Structural Characterisation of Non-Deamidated Acidic Variants of Erwinia chrysanthemi L-asparaginase Using Small-Angle X-ray Scattering and Ion-Mobility Mass Spectrometry.

PURPOSE: Erwinia chrysanthemi L-asparaginase (ErA) is an enzyme commonly used in the treatment regimen for Acute Lymphoblastic Leukaemia (ALL). Biopharmaceutical products such as ErA must be monitored for modifications such as deamidation, typically using ion-exchange chromatography (IEX). Analysis of clinical-grade ErA using native IEX resolves a number of enzymatically-active, acidic variants that were poorly characterised. METHODS: ErA IEX variants were isolated and fully characterised using capillary electrophoresis (cIEF), LC-MS and LC-MS/MS of proteolytic digests, and structural techniques including circular dichroism, small-angle X-ray scattering (SAXS) and ion-mobility mass spectrometry (IM-MS). RESULTS: LC-MS, MS/MS and cIEF demonstrated that all ErA isolates consist mainly of enzyme lacking primary-sequence modifications (such as deamidation). Both SAXS and IM-MS revealed a different conformational state in the most prominent acidic IEX peak. However, SAXS data also suggested conformational differences between the main peak and major acidic variant were minor, based on comparisons with crystal structures. CONCLUSIONS: IEX data for biopharmaceuticals such as ErA should be thoroughly characterised, as the most common modifications, such as deamidation, may be absent.

Capillary isoelectric focusing of a difficult-to-denature tetrameric enzyme using alkylurea-urea mixtures.

Capillary isoelectric focusing (cIEF) is normally run under denaturing conditions using urea to expose any buried protein residues that may contribute to the overall charge. However, urea does not completely denature some proteins, such as the tetrameric enzyme Erwinia chrysanthemil-asparaginase (ErA), in which case electrophoresis-compatible alternative denaturants are required. Here, we show that alkylureas such as N-ethylurea provide increased denaturation during cIEF. The cIEF analysis of ErA in 8 M urea alone resulted in a cluster of ill-resolved peaks with isoelectric points (pI values) in the range 7.4 to 8.5. A combination of 2.0 to 2.2 M N-ethylurea and 8M urea provided sufficient denaturation of ErA, resulting in a main peak with a pI of 7.35 and an acidic species minor peak at 7.0, both comparing well with predicted pI values based on the sum of protein residue pKa values. Recombinant deamidated ErA mutants were also demonstrated to migrate to pI values consistent with predictions (pI 7.0 for one deamidation). The quantitation of ErA acidic species in samples from full-scale manufacturing (1.0-3.5% of total peak area) was found to be reproducible and linear. Use of alkylureas as denaturing agents in capillary electrophoresis and cIEF should be considered during biopharmaceutical assay development.

Robust quantitation of basic-protein higher-order aggregates using size-exclusion chromatography.

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.