Temperature and pH-Dependent Behaviors of mAb Drugs: A Case Study for Trastuzumab

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Sert F., Hız D., Gülmez M., Cankurtaran S. E., Kayalan C. I., KURT H., ...More

Scientia Pharmaceutica, vol.90, no.1, 2022 (ESCI) identifier identifier

  • Publication Type: Article / Article
  • Volume: 90 Issue: 1
  • Publication Date: 2022
  • Doi Number: 10.3390/scipharm90010021
  • Journal Name: Scientia Pharmaceutica
  • Journal Indexes: Emerging Sources Citation Index (ESCI), Scopus, Academic Search Premier, CAB Abstracts, Chemical Abstracts Core, Chimica, EMBASE, International Pharmaceutical Abstracts, Veterinary Science Database, Directory of Open Access Journals
  • Keywords: thermal stability, conformational stability, colloidal stability, biophysical characterization, complementary tools
  • Istanbul Medipol University Affiliated: Yes


The distortions in the high-order structure of therapeutic monoclonal antibodies (mAbs) under different environmental conditions acutely affect mAb stability, resulting in altered safety, efficacy, and shelf-life profiles. The overall stability of mAbs depends on many factors, and it re-quires complementary techniques for an in-depth analysis. The stability of mAbs can be character-ized by differential centrifugal sedimentation (DCS), differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), and size exclusion chromatography (SEC) techniques. In this report, temperature-ramped dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy were employed as complementary tools to show how temperature and pH affect the aggregation of a model mAb, trastuzumab, in solution. The results showed that the aggregation onset temperature of trastuzumab defined by DLS was 75 °C, which decreases the amount of β-sheets and causes a slight increase in helix structures. Moreover, the melting temperature of trastuzumab was determined to be between 80–83 °C by temperature-ramped CD spectrophotometry, which is in line with the Tm of trastuzumab’s Fab region tested with DSC. Thus, unfolding and aggregation of trastuzumab start simultaneously at 75 °C, and unfolding triggers the aggregation. The tempera-ture-ramped CD and DLS methods are robust tools to determine the thermal behavior of biosimilars in various solution conditions. Their complementary usage provides solid scientific background for regulatory applications and a better understanding of mAb instability and its relationship with structural changes.