title: Robust quantitation of basic-protein higher-order aggregates using size-exclusion chromatography.
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.
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom. Electronic address: dave.gervais@portonbiopharma.com.
authors.forename: David
authors.initials: D
authors.lastname: Gervais
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom.
authors.forename: Andrew
authors.initials: A
authors.lastname: Downer
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom.
authors.forename: Darryl
authors.initials: D
authors.lastname: King
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom.
authors.forename: Patrick
authors.initials: P
authors.lastname: Kanda
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Foote
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom.
authors.forename: Stuart
authors.initials: S
authors.lastname: Smith
mesh_terms: D001215:Asparaginase / Q000032:analysis* / Q000378:metabolism*; D002850:Chromatography, Gel / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D004885:Erwinia / Q000201:enzymology*; D066329:Protein Aggregates / Q000502:physiology*; D017433:Protein Structure, Secondary
chemical_list: D066329:Protein Aggregates; D001215:Asparaginase
keywords: Higher-order aggregates; Non-specific interactions; Size-exclusion chromatography; l-Asparaginase