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Raman spectroscopy characterization of antibody phases in serum

When administered in serum, an efficacious therapeutic antibody should be homogeneous to minimize immune reactions or injection site irritation during administration. Monoclonal antibody (mAb) phase separation is one type of inhomogeneity observed in serum, and thus screening potential phase separat...

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Autores principales: Baker, Audrey E, Mantz, Amber R, Chiu, Mark L
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622053/
https://www.ncbi.nlm.nih.gov/pubmed/25484036
http://dx.doi.org/10.4161/19420862.2014.975100
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author Baker, Audrey E
Mantz, Amber R
Chiu, Mark L
author_facet Baker, Audrey E
Mantz, Amber R
Chiu, Mark L
author_sort Baker, Audrey E
collection PubMed
description When administered in serum, an efficacious therapeutic antibody should be homogeneous to minimize immune reactions or injection site irritation during administration. Monoclonal antibody (mAb) phase separation is one type of inhomogeneity observed in serum, and thus screening potential phase separation of mAbs in serum could guide lead optimization. However, serum contains numerous components, making it difficult to resolve mAb/serum mixtures at a scale amenable to analysis in a discovery setting. To address these challenges, a miniaturized assay was developed that combined confocal microscopy with Raman spectroscopy. The method was examined using CNTO607, a poorly-soluble anti-interleukin-13 human mAb, and CNTO3930, a soluble anti-respiratory syncytial virus humanized mAb. When CNTO607 was diluted into serum above 4.5 mg/mL, phase separation occurred, resulting in droplet formation. Raman spectra of droplet phases in mixtures included bands at 1240 and 1670 cm(−1), which are typical of mAb β-sheets, and lacked bands at 1270 and 1655 cm(−1), which are typical of α-helices. The continuous phases included bands at 1270 and 1655 cm(−1) and lacked those at 1240 and 1670 cm(−1). Therefore, CNTO607 appeared to be sequestered within the droplets, while albumin and other α-helix-forming serum proteins remained within the continuous phases. In contrast, CNTO3930 formed only one phase, and its Raman spectra contained bands at 1240, 1670, 1270 and 1655 cm,(−1) demonstrating homogeneous distribution of components. Our results indicate that this plate-based method utilizing confocal Raman spectroscopy to probe liquid-liquid phases in mAb/serum mixtures can provide a screen for phase separation of mAb candidates in a discovery setting.
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spelling pubmed-46220532015-11-12 Raman spectroscopy characterization of antibody phases in serum Baker, Audrey E Mantz, Amber R Chiu, Mark L MAbs Reports When administered in serum, an efficacious therapeutic antibody should be homogeneous to minimize immune reactions or injection site irritation during administration. Monoclonal antibody (mAb) phase separation is one type of inhomogeneity observed in serum, and thus screening potential phase separation of mAbs in serum could guide lead optimization. However, serum contains numerous components, making it difficult to resolve mAb/serum mixtures at a scale amenable to analysis in a discovery setting. To address these challenges, a miniaturized assay was developed that combined confocal microscopy with Raman spectroscopy. The method was examined using CNTO607, a poorly-soluble anti-interleukin-13 human mAb, and CNTO3930, a soluble anti-respiratory syncytial virus humanized mAb. When CNTO607 was diluted into serum above 4.5 mg/mL, phase separation occurred, resulting in droplet formation. Raman spectra of droplet phases in mixtures included bands at 1240 and 1670 cm(−1), which are typical of mAb β-sheets, and lacked bands at 1270 and 1655 cm(−1), which are typical of α-helices. The continuous phases included bands at 1270 and 1655 cm(−1) and lacked those at 1240 and 1670 cm(−1). Therefore, CNTO607 appeared to be sequestered within the droplets, while albumin and other α-helix-forming serum proteins remained within the continuous phases. In contrast, CNTO3930 formed only one phase, and its Raman spectra contained bands at 1240, 1670, 1270 and 1655 cm,(−1) demonstrating homogeneous distribution of components. Our results indicate that this plate-based method utilizing confocal Raman spectroscopy to probe liquid-liquid phases in mAb/serum mixtures can provide a screen for phase separation of mAb candidates in a discovery setting. Taylor & Francis 2014-11-03 /pmc/articles/PMC4622053/ /pubmed/25484036 http://dx.doi.org/10.4161/19420862.2014.975100 Text en © 2014 The Author(s). Published with license by Taylor & Francis Group, LLC http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
spellingShingle Reports
Baker, Audrey E
Mantz, Amber R
Chiu, Mark L
Raman spectroscopy characterization of antibody phases in serum
title Raman spectroscopy characterization of antibody phases in serum
title_full Raman spectroscopy characterization of antibody phases in serum
title_fullStr Raman spectroscopy characterization of antibody phases in serum
title_full_unstemmed Raman spectroscopy characterization of antibody phases in serum
title_short Raman spectroscopy characterization of antibody phases in serum
title_sort raman spectroscopy characterization of antibody phases in serum
topic Reports
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622053/
https://www.ncbi.nlm.nih.gov/pubmed/25484036
http://dx.doi.org/10.4161/19420862.2014.975100
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