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Joint Plenary Session - A Paradigm Shift – Characterisation, Stability and Analytical Method Development for Next Generation Therapeutics
Heightened characterization of protein biotherapeutics typically involves various ultrahigh resolution LC/MS methods to confirm the expected primary structure and posttranslational modifications. However, application of these MS-based methods during clone selection and cell culture process development has yielded vital product quality information at the molecular level that helped steer process development, enabling the selection of more robust cell lines and increased process understanding and consistency. In this presentation, we will share several experiences and lessons we have learned in the application of ultrahigh-resolution MS-based methods for bioprocess development.
Late Morning Session
Imaged capillary isoelectric focusing (iCIEF) has quickly arisen as a charge profiling technique. It is quicker and more robust than its conventional counterpart, cIEF. Nevertheless, further characterization of the isoforms on MS remained an issue. By combining precise mobilization and a novel cartridge design, the CEInfinite addresses fractionation and allows the accurate recuperation of isoforms for downstream analyses.
Poster 1: FTIR spectroscopy as a multi-parameter analytical tool for stability studies and batch consistency testing of therapeutic proteins
Allison Derenne, Spin-Off Developer, Universite libre de Bruxelles
Early Afternoon Session
Talk 1: An Integrated LC-MS/MS Strategy for Comprehensive Detection and Quantification of Host Cell Proteins in Biopharmaceuticals
Donald Walker, Genentech-A Member of the Roche Group
Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has assumed an increasingly important role for characterization of host cell protein (HCP) impurities in biopharmaceuticals, with a variety of options now available to deal with the specific challenges of HCP identification and quantification. We present an integrated LC-MS/MS strategy that is comprehensive, flexible, and robust and can easily be adapted to meet the specific sensitivity and throughput requirements needed for today’s competitive bioprocess development environment. Beginning with HCP identification, we describe a robust 1D LC –MS/MS method with the speed necessary to support process development. Next we describe an offline 2D LC-MS/MS method that can be easily optimized to achieve higher sensitivity levels. Following HCP identification, targeted analysis can be explored which use comprehensive ion libraries to track and quantify all HCPs through in-process purification pools. Finally, absolute quantification is discussed using specific examples to show high sensitivity methods for tracking levels of specific HCPs using known amounts of protein or peptide standards. These methods in combination provide the necessary tools for generation of timely, reliable data, and allow development of purification processes which result in products with low and consistent levels of HCPs.
Talk 1: Forced Degradation Studies to Demonstrate Formulation Limits
Olivier Brass, Formulation Development Unit Head, Sanofi Pasteur, France
Talk 2: Determination of the forced degradation conditions for generating antibody and ADC samples with desired degradation levels
Forced degradation is used for multiple purposes during drug development, including the evaluation of the stability indicating nature of the analytical methods. The typical stress conditions applied to protein therapeutics are exposures to low and high pH, UV and white fluorescent light, heat and oxidizing agents. To achieve the desired degradation level, the stressing conditions need to be established. The presentation will discuss the methodologies for forced degradation applied to an antibody and its conjugate (ADC).
Paul Weisbach, Scientist I, Analytical Pharmaceutical Sciences, Immunogen, USA
Multi-angle and dynamic light scattering have become essential for the characterization of biotherapeutics, providing key biophysical properties and stability evaluation. This talk will focus on advanced light scattering tools for high-throughput, low-volume applications such as early stage characterization, developability assessment and formulation. A common microwell-plate format streamlines the workflow between these and other measurement technologies.
Late Afternoon Session
Glycosylation is frequently a critical quality attribute of biotherapeutics, making the characterization of N-glycans an essential part of the development process. We present a rapid N-glycan sample preparation platform with a choice of labels for LC, LC-MS and CE applications. The Gly-Q CE platform enables relative N-glycan quantification for up to 96 cell culture samples within a single workday.
Multi-spectral imaging flow cytometry (MIFC) is an established analytical method for cellular analysis, however has only recently been evaluated for characterization of sub-visible particles in therapeutic formulations1 despite numerous favorable attributes including:
• Simultaneous collection of bright-field, side-scatter, and fluorescent imagery
• Sensitive detection of particles 100 nm-100 μm
• High image quality using 20X-60X magnification objectives
• 100% sampling efficiency using hydrodynamic focusing
• Small sample volume requirement (20 μL)
• Linear concentration range up to 100 million/mL
• Wide flow cell (250 μm) minimizes clogs
Assorted case studies using MIFC for analysis of protein and vaccine formulations will be presented, with an emphasis on measurements and samples that pose challenges for current techniques. First, MIFC sensitivity was compared to dynamic imaging. While concentration measurements matched for polystyrene beads 2 µm and up, MIFC measured higher concentrations of protein aggregates, especially for particles smaller than 10 µm. Interestingly, one sample containing aggregated lysozyme including particles >25 µm were undetected by dynamic imaging but detected using MIFC by labeling particles with ProteoStat fluorescence dye. Next, protein aggregates and silicone oil droplets were classified using MIFC by labeling samples with fluorescent dyes specific to each particle type (ProteoStat and BODIPY, respectively), demonstrating that particles can be classified independent of size. This approach also allowed measurement of protein aggregate-silicone oil complexes and protein adsorption to the surface of silicone oil droplets. Finally, MIFC was used to directly measure highly concentrated formulations. In one case, concentrated glucagon fibrils were evaluated for amyloid conformation using ThioFlavinT fluorescent dye, and size-distribution analysis revealed that large glucagon fibrils disassociate upon sample dilution. In a second case, interaction of adjuvant particles with protein was evaluated using Nile Red, which showed protein adsorbs to large adjuvant particles and causes disassociation. For the third case, concentrated virosomes were detected using MIFC using Nile Red, and swarm detection could be visualized in collected imagery, indicating samples must be diluted for accurate concentration measurements. The results demonstrate that MIFC overcomes important challenges faced by current analytical methods for analysis of therapeutic formulations, including detection of small and transparent particles, direct analysis of highly concentrated formulations, as well as fluorescence characterization of particle type, heterogeneous interactions, and chemical composition.
Philae is a comet Lander, part of the ESA Rosetta Mission to comet 67P/Churyumov-Gerasimenko. After about ten years of development and a ten-year cruise through the solar system it successfully landed on the nucleus of the comet on November 12th, 2014.
Since the anchoring harpoons, which were expected to fix the lander to ground, did not work, Philae bounced in the low gravity environment, and only came to rest after a 2 hour’s “hop” in an unforeseen area on the comet surface. Fortunately, the scientific instruments, including cameras, mass spectrometers, a magnetometer and a radar instrument could be operated, and fascinating, unprecedented scientific results have been obtained from the surface of an active comet.
Interplanetary space missions, including a long development phase and elaborated qualification tests do have similarities with pharmaceutical research, where time-spans between the concepts for a new drug until readiness for marketing can be similar.
Rosetta is an ESA mission with contributions from its member states and NASA. Rosetta's Philae Lander is provided by a consortium led by DLR, MPS, CNES and ASI with additional contributions from Hungary, UK, Finland, Ireland and Austria.