enable fast and efficient online deglycosylation
at harsh quench conditions to provide comprehensive analysis of complex
glycoproteins