LAPSE:2023.2993
Published Article
LAPSE:2023.2993
An Optimized Purification Design for Extracting Active ADAMTS13 from Conditioned Media
February 21, 2023
Abstract
ADAMTS13 is a hemostatic enzyme that breaks down pro-thrombotic ultra-large multimers of von Willebrand factor (VWF). The deficiency of ADAMTS13 increases VWF-mediated thrombogenic potential and may lead to thrombotic thrombocytopenic purpura (TTP). Recently, clinical studies have shown the development of acquired TTP after COVID-19 infection and a correlation between low ADAMTS13 plasma levels and increased mortality. As a result, investigating ADAMTS13 as a potential recombinant therapeutic is of broad interest in the field of hematology. ADAMTS13 is considered challenging to purify in its biologically active state. Current purification methods utilize immobilized metal ions, which can interfere with ADAMTS13 metalloprotease activity. For this reason, we optimized an alternative strategy to isolate milligram quantities of highly active recombinant ADAMTS13 (rADAMTS13) from conditioned media after exogenous expression in human cell line, HEK293. HEK293 cells stably expressing C-terminal V5-His-tagged ADAMTS13 were grown in two parallel systems, culture bottles and flasks, for identifying an optimal cultivation strategy. Subsequently, we employed anion exchange followed by anti-V5-tag affinity chromatography to purify rADAMTS13, and extracted rADAMTS13 of high specific activity while preserving its native post-translational modifications. In addition, this process has been optimized and scaled up to produce active rADAMTS13 at levels sufficient for laboratory-scale structural, enzymatic, and biochemical studies.
ADAMTS13 is a hemostatic enzyme that breaks down pro-thrombotic ultra-large multimers of von Willebrand factor (VWF). The deficiency of ADAMTS13 increases VWF-mediated thrombogenic potential and may lead to thrombotic thrombocytopenic purpura (TTP). Recently, clinical studies have shown the development of acquired TTP after COVID-19 infection and a correlation between low ADAMTS13 plasma levels and increased mortality. As a result, investigating ADAMTS13 as a potential recombinant therapeutic is of broad interest in the field of hematology. ADAMTS13 is considered challenging to purify in its biologically active state. Current purification methods utilize immobilized metal ions, which can interfere with ADAMTS13 metalloprotease activity. For this reason, we optimized an alternative strategy to isolate milligram quantities of highly active recombinant ADAMTS13 (rADAMTS13) from conditioned media after exogenous expression in human cell line, HEK293. HEK293 cells stably expressing C-terminal V5-His-tagged ADAMTS13 were grown in two parallel systems, culture bottles and flasks, for identifying an optimal cultivation strategy. Subsequently, we employed anion exchange followed by anti-V5-tag affinity chromatography to purify rADAMTS13, and extracted rADAMTS13 of high specific activity while preserving its native post-translational modifications. In addition, this process has been optimized and scaled up to produce active rADAMTS13 at levels sufficient for laboratory-scale structural, enzymatic, and biochemical studies.
Record ID
Keywords
ADAMTS13, purification, V5-tag, von Willebrand factor
Subject
Suggested Citation
Jankowska KI, Katneni U, Lin BC, Amarasinghe R, Phue JN, Wu WW, Hamasaki-Katagiri N, Jankowski W, Shen RF, Kimchi-Sarfaty C. An Optimized Purification Design for Extracting Active ADAMTS13 from Conditioned Media. (2023). LAPSE:2023.2993
Author Affiliations
Jankowska KI: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA [ORCID]
Katneni U: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA
Lin BC: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA [ORCID]
Amarasinghe R: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA
Phue JN: Facility for Biotechnology Resources, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA [ORCID]
Wu WW: Facility for Biotechnology Resources, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA [ORCID]
Hamasaki-Katagiri N: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA [ORCID]
Jankowski W: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA
Shen RF: Facility for Biotechnology Resources, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA
Kimchi-Sarfaty C: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA
Katneni U: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA
Lin BC: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA [ORCID]
Amarasinghe R: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA
Phue JN: Facility for Biotechnology Resources, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA [ORCID]
Wu WW: Facility for Biotechnology Resources, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA [ORCID]
Hamasaki-Katagiri N: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA [ORCID]
Jankowski W: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA
Shen RF: Facility for Biotechnology Resources, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA
Kimchi-Sarfaty C: Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US FDA, Silver Spring, MD 20993, USA
Journal Name
Processes
Volume
10
Issue
2
First Page
322
Year
2022
Publication Date
2022-02-08
ISSN
2227-9717
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Original Submission
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PII: pr10020322, Publication Type: Journal Article
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LAPSE:2023.2993
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https://doi.org/10.3390/pr10020322
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Feb 21, 2023
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Feb 21, 2023
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