Effects of precipitation process on the biophysical properties of highly concentrated proteins
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Journal of Pharmaceutical Investigation https://doi.org/10.1007/s40005-020-00471-2
ORIGINAL ARTICLE
Effects of precipitation process on the biophysical properties of highly concentrated proteins Dae Gon Lim1 · Jae Chul Lee1 · Dong Jun Kim1 · Seong Jun Kim1 · Hyun Woo Yu1 · Seong Hoon Jeong1 Received: 25 November 2019 / Accepted: 22 January 2020 © The Korean Society of Pharmaceutical Sciences and Technology 2020
Abstract Purpose One of the primary challenges in developing highly concentrated protein formulations is ensuring adequate stability of the protein product. Among the many available methods, cold solvent precipitation is useful, and it has been adopted for protein purification. This study aimed to characterize protein stability under various precipitation conditions and to determine the most stable precipitation conditions. Methods Lysozyme was used as a model protein to investigate the effects of various solvents and temperature conditions on the physicochemical properties. Size exclusion chromatography, dynamic light scattering, circular dichroism, and thermodynamic analysis are the primary tools for the protein characterization. Results Changes in the rehydrated particle size, secondary structure, thermodynamic stability, and in vitro activity of lysozyme were observed under various conditions. By analyzing the effect of temperature on protein characteristics, it has been discovered that protein precipitation at a temperature of 4 °C or below is optimal. During the precipitation at – 20 °C, the secondary structures of proteins are adversely affected by the formation of ice crystals. None of the solvents used as precipitants in this study affected the activity of the protein. However, changes in the secondary structure were minimal when ethanol and octanol were used. Conclusion In this study, the feasibility of various precipitation conditions was evaluated for stable reversible precipitation formation. Although further studies are needed, precipitation of proteins using various organic solvents could be used in the development of formulations that purify and concentrate proteins. Keywords Precipitation · Biophysical properties · Protein stability · High concentration
Introduction Therapeutic proteins such as monoclonal antibodies are often administered in high doses (several mg/kg). Especially, subcutaneous injection (volume limit 100 μm) and a large amount of small precipitates (0–20 μm) were observed (Table 2). Since ethanol and IPA are water-miscible, the lysozyme was exposed to a rapid environmental change, leading to fast precipitation and the formation of large polygon-like precipitates. However, when pentanol and octanol were used, lysozyme precipitated into relatively uniform sphere-like shapes (Fig. 2 c and d), and the distribution of the particles was relatively homogeneous when compared to those formed by ethanol and IPA. The average particle size of the precipitated lysozyme was 22 μm when precipitated with pentanol (Pen-lysozyme) and 29 μm when precipitated with octanol (Oct-lysozym
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