Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics
Oksana Tyshchuk,
Christoph Gstöttner,
Dennis Funk,
Simone Nicolardi,
Stefan Frost,
Stefan Klostermann,
Tim Becker,
Elena Jolkver,
Felix Schumacher,
Claudia Ferrara Koller,
Hans Rainer Völger,
Manfred Wuhrer,
Patrick Bulau,
Michael Mølhøj
Affiliations
Oksana Tyshchuk
Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, Germany
Christoph Gstöttner
Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands
Dennis Funk
Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, Germany
Simone Nicolardi
Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands
Stefan Frost
Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, Germany
Stefan Klostermann
Roche Pharma Research and Early Development Informatics, Roche Innovation Center Munich, Penzberg, Germany
Tim Becker
xValue GmbH, Willich, Germany
Elena Jolkver
xValue GmbH, Willich, Germany
Felix Schumacher
Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, Germany
Claudia Ferrara Koller
Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Zurich, Schlieren, Switzerland
Hans Rainer Völger
Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, Germany
Manfred Wuhrer
Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands
Patrick Bulau
Roche Pharma Technical Development Penzberg, Penzberg, Germany
Michael Mølhøj
Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, Germany
Biotherapeutics may contain a multitude of different post-translational modifications (PTMs) that need to be assessed and possibly monitored and controlled to ensure reproducible product quality. During early development of biotherapeutics, unexpected PTMs might be prevented by in silico identification and characterization together with further molecular engineering. Mass determinations of a human IgG1 (mAb1) and a bispecific IgG-ligand fusion protein (BsAbA) demonstrated the presence of unusual PTMs resulting in major +80 Da, and +16/+32 Da chain variants, respectively. For mAb1, analytical cation exchange chromatography demonstrated the presence of an acidic peak accounting for 20%. A + 79.957 Da modification was localized within the light chain complementarity-determining region-2 and identified as a sulfation based on accurate mass, isotopic distribution, and a complete neutral loss reaction upon collision-induced dissociation. Top-down ultrahigh resolution MALDI-ISD FT-ICR MS of modified and unmodified Fabs allowed the allocation of the sulfation to a specific Tyr residue. An aspartate in amino-terminal position-3 relative to the affected Tyr was found to play a key role in determining the sulfation. For BsAbA, a + 15.995 Da modification was observed and localized to three specific Pro residues explaining the +16 Da chain A, and +16 Da and +32 Da chain B variants. The BsAbA modifications were verified as 4-hydroxyproline and not 3-hydroxyproline in a tryptic peptide map via co-chromatography with synthetic peptides containing the two isomeric forms. Finally, our approach for an alert system based on in-house in silico predictors is presented. This system is designed to prevent these PTMs by molecular design and engineering during early biotherapeutic development.
