Production of a novel N‐terminal PEGylated crisantaspase

Crisantaspase is an asparaginase enzyme produced by Erwinia chrysanthemi and used to treat acute lymphoblastic leukemia (ALL) in case of hypersensitivity to Escherichia coli l‐asparaginase (ASNase). The main disadvantages of crisantaspase are the short half‐life (10 H) and immunogenicity. In this se...

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Bibliographic Details
Published in:Biotechnology and applied biochemistry 2019-05, Vol.66 (3), p.281-289
Main Authors: Torres‐Obreque, Karin, Meneguetti, Giovanna Pastore, Custódio, Débora, Monteiro, Gisele, Pessoa‐Junior, Adalberto, Rangel‐Yagui, Carlota
Format: Article
Language:English
Subjects:
Acute lymphoblastic leukemia
Asparaginase
Asparaginase - biosynthesis
Asparaginase - chemistry
Asparaginase - metabolism
biobetter
Bioreactors
Cation exchange
Cation exchanging
Chromatography
E coli
Enzymes
High temperature
Humans
Hypersensitivity
Immunogenicity
Ions
Kinetics
Leukemia
Lymphatic leukemia
l‐asparaginase
N‐terminal pegylation
Osmotic shock
pH effects
Polyethylene glycol
Polyethylene Glycols - chemistry
Polyethylene Glycols - metabolism
Precursor Cell Lymphoblastic Leukemia-Lymphoma - metabolism
Precursor Cell Lymphoblastic Leukemia-Lymphoma - therapy
Purification
site‐specific pegylation
Size exclusion chromatography
Video compression
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Summary:Crisantaspase is an asparaginase enzyme produced by Erwinia chrysanthemi and used to treat acute lymphoblastic leukemia (ALL) in case of hypersensitivity to Escherichia coli l‐asparaginase (ASNase). The main disadvantages of crisantaspase are the short half‐life (10 H) and immunogenicity. In this sense, its PEGylated form (PEG‐crisantaspase) could not only reduce immunogenicity but also improve plasma half‐life. In this work, we developed a process to obtain a site‐specific N‐terminal PEGylated crisantaspase (PEG‐crisantaspase). Crisantaspase was recombinantly expressed in E. coli BL21(DE3) strain cultivated in a shaker and in a 2‐L bioreactor. Volumetric productivity in bioreactor increased 37% compared to shaker conditions (460 and 335 U L−1 H−1, respectively). Crisantaspase was extracted by osmotic shock and purified by cation exchange chromatography, presenting specific activity of 694 U mg−1, 21.7 purification fold, and yield of 69%. Purified crisantaspase was PEGylated with 10 kDa methoxy polyethylene glycol‐N‐hydroxysuccinimidyl (mPEG‐NHS) at different pH values (6.5–9.0). The highest N‐terminal pegylation yield (50%) was at pH 7.5 with the lowest poly‐PEGylation ratio (7%). PEG‐crisantaspase was purified by size exclusion chromatography and presented a KM value three times higher than crisantaspase (150 and 48.5 µM, respectively). Nonetheless, PEG‐crisantaspase was found to be more stable at high temperatures and over longer periods of time. In 2 weeks, crisantaspase lost 93% of its specific activity, whereas PEG‐crisantaspase was stable for 20 days. Therefore, the novel PEG‐crisantaspase enzyme represents a promising biobetter alternative for the treatment of ALL.
ISSN:0885-4513
1470-8744
DOI:10.1002/bab.1723