Low level sequence variant analysis of recombinant proteins: an optimized approach.

Sequence variants in recombinant biopharmaceuticals may have a relevant and unpredictable impact on clinical safety and efficacy. Hence, their sensitive analysis is important throughout bioprocess development. The two stage analytical approach presented here provides a quick multi clone comparison o...

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Autores principales:	Anne Zeck, Jörg Thomas Regula, Vincent Larraillet, Björn Mautz, Oliver Popp, Ulrich Göpfert, Frank Wiegeshoff, Ulrike E E Vollertsen, Ingo H Gorr, Hans Koll, Apollon Papadimitriou
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c60da79177b54a82b4fdb2888fb9f3e0
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spelling	oai:doaj.org-article:c60da79177b54a82b4fdb2888fb9f3e02021-11-18T07:13:12ZLow level sequence variant analysis of recombinant proteins: an optimized approach.1932-620310.1371/journal.pone.0040328https://doaj.org/article/c60da79177b54a82b4fdb2888fb9f3e02012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22792284/?tool=EBIhttps://doaj.org/toc/1932-6203Sequence variants in recombinant biopharmaceuticals may have a relevant and unpredictable impact on clinical safety and efficacy. Hence, their sensitive analysis is important throughout bioprocess development. The two stage analytical approach presented here provides a quick multi clone comparison of candidate production cell lines as a first stage, followed by an in-depth analysis including identification and quantitation of aberrant sequence variants of selected clones as a second stage. We show that the differential analysis is a suitable tool for sensitive and fast batch to batch comparison of recombinant proteins. The optimized approach allows for detection of not only single amino acid substitutions in unmodified peptides, but also substitutions in posttranslational modified peptides such as glycopeptides, for detection of truncated or elongated sequence variants as well as double amino acid substitutions or substitution with amino acid structural isomers within one peptide. In two case studies we were able to detect sequence variants of different origin down to a sub percentage level. One of the sequence variants (Thr → Asn) could be correlated to a cytosine to adenine substitution at DNA (desoxyribonucleic acid) level. In the second case we were able to correlate the sub percentage substitution (Phe → Tyr) to amino acid limitation in the chemically defined fermentation medium.Anne ZeckJörg Thomas RegulaVincent LarrailletBjörn MautzOliver PoppUlrich GöpfertFrank WiegeshoffUlrike E E VollertsenIngo H GorrHans KollApollon PapadimitriouPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 7, p e40328 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Anne Zeck Jörg Thomas Regula Vincent Larraillet Björn Mautz Oliver Popp Ulrich Göpfert Frank Wiegeshoff Ulrike E E Vollertsen Ingo H Gorr Hans Koll Apollon Papadimitriou Low level sequence variant analysis of recombinant proteins: an optimized approach.
description	Sequence variants in recombinant biopharmaceuticals may have a relevant and unpredictable impact on clinical safety and efficacy. Hence, their sensitive analysis is important throughout bioprocess development. The two stage analytical approach presented here provides a quick multi clone comparison of candidate production cell lines as a first stage, followed by an in-depth analysis including identification and quantitation of aberrant sequence variants of selected clones as a second stage. We show that the differential analysis is a suitable tool for sensitive and fast batch to batch comparison of recombinant proteins. The optimized approach allows for detection of not only single amino acid substitutions in unmodified peptides, but also substitutions in posttranslational modified peptides such as glycopeptides, for detection of truncated or elongated sequence variants as well as double amino acid substitutions or substitution with amino acid structural isomers within one peptide. In two case studies we were able to detect sequence variants of different origin down to a sub percentage level. One of the sequence variants (Thr → Asn) could be correlated to a cytosine to adenine substitution at DNA (desoxyribonucleic acid) level. In the second case we were able to correlate the sub percentage substitution (Phe → Tyr) to amino acid limitation in the chemically defined fermentation medium.
format	article
author	Anne Zeck Jörg Thomas Regula Vincent Larraillet Björn Mautz Oliver Popp Ulrich Göpfert Frank Wiegeshoff Ulrike E E Vollertsen Ingo H Gorr Hans Koll Apollon Papadimitriou
author_facet	Anne Zeck Jörg Thomas Regula Vincent Larraillet Björn Mautz Oliver Popp Ulrich Göpfert Frank Wiegeshoff Ulrike E E Vollertsen Ingo H Gorr Hans Koll Apollon Papadimitriou
author_sort	Anne Zeck
title	Low level sequence variant analysis of recombinant proteins: an optimized approach.
title_short	Low level sequence variant analysis of recombinant proteins: an optimized approach.
title_full	Low level sequence variant analysis of recombinant proteins: an optimized approach.
title_fullStr	Low level sequence variant analysis of recombinant proteins: an optimized approach.
title_full_unstemmed	Low level sequence variant analysis of recombinant proteins: an optimized approach.
title_sort	low level sequence variant analysis of recombinant proteins: an optimized approach.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/c60da79177b54a82b4fdb2888fb9f3e0
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_version_	1718423815257587712

Low level sequence variant analysis of recombinant proteins: an optimized approach.

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