Abstract:
:Detection of higher-order aggregates (HOA) using size-exclusion chromatography (SEC) was found to be variable for a basic protein, using exposed-silanol or diol-silica-based SEC columns. Preparations of the tetrameric biopharmaceutical enzyme Erwinia chrysanthemil-asparaginase (ErA), which has an isoelectric point of 8.6, were analysed using a diol-silica SEC column. Although the proportions of ErA main peak and octamer species were unaffected, HOA recovery and detection were extremely variable and had poor agreement with an orthogonal measurement technique, analytical ultracentrifugation (AUC). The observation that only HOA was selectively affected by non-specific silanol interactions was unexpected, so alternatives were sought. Coated-silica SEC columns improved the resolution and reproducibility of HOA detection for this alkaline-pI protein, and improved the agreement of HOA with the AUC method. Basic proteins, such as ErA, should be thoroughly evaluated in SEC method development, to ensure that resolution of larger aggregate species is not compromised.
journal_name
J Pharm Biomed Analjournal_title
Journal of pharmaceutical and biomedical analysisauthors
Gervais D,Downer A,King D,Kanda P,Foote N,Smith Sdoi
10.1016/j.jpba.2017.03.004subject
Has Abstractpub_date
2017-05-30 00:00:00pages
215-220eissn
0731-7085issn
1873-264Xpii
S0731-7085(16)31455-8journal_volume
139pub_type
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