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Scalable manufacturing platform for the production of PEGylated heme albumin
Cell‐free heme, which was previously shown to have adverse effects on the innate immune system, does not induce inflammation when bound to a protein carrier via overexpression of the enzyme heme‐oxygenase 1 (HO‐1). Studies in mouse macrophage cell culture and human endothelial cells have confirmed H...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9669187/ https://www.ncbi.nlm.nih.gov/pubmed/36111455 http://dx.doi.org/10.1002/bit.28237 |
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author | Savla, Chintan Palmer, Andre F. |
author_facet | Savla, Chintan Palmer, Andre F. |
author_sort | Savla, Chintan |
collection | PubMed |
description | Cell‐free heme, which was previously shown to have adverse effects on the innate immune system, does not induce inflammation when bound to a protein carrier via overexpression of the enzyme heme‐oxygenase 1 (HO‐1). Studies in mouse macrophage cell culture and human endothelial cells have confirmed HO‐1 catalyzed breakdown of protein bound heme into biliverdin, iron, and carbon monoxide (CO), which elicits anti‐inflammatory effects. However, to fully realize the anti‐inflammatory therapeutic effects of heme, a colloidally stable heme protein carrier must be developed. To accomplish this goal, we incorporated multiple heme molecules into human serum albumin (HSA) via partial unfolding of HSA at basic pH followed by refolding at neutral pH, and subsequently conjugated the surface of the heme‐HSA complex with polyethylene glycol (PEG) to stabilize heme‐HSA. Quantification studies confirmed that a maximum of 5−6 hemes could be bound to HSA without precipitation or degradation of heme‐HSA. Dynamic light scattering, size exclusion‐high performance liquid chromatography (SEC‐HPLC), and matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry confirmed the increase in hydrodynamic diameter and molecular weight (MW), respectively, upon PEGylation of heme‐HSA. Furthermore, PEG‐heme‐HSA was stable upon exposure to different pH environments, freeze‐thaw cycles, and storage at 4°C. Taken together, we devised a synthesis and purification platform for the production of PEGylated heme‐incorporated HSA that can be used to test the potential anti‐inflammatory effects of heme in vivo. |
format | Online Article Text |
id | pubmed-9669187 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-96691872023-01-10 Scalable manufacturing platform for the production of PEGylated heme albumin Savla, Chintan Palmer, Andre F. Biotechnol Bioeng ARTICLES Cell‐free heme, which was previously shown to have adverse effects on the innate immune system, does not induce inflammation when bound to a protein carrier via overexpression of the enzyme heme‐oxygenase 1 (HO‐1). Studies in mouse macrophage cell culture and human endothelial cells have confirmed HO‐1 catalyzed breakdown of protein bound heme into biliverdin, iron, and carbon monoxide (CO), which elicits anti‐inflammatory effects. However, to fully realize the anti‐inflammatory therapeutic effects of heme, a colloidally stable heme protein carrier must be developed. To accomplish this goal, we incorporated multiple heme molecules into human serum albumin (HSA) via partial unfolding of HSA at basic pH followed by refolding at neutral pH, and subsequently conjugated the surface of the heme‐HSA complex with polyethylene glycol (PEG) to stabilize heme‐HSA. Quantification studies confirmed that a maximum of 5−6 hemes could be bound to HSA without precipitation or degradation of heme‐HSA. Dynamic light scattering, size exclusion‐high performance liquid chromatography (SEC‐HPLC), and matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry confirmed the increase in hydrodynamic diameter and molecular weight (MW), respectively, upon PEGylation of heme‐HSA. Furthermore, PEG‐heme‐HSA was stable upon exposure to different pH environments, freeze‐thaw cycles, and storage at 4°C. Taken together, we devised a synthesis and purification platform for the production of PEGylated heme‐incorporated HSA that can be used to test the potential anti‐inflammatory effects of heme in vivo. John Wiley and Sons Inc. 2022-09-26 2022-12 /pmc/articles/PMC9669187/ /pubmed/36111455 http://dx.doi.org/10.1002/bit.28237 Text en © 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | ARTICLES Savla, Chintan Palmer, Andre F. Scalable manufacturing platform for the production of PEGylated heme albumin |
title | Scalable manufacturing platform for the production of PEGylated heme albumin |
title_full | Scalable manufacturing platform for the production of PEGylated heme albumin |
title_fullStr | Scalable manufacturing platform for the production of PEGylated heme albumin |
title_full_unstemmed | Scalable manufacturing platform for the production of PEGylated heme albumin |
title_short | Scalable manufacturing platform for the production of PEGylated heme albumin |
title_sort | scalable manufacturing platform for the production of pegylated heme albumin |
topic | ARTICLES |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9669187/ https://www.ncbi.nlm.nih.gov/pubmed/36111455 http://dx.doi.org/10.1002/bit.28237 |
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