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Control of process-induced asparaginyl deamidation during manufacture of Erwinia chrysanthemil-asparaginase

Paper ID Volume ID Publish Year Pages File Format Full-Text
34624 45037 2013 6 PDF Available
Title
Control of process-induced asparaginyl deamidation during manufacture of Erwinia chrysanthemil-asparaginase
Abstract

•Erwinia chrysanthemil-asparaginase tetramer partially dissociates on freeze–thaw.•Tetramer dissociation exposes protein residues susceptible to deamidation, but this dissociation is reversible.•The extent of deamidation may be measured by HPLC, capillary electrophoresis, and peptide mapping.•We show how to protect l-asparaginase residues from deamidation by allowing tetramer reassembly before alkaline cell lysis.

During the manufacture of the chemotherapeutic enzyme Erwinia chrysanthemil-asparaginase, a small proportion (approximately 5–15%) of acidic variants, including deamidated species, are observed. Although the deamidated forms appear to have similar specific activity and quaternary structure to the unmodified enzyme, monitoring and control of these forms is important from a regulatory perspective. The extent of Asn to Asp deamidation directly correlates with the time taken to thaw the Erwinia cells. Erwinial-asparaginase is a tetrameric enzyme containing one site, Asn281, theoretically very labile to deamidation due to the sequence Asn-Gly. Structurally, this part of the protein sequence is completely buried inside the tetramer, but solvent-exposed upon tetramer dissociation. During the cell thawing and alkaline lysis sequence of the process, lengthening the cell thaw times by up to 24 h allowed tetramer to reassociate, protected Asn281 from deamidation and reduced the acidic species content of the l-asparaginase from approximately 17% to 9% as measured by weak cation-exchange (WCX) HPLC. The correlation of cell thaw time with acidic species content was also confirmed using capillary zone electrophoresis (CZE) and peptide mapping. These studies demonstrate that cell thaw time is an important, if unexpected, control variable for l-asparaginase deamidation.

Keywords
Deamidation; l-Asparaginase; Erwinia; Cell freeze/thaw; Tetramer; Capillary electrophoresis
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Control of process-induced asparaginyl deamidation during manufacture of Erwinia chrysanthemil-asparaginase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 48, Issue 9, September 2013, Pages 1311–1316
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us