Abstract
Formulation conditions have a significant influence on the degree of freeze/thaw (FT) stress-induced protein instabilities. Adding cryoprotectants might stabilize the induced FT stress instabilities. However, a simple preservation of protein stability might be insufficient and further methods are necessary. This study aims to evaluate the addition of a heat cycle following FT application as a function of different cryoprotectants with lysozyme as exemplary protein. Sucrose and glycerol were shown to be the most effective cryoprotectants when compared to PEG200 and Tween20. In terms of heat-induced reversibility of aggregates, glycerol showed the best performance followed by sucrose, NaCl and Tween20 systems. The analysis was performed using a novel approach to visualize complex interplays by a clustering and data reduction scheme. In addition, solubility and structural integrity were measured and confirmed the obtained results.Graphic abstract
Highlights
Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users. Freezing and thawing enable a higher degree of flexibility during manufacturing and improve long-term stability during storage [1,2,3]
An investigation of the reversibility/dissociability of FT stress-induced protein aggregates as a function of different excipients using heat cycling is currently missing and promises new insights when dealing with reversible aggregation during bioprocessing
The curves were integrated, using the function integral available in MATLAB (Version 2019a), and the areas from 0 M to 2.5 M salt and from 0 mg/mL to 25 mg/mL protein were calculated. This aim of the study was to evaluate the following: (a) whether the influence of different excipients on the longterm protein stability of FT stressed formulations can be followed by the creation of multidimensional protein phase diagram (MPPD) and (b) whether the induced instabilities are reversible by a simple heat treatment
Summary
Protein aggregates can appear through different mechanisms [21], depending on the protein surface charge, conformational changes, and excipients in the solutions. These parameters influence intermolecular and intramolecular interactions of proteins and/or excipients. It was shown to be possible to dissociate reversible protein aggregates by special treatments, for example through the application of heat [26] This said, heat might cause protein denaturation and/or induce irreversible aggregation [21]. An investigation of the reversibility/dissociability of FT stress-induced protein aggregates as a function of different excipients using heat cycling is currently missing and promises new insights when dealing with reversible aggregation during bioprocessing
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