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标题：A stepwise mechanism for aqueous two-phase system formation in concentrated antibody solutions
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作者：Bradley A. Rogers ; Bradley A. Rogers ; Kelvin B. Rembert 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2019
卷号：116
期号：32
页码：15784-15791
DOI：10.1073/pnas.1900886116
出版社：The National Academy of Sciences of the United States of America
摘要：Aqueous two-phase system (ATPS) formation is the macroscopic completion of liquid–liquid phase separation (LLPS), a process by which aqueous solutions demix into 2 distinct phases. We report the temperature-dependent kinetics of ATPS formation for solutions containing a monoclonal antibody and polyethylene glycol. Measurements are made by capturing dark-field images of protein-rich droplet suspensions as a function of time along a linear temperature gradient. The rate constants for ATPS formation fall into 3 kinetically distinct categories that are directly visualized along the temperature gradient. In the metastable region, just below the phase separation temperature, T ph , ATPS formation is slow and has a large negative apparent activation energy. By contrast, ATPS formation proceeds more rapidly in the spinodal region, below the metastable temperature, T meta , and a small positive apparent activation energy is observed. These region-specific apparent activation energies suggest that ATPS formation involves 2 steps with opposite temperature dependencies. Droplet growth is the first step, which accelerates with decreasing temperature as the solution becomes increasingly supersaturated. The second step, however, involves droplet coalescence and is proportional to temperature. It becomes the rate-limiting step in the spinodal region. At even colder temperatures, below a gelation temperature, T gel , the proteins assemble into a kinetically trapped gel state that arrests ATPS formation. The kinetics of ATPS formation near T gel is associated with a remarkably fragile solid-like gel structure, which can form below either the metastable or the spinodal region of the phase diagram.
关键词：liquid;liquid phase separation ; upper critical solution temperature ; gelation ; monoclonal antibody ; temperature gradient microfluidics