Biophysical characterisation of thermal-induced precipitates of recombinant anthrax protective antigen: evidence for kinetically trapped unfolding domains in solid-state

Ashok Ganesan, Allan Watkinson, Barry Moore

Research output: Contribution to journalArticle

12 Citations (Scopus)


Insoluble aggregation or precipitation is one of the most common degradation pathways observed for biotherapeutics; despite this, the structural mechanisms by which this occurs remain poorly understood due to difficulties associated with biophysical characterisation of protein particulates. To address this knowledge gap, we developed a solid-state circular dichroism (CD) technique, which allows in situ measurements of the secondary and tertiary structural changes associated with the formation of visible therapeutic protein aggregates. We demonstrate how solid-state CD, in conjunction with other biophysical and computational methods can aid in gaining valuable insights into the mechanisms and pathways of thermal-induced precipitation of Bacillus anthracis recombinant protective antigen (rPA), the primary immunogen of anthrax subunit vaccine. Using these methods, we show the domains d3 and d4 are the most labile of the four structurally distinct domains of rPA and play the critical role in nucleating the cascade of unfolding and aggregation. During the assembly process, the domains d1 and d2 become kinetically trapped within the insoluble aggregate and reveal previously intractable distinct tertiary structural elements of the rPA native structure. These findings reveal a uniquely detailed insight into the role of rPA domains on protein stability and provide a mechanistic framework for thermal-induced unfolding and precipitation. It also shows that solid-state CD provides a novel approach in characterising protein precipitation that may facilitate rational improvements to the stability of biopharmaceuticals.
Original languageEnglish
Pages (from-to)475–484
Number of pages10
JournalEuropean Journal of Pharmaceutics and Biopharmaceutics
Issue number3
Early online date7 Jun 2012
Publication statusPublished - Nov 2012


  • biophysical characterisation
  • thermal-induced
  • precipitates
  • recombinant anthrax
  • protective antigen
  • unfolding domains
  • solid-state
  • evidence
  • kinetically trapped

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