Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.

The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based syst...

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Autores principales:	Grant E Frahm, Daryl G S Smith, Anita Kane, Barry Lorbetskie, Terry D Cyr, Michel Girard, Michael J W Johnston
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a8433f41fbf446fdb4d542f13b7782e7
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spelling	oai:doaj.org-article:a8433f41fbf446fdb4d542f13b7782e72021-11-25T05:57:18ZDetermination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.1932-620310.1371/journal.pone.0109893https://doaj.org/article/a8433f41fbf446fdb4d542f13b7782e72014-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0109893https://doaj.org/toc/1932-6203The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.Grant E FrahmDaryl G S SmithAnita KaneBarry LorbetskieTerry D CyrMichel GirardMichael J W JohnstonPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 10, p e109893 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Grant E Frahm Daryl G S Smith Anita Kane Barry Lorbetskie Terry D Cyr Michel Girard Michael J W Johnston Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.
description	The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.
format	article
author	Grant E Frahm Daryl G S Smith Anita Kane Barry Lorbetskie Terry D Cyr Michel Girard Michael J W Johnston
author_facet	Grant E Frahm Daryl G S Smith Anita Kane Barry Lorbetskie Terry D Cyr Michel Girard Michael J W Johnston
author_sort	Grant E Frahm
title	Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.
title_short	Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.
title_full	Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.
title_fullStr	Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.
title_full_unstemmed	Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.
title_sort	determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in oryza sativa.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/a8433f41fbf446fdb4d542f13b7782e7
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Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.

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