4.7 Article

Protein Engineering and HDX Identify Structural Regions of G-CSF Critical to Its Stability and Aggregation

Journal

MOLECULAR PHARMACEUTICS
Volume 19, Issue 2, Pages 616-629

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acs.molpharmaceut.1c00754

Keywords

aggregation; formulation; protein engineering; stability; intermediate

Funding

  1. Engineering and Physical Sciences Research Council (EPSRC) Centre for Doctoral Training in Emergent Macromolecular Therapies [EP/L015218/1]
  2. EPSRC Future Targeted Healthcare Manufacturing Hub [EP/P006485/1, EP/I033270/1]
  3. EPSRC [EP/I033270/1, EP/P006485/1] Funding Source: UKRI

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Protein engineering and formulation for therapeutic proteins present a major challenge in the biopharmaceutical industry due to the need for prolonged shelf-life. Understanding the impact of mutations and formulations on protein structure and dynamics can lead to more predictive approaches for improvement. Studies using intrinsic fluorescence analysis and hydrogen deuterium exchange mass spectrometry have revealed insights into the reorganization and stabilization of specific protein loops, providing valuable information for enhancing protein stability and reducing aggregation rates.
The protein engineering and formulation of therapeutic proteins for prolonged shelf- life remain a major challenge in the biopharmaceutical industry. Understanding the influence of mutations and formulations on the protein structure and dynamics could lead to more predictive approaches to their improvement. Previous intrinsic fluorescence analysis of the chemically denatured granulocyte colony-stimulating factor (G-CSF) suggested that loop AB could subtly reorganize to form an aggregation-prone intermediate state. Hydrogen deuterium exchange mass spectrometry (HDX-MS) has also revealed that excipient binding increased the thermal unfolding transition midpoint (Tm) by stabilizing loop AB. Here, we have combined protein engineering with biophysical analyses and HDX-MS to reveal that increased exchange in a core region of the G-CSF comprising loop AB (ABI, a small helix, ABII) and loop CD packed onto helix B and the beginning of loop BC leads to a decrease in Tm and higher aggregation rates. Furthermore, some mutations can increase the population of the aggregation-prone conformation within the native ensemble, as measured by the greater local exchange within this core region.

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