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The Missing Link: Connecting Cultivation Conditions and Refolding Performance via Inclusion Body Biophysical

Matthias Rüdt1, Aymerick Bussien1, Joan Cortada-Garcia2

  • 1Institute of Life Sciences, School of Engineering, HES-SO University of Applied Sciences and Arts Western, Delémont, Switzerland.

Biotechnology and Bioengineering
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PubMed
Summary

Cultivation conditions impact inclusion body (IB) properties, influencing therapeutic protein refolding. Optimizing IB size and minimizing amyloid structures enhances bioprocess efficiency in pharmaceutical production.

Keywords:
biophysical characterizationcultivation conditionsdesign of experimentsinclusion bodiesprotein refoldingspectroscopystatistical modeling

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Area of Science:

  • Biotechnology
  • Bioprocess Engineering
  • Protein Chemistry

Background:

  • Inclusion bodies (IBs) are common in recombinant protein production using Escherichia coli.
  • Cultivation conditions are known to affect protein production but their link to refolding performance is not well understood.

Purpose of the Study:

  • To investigate how cultivation conditions influence the biophysical properties of inclusion bodies.
  • To establish the relationship between inclusion body properties and refolding performance for therapeutic proteins.

Main Methods:

  • A design of experiments approach was used to study cultivation parameters (temperature, pH, feed rate).
  • Biophysical properties of inclusion bodies (hydrophobicity, secondary structure, particle size) were characterized.
  • The impact of these properties on refolding efficiency was analyzed.

Main Results:

  • Higher feed rates and temperatures increased product titer and inclusion body size.
  • Larger inclusion bodies improved refolding, while amyloid structures hindered solubilization.
  • Increased protein content in inclusion bodies negatively affected refolding yield.

Conclusions:

  • Inclusion body biophysical properties are critical for linking upstream cultivation and downstream refolding.
  • Understanding these properties offers insights to improve the robustness and efficiency of bioprocesses.
  • Optimizing cultivation conditions can enhance therapeutic protein production via inclusion body engineering.