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Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes

CD19 is an essential protein in personalized CD19-targeting chimeric antigen receptor (CAR)-T cell-based cancer immunotherapies and CAR-T cell functionality evaluation. However, the recombinant expression of this “difficult to-express” (DTE) protein is challenging, and therefore, commercial access t...

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Autores principales: Billerhart, Magdalena, Hunjadi, Monika, Hawlin, Vanessa, Grünwald-Gruber, Clemens, Maresch, Daniel, Mayrhofer, Patrick, Kunert, Renate
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10341778/
https://www.ncbi.nlm.nih.gov/pubmed/37446069
http://dx.doi.org/10.3390/ijms241310891
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author Billerhart, Magdalena
Hunjadi, Monika
Hawlin, Vanessa
Grünwald-Gruber, Clemens
Maresch, Daniel
Mayrhofer, Patrick
Kunert, Renate
author_facet Billerhart, Magdalena
Hunjadi, Monika
Hawlin, Vanessa
Grünwald-Gruber, Clemens
Maresch, Daniel
Mayrhofer, Patrick
Kunert, Renate
author_sort Billerhart, Magdalena
collection PubMed
description CD19 is an essential protein in personalized CD19-targeting chimeric antigen receptor (CAR)-T cell-based cancer immunotherapies and CAR-T cell functionality evaluation. However, the recombinant expression of this “difficult to-express” (DTE) protein is challenging, and therefore, commercial access to the protein is limited. We have previously described the successful stable expression of our soluble CD19-AD2 fusion protein of the CD19 extracellular part fused with human serum albumin domain 2 (AD2) in CHO-K1 cells. The function, stability, and secretion rate of DTE proteins can be improved by culture conditions, such as reduced temperature and a shorter residence time. Moreover, glycosylation, as one of the most important post-translational modifications, represents a critical quality attribute potentially affecting CAR-T cell effector function and thus impacting therapy’s success. In this study, we increased the production rate of CD19-AD2 by 3.5-fold through applying hypothermic culture conditions. We efficiently improved the purification of our his-tagged CD19-AD2 fusion protein via a Ni-NTA-based affinity column using a stepwise increase in the imidazole concentration. The binding affinity to commercially available anti-CD19 antibodies was evaluated via Bio-Layer Interferometry (BLI). Furthermore, we revealed glycosylation patterns via Electrospray Ionization Mass Spectrometry (ESI–MS), and five highly sialylated and multi-antennary N-glycosylation sites were identified. In summary, we optimized the CD19-AD2 production and purification process and were the first to characterize five highly complex N-glycosylation sites.
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spelling pubmed-103417782023-07-14 Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes Billerhart, Magdalena Hunjadi, Monika Hawlin, Vanessa Grünwald-Gruber, Clemens Maresch, Daniel Mayrhofer, Patrick Kunert, Renate Int J Mol Sci Article CD19 is an essential protein in personalized CD19-targeting chimeric antigen receptor (CAR)-T cell-based cancer immunotherapies and CAR-T cell functionality evaluation. However, the recombinant expression of this “difficult to-express” (DTE) protein is challenging, and therefore, commercial access to the protein is limited. We have previously described the successful stable expression of our soluble CD19-AD2 fusion protein of the CD19 extracellular part fused with human serum albumin domain 2 (AD2) in CHO-K1 cells. The function, stability, and secretion rate of DTE proteins can be improved by culture conditions, such as reduced temperature and a shorter residence time. Moreover, glycosylation, as one of the most important post-translational modifications, represents a critical quality attribute potentially affecting CAR-T cell effector function and thus impacting therapy’s success. In this study, we increased the production rate of CD19-AD2 by 3.5-fold through applying hypothermic culture conditions. We efficiently improved the purification of our his-tagged CD19-AD2 fusion protein via a Ni-NTA-based affinity column using a stepwise increase in the imidazole concentration. The binding affinity to commercially available anti-CD19 antibodies was evaluated via Bio-Layer Interferometry (BLI). Furthermore, we revealed glycosylation patterns via Electrospray Ionization Mass Spectrometry (ESI–MS), and five highly sialylated and multi-antennary N-glycosylation sites were identified. In summary, we optimized the CD19-AD2 production and purification process and were the first to characterize five highly complex N-glycosylation sites. MDPI 2023-06-30 /pmc/articles/PMC10341778/ /pubmed/37446069 http://dx.doi.org/10.3390/ijms241310891 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Billerhart, Magdalena
Hunjadi, Monika
Hawlin, Vanessa
Grünwald-Gruber, Clemens
Maresch, Daniel
Mayrhofer, Patrick
Kunert, Renate
Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes
title Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes
title_full Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes
title_fullStr Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes
title_full_unstemmed Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes
title_short Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes
title_sort recombinant human cd19 in cho-k1 cells: glycosylation patterns as a quality attribute of high yield processes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10341778/
https://www.ncbi.nlm.nih.gov/pubmed/37446069
http://dx.doi.org/10.3390/ijms241310891
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