Determination of glycation levels in Erwinia chrysanthemi asparaginase drug product by liquid chromatography - mass spectrometry.

Erwinase (Erwinia chrysanthemi L-asparaginase) Drug Product (DP) is a freeze-dried formulation with a three-year shelf life at 2-8 °C, and an established safety, stability and efficacy profile over the more than three decades of clinical use. Seven Erwinase® DP batches, released over a 7-year period, were screened by reversed-phase liquid chromatography coupled to time-of-flight mass spectrometry for glycation levels. This modification is a known and natural consequence of exposure of Erwinase Drug Product to glucose excipients in stabilizing formulations. Although glycation is detected in current release and stability methods, glycation, including the conditions under which this reaction occurs, has not been previously characterised in detail. We have found that glycation levels of different DP lots generally correlated with age, when they were stored at low temperature. This suggests that the glycation reaction continues over time within the Drug Product formulation in the lyophilised state, even under low temperature (+2-8 °C) conditions. We were also able to examine glycation levels of one DP lot, Lot D, held under long term stability at 3 different temperatures over a 5-year period. The 2 samples held at -20 °C and -80 °C, were glycated to levels of 12% and 17%, respectively. However, the DP Lot D sample held at +2-8 oC in this time period was found to be glycated to a level of 35.6%, with multiple glycations of individual subunits observed. For analytical reference materials, it is important to keep parameters such as glycation levels as constant as possible, to avoid a 'moving target' with respect to comparisons with release and stability testing. These data suggest that storage of DP as reference standards at a lower temperature (e.g., -20 °C) can significantly reduce levels of glycation over the longer time periods required for analytical reference standards.

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.

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.