Bioprocess and Biosystems Engineering

, Volume 43, Issue 2, pp 179–192 | Cite as

A phase diagram-based toolbox to assess the impact of freeze/thaw ramps on the phase behavior of proteins

  • Anna Katharina Wöll
  • Monika Desombre
  • Lena Enghauser
  • Jürgen HubbuchEmail author
Research Paper


The influence of process parameters during freeze/thaw (FT) operations is essential for the preservation of the protein stability/activity during production and storage processes in the biopharmaceutical industry. Process parameters, such as FT ramps, the final storage time and temperature, affect the occurring FT stress onto the target protein in different ways. FT stress includes cold denaturation, freeze concentration, and ice crystal formation which can result in protein aggregation. To visualize the impact of variations in FT ramps, descriptors such as solubility, phase behavior and crystal morphology were evaluated. The phase diagram-based toolbox in combination with an HTS-compatible cryo-device allowed the identification of suitable ramping schemes during FT operations. It could be clearly shown that rapid operations are needed above the glass transition temperature of the target protein to circumvent precipitation during FT cycles. Finally, a stability index is introduced which allows ranking of the systems investigated.

Graphic abstract


Temperature ramps Phase diagram Stability index Lysozyme 


Compliance with ethical standards

Conflict of interest

The authors report no conflict of interest.

Supplementary material

449_2019_2215_MOESM1_ESM.docx (489 kb)
Supplementary file1 (DOCX 489 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Anna Katharina Wöll
    • 1
  • Monika Desombre
    • 1
  • Lena Enghauser
    • 1
  • Jürgen Hubbuch
    • 1
    Email author
  1. 1.Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation EngineeringKarlsruhe Institute of Technology (KIT)KarlsruheGermany

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