The effort to fully characterize and release a biotherapeutic to the market can be onerous. Typically, many tests are required to identify and monitor various attributes of the final product in order to ensure the safety and efficacy of the drug. These product quality attributes, or PQAs, consist of things such as the extent and type of glycosylation, or the existence and level of other post-translational modifications. Due to the diverse nature of these PQAs, many assays are often required which can be time-consuming and burdensome.
Liquid Chromatography Mass Spectrometry (LC-MS) can help. With LC-MS, a Multiple Attribute Methodology (MAM) can enable the characterization and monitoring of many different PQAs within a single assay. One assay can measure sequence identity, modification levels, impurities, and more. Additionally, while many conventional assays indirectly measure the PQA of interest, the LC-MS workflow for MAM is a direct measure of each attribute. Every peak points to a distinct PQA so you can quickly determine which attribute is changing from sample to sample.
The basic premise of the LC-MS workflow for MAM involves comparison of a biotherapeutic sample with a reference standard and relies upon a technique called peptide mapping used extensively in protein and biotherapeutic research. To start, the biotherapeutic reference or sample is first enzymatically digested into a mixture of peptides. This mixture is then injected onto an LC column to separate the peptides into individual peaks using a high-resolution system such as the SCIEX ExionLC AD. The effluent from the column is fed directly into a high mass accuracy mass spectrometer, such as the SCIEX TripleTOF® 6600 System or X500B QTOF System where the accurate molecular weights of the components within each LC peak are measured. For the reference standard, MS/MS fragment data are also acquired in order to initially identify each component. The resultant data files contain the retention times, molecular weights, fragment data (for the reference standard), and quantities of each component in the digest. A software comparison of the reference data file with the sample data file highlights any differences between the two.
A key aspect for success with the LC-MS workflow for MAM is the accurate and comprehensive characterization of the reference standard. SCIEX uses a technique called SWATH® Acquisition to exhaustively detect every component within the reference standard so that a comprehensive reference archive can be created. As a data independent technique, SCIEX SWATH Acquisition has been shown to not only detect more components within proteolytic digests, but to also be more reproducible than conventional MS/MS strategies used elsewhere – even across different laboratories.1
Another key aspect for success is the power and ease of the data processing component. The SCIEX LC-MS workflow for MAM uses one software package, BioPharmaView™ Software, to both characterize the reference standard and perform comparison studies. BioPharmaView enables the identification of attributes, definition of acceptance criteria, and creation of standard methods for implementation. BioPharmaView is then used to monitor PQAs, known impurities, and can detect and flag new unspecified impurities within samples. A report is then created of all PQAs and impurities and whether they “passed” or “failed” based on the acceptance criteria. The use of one intuitive and easy-to-use package for all data processing greatly simplifies and streamlines a workflow for MAM. Additionally, the ability to detect new and unspecified impurities is a valuable and vital feature included with the SCIEX LC-MS workflow.
The pressure to meet timelines and provide new and more detailed information to regulatory agencies continues to place enormous demands on manufacturing and development. An LC-MS workflow for biotherapeutic characterization can greatly simplify the overall number of required assays while delivering a more comprehensive analysis of the product quality.
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- Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry. Ben Collins, Christie Hunter, Yansheng Liu, Birgit Schilling, George Rosenberger, Samuel Bader, Daniel Chan, Bradford W. Gibson, Anne Claude Gingras, Jason Held, Mio Hirayama-Kurogi, Guixue Hou, Christoph Krisp, Brett Larsen, Liang Lin, Siqi Liu, Mark Molloy, Robert Moritz, Sumio Ohtsuki, Ralph Schlapbach, Nathalie Selevsek, Stefani Thomas, Shin Chen Tzeng, Hui Zhang, and Ruedi Aebersold, Nature Communications. 2017 Jun; 8 (291)