Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics
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 identificat...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2019-10-01
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| Series: | mAbs |
| Subjects: | |
| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2019.1635865 |
| _version_ | 1798038579918667776 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Oksana Tyshchuk |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T21:42:07Z |
| format | Article |
| id | doaj.art-7a56bb8a59684d8daaabf729937341e7 |
| institution | Directory Open Access Journal |
| issn | 1942-0862 1942-0870 |
| language | English |
| last_indexed | 2024-04-11T21:42:07Z |
| publishDate | 2019-10-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | mAbs |
| spelling | doaj.art-7a56bb8a59684d8daaabf729937341e72022-12-22T04:01:33ZengTaylor & Francis GroupmAbs1942-08621942-08702019-10-011171219123210.1080/19420862.2019.1635865Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeuticsOksana Tyshchuk0Christoph Gstöttner1Dennis Funk2Simone Nicolardi3Stefan Frost4Stefan Klostermann5Tim Becker6Elena Jolkver7Felix Schumacher8Claudia Ferrara Koller9Hans Rainer Völger10Manfred Wuhrer11Patrick Bulau12Michael Mølhøj13Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, GermanyCenter for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, NetherlandsRoche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, GermanyCenter for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, NetherlandsRoche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, GermanyRoche Pharma Research and Early Development Informatics, Roche Innovation Center Munich, Penzberg, GermanyxValue GmbH, Willich, GermanyxValue GmbH, Willich, GermanyRoche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, GermanyRoche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Zurich, Schlieren, SwitzerlandRoche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, GermanyCenter for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, NetherlandsRoche Pharma Technical Development Penzberg, Penzberg, GermanyRoche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, GermanyBiotherapeutics 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.https://www.tandfonline.com/doi/10.1080/19420862.2019.1635865Biotherapeuticsfab purificationMALDI-ISD FT-ICRhydroxyprolinemass spectrometryPTM |
| spellingShingle | 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 Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics mAbs Biotherapeutics fab purification MALDI-ISD FT-ICR hydroxyproline mass spectrometry PTM |
| title | Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics |
| title_full | Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics |
| title_fullStr | Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics |
| title_full_unstemmed | Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics |
| title_short | Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics |
| title_sort | characterization and prediction of positional 4 hydroxyproline and sulfotyrosine two post translational modifications that can occur at substantial levels in cho cells expressed biotherapeutics |
| topic | Biotherapeutics fab purification MALDI-ISD FT-ICR hydroxyproline mass spectrometry PTM |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2019.1635865 |
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