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Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies
A ubiquitous post-translational modification observed in proteins is isomerization of aspartic acid to isoaspartic acid (isoAsp). This non-enzymatic post-translational modification occurs spontaneously in proteins and plays a role in aging, autoimmune response, cancer, neurodegeneration, and other d...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010776/ https://www.ncbi.nlm.nih.gov/pubmed/24808864 http://dx.doi.org/10.3389/fphar.2014.00087 |
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author | Eakin, Catherine M. Miller, Amanda Kerr, Jennifer Kung, James Wallace, Alison |
author_facet | Eakin, Catherine M. Miller, Amanda Kerr, Jennifer Kung, James Wallace, Alison |
author_sort | Eakin, Catherine M. |
collection | PubMed |
description | A ubiquitous post-translational modification observed in proteins is isomerization of aspartic acid to isoaspartic acid (isoAsp). This non-enzymatic post-translational modification occurs spontaneously in proteins and plays a role in aging, autoimmune response, cancer, neurodegeneration, and other diseases. Formation of isoAsp is also a significant issue for recombinant monoclonal antibody based protein therapeutics particularly when isomerization occurs in a complementarity-determining region due to potential impact to the clinical efficacy. Here, we present and compare three analytical methods to monitor and/or quantify isoAsp formation in a monoclonal antibody. The methods include two peptide map based technologies with quantitation from either UV integration or total ion peak areas, as well as an alternative approach using IdeS digestion to generate Fc/2 and Fab’2 regions, followed by hydrophobic interaction chromatography (HIC) to separate the population of Fab’2 containing an isoAsp. The level of isoAsp detected by the peptide map and the digested-HIC methods presented here show similar trends although sample throughput varies by method. |
format | Online Article Text |
id | pubmed-4010776 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-40107762014-05-07 Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies Eakin, Catherine M. Miller, Amanda Kerr, Jennifer Kung, James Wallace, Alison Front Pharmacol Pharmacology A ubiquitous post-translational modification observed in proteins is isomerization of aspartic acid to isoaspartic acid (isoAsp). This non-enzymatic post-translational modification occurs spontaneously in proteins and plays a role in aging, autoimmune response, cancer, neurodegeneration, and other diseases. Formation of isoAsp is also a significant issue for recombinant monoclonal antibody based protein therapeutics particularly when isomerization occurs in a complementarity-determining region due to potential impact to the clinical efficacy. Here, we present and compare three analytical methods to monitor and/or quantify isoAsp formation in a monoclonal antibody. The methods include two peptide map based technologies with quantitation from either UV integration or total ion peak areas, as well as an alternative approach using IdeS digestion to generate Fc/2 and Fab’2 regions, followed by hydrophobic interaction chromatography (HIC) to separate the population of Fab’2 containing an isoAsp. The level of isoAsp detected by the peptide map and the digested-HIC methods presented here show similar trends although sample throughput varies by method. Frontiers Media S.A. 2014-04-29 /pmc/articles/PMC4010776/ /pubmed/24808864 http://dx.doi.org/10.3389/fphar.2014.00087 Text en Copyright © 2014 Eakin, Miller, Kerr, Kung and Wallace. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Pharmacology Eakin, Catherine M. Miller, Amanda Kerr, Jennifer Kung, James Wallace, Alison Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies |
title | Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies |
title_full | Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies |
title_fullStr | Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies |
title_full_unstemmed | Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies |
title_short | Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies |
title_sort | assessing analytical methods to monitor isoasp formation in monoclonal antibodies |
topic | Pharmacology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010776/ https://www.ncbi.nlm.nih.gov/pubmed/24808864 http://dx.doi.org/10.3389/fphar.2014.00087 |
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