Webinar Description:

This webinar will present workflows for biotherapeutic characterization, using Microfluidic Modulation Spectroscopy.

Workflows:

• Higher order structure similarity assessment in bioformulation
• Stability assessment workflow in biopharmaceuticals

We will feature speakers from Acceleron and University of Delaware with case studies

Acceleron Pharma: Analytical Strategies for the Characterization of the Higher-Order Structure of a Bi-Specific Fusion Protein
Robust analytical methods and regulatory guidelines help to control critical quality attributes (CQAs) that could potentially impact safety and efficacy of heterogeneous biotherapeutics. Structurally misfolded impurities in a biotherapeutic process are critical quality attributes (CQAs) that require analytical strategies to detect subtle differences in higher-order structure (HOS) A further challenge is that these methods must be able to robustly measure these differences in an ensemble of heterogeneous product variants. In this study we have purified a misfolded impurity existing at low abundance in an early stage purification process, for the purposes of structural elucidation and to evaluate optimal method development strategies for this product variant.
Analysis was done to structurally characterize the size, charge and post translational modifications using methods typically applied as critical quality attribute control strategies. We have also applied methods specifically targeting higher-order structure (HOS) to evaluate their utility at differentiating the misfolded conformer and the desired product. Results from this comprehensive characterization of a misfolded conformer help inform which analytical methods are best at measuring impactful HOS differences in biopharmaceutical processes. We found that specific methods including CD, Trp-fluorescence, and a novel FT-IR technique (Microfluidic Modulation Spectroscopy) were necessary to resolve this misfolded variant and should be implemented in early development control strategies for product impurities. High resolution techniques such as microfluidic modulation spectroscopy (MMS) and HDX-MS methods have potential for robust and quantitative measures of HOS changes during process development and product lifecycle management.

University of Delaware: Impact of low temperatures and high pressures on monoclonal antibody higher order structure
Aggregation of monoclonal antibodies at any stage of manufacturing, storage, or patient delivery can negatively affect product quality, including safety and efficacy, and thus needs to be detected if it occurs. Current methods of forced degradation at high temperatures and extreme pH can be insufficient due to potentially different aggregation mechanisms at conditions of interest. An isochoric method was developed to study protein aggregation below the freezing temperature, and closer to temperatures of interest (below freezing). Toward the continued development and improvement of this method that is used to predict long term stability, it is critical to characterize irreversible changes in secondary structure induced by cold temperatures and high pressures. An understanding of secondary structure perturbations would provide greater insight into how such changes cause aggregation at these conditions. This may improve empirical kinetic modeling used to make long term predictions of stability.
It remains an open question as how best to apply biophysical methods that characterize higher order structure and use the resulting data to make technical decisions during commercialization. The present work uses a next-generation infrared spectroscopy technique, Microfluidic Modulation Spectroscopy (MMS), to provide intermediate-level resolution structural information. Intrinsic fluorescence was used to observe the in situ effects of isochoric conditions in an effort to characterize aggregation prone intermediates. In this webinar, we will review cold temperature and pressure effects on monoclonal antibody secondary structure using MMS. Monitoring irreversible changes in protein structure after cold temperature and high-pressure incubation can enhance formulation development, and aid in the understanding of mechanisms of aggregation that are accelerated or probed by cold temperature and elevated pressure.

Learning Objectives:

• Higher order structure of fusion proteins
• Higher order structure of monoclonal antibodies
• Higher order structure similarity assessment in bioformulation
• Stability assessment workflow in biopharmaceuticals

Speakers:

Dr. Timothy Keefe
Sr. Analytical Development Scientist and Manager
Acceleron Pharma

Tim Keefe is a Sr. Scientist at Acceleron Pharma focused on analytical method development and characterization of protein therapeutics. In the early years of his career Tim applied his experience in mass spectrometry approaches to protein identifications and structural characterization. Throughout his career Tim has taken interest in orthogonal characterization techniques and is now interested in structural elucidation of protein HOS. Tim recently received his Ph.D. in Chemistry from UMass Lowell during which time he was involved in structural elucidation of the HOS of a bi-specific fusion protein. Tim lives in Arlington, MA along with his wife and twins of 11 years.

Jordan E. Berger
Department of Chemical & Biomolecular Engineering
University of Delaware

Jordan E. Berger is a graduate student at the University of Delaware, Department of Chemical and Biomolecular Engineering and is pursuing a PhD. He works with his advisor Christopher J. Roberts on developing methods to predict protein aggregation as well as understand the fundamental phenomena at low temperatures and high pressures that contribute to protein (in)stability.
For more information, please visit: http://research.che.udel.edu/research_groups/roberts/index.html#top

Valerie Ivancic
Application Scientist
RedShifBio

Valerie Ivancic is an Application Scientist at RedShifBio. She was involved in one of the first Beta tests for the AQS3pro while in graduate school at Clark University in Worcester, MA. In graduate school, Valerie worked in a Biophysical Chemistry lab gaining experience in circular dichroism, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, mass spectrometry, fast-performance liquid chromatography, transmission electron microscopy, and computer simulations in order to address new ways of detecting, inhibiting, and degrading amyloid assemblies.