Bye Jordan, Murray Kiah, Curtis Robin
Department of Chemical Engineering and Analytical Science, University of Manchester, Manchester M1 7DN, UK.
Biomedicines. 2021 Nov 9;9(11):1646. doi: 10.3390/biomedicines9111646.
A common strategy to increase aggregation resistance is through rational mutagenesis to supercharge proteins, which leads to high colloidal stability, but often has the undesirable effect of lowering conformational stability. We show this trade-off can be overcome by using small multivalent polyphosphate ions, adenosine triphosphate (ATP) and tripolyphosphate (TPP) as excipients. These ions are equally effective at suppressing aggregation of ovalbumin and bovine serum albumin (BSA) upon thermal stress as monitored by dynamic and static light scattering. Monomer loss kinetic studies, combined with measurements of native state protein-protein interactions and ζ-potentials, indicate the ions reduce aggregate growth by increasing the protein colloidal stability through binding and overcharging the protein. Out of three additional proteins studied, ribonuclease A (RNaseA), α-chymotrypsinogen (α-Cgn), and lysozyme, we only observed a reduction in aggregate growth for RNaseA, although overcharging by the poly-phosphate ions still occurs for lysozyme and α-Cgn. Because the salts do not alter protein conformational stability, using them as excipients could be a promising strategy for stabilizing biopharmaceuticals once the protein structural factors that determine whether multivalent ion binding will increase colloidal stability are better elucidated. Our findings also have biological implications. Recently, it has been proposed that ATP also plays an important role in maintaining intracellular biological condensates and preventing protein aggregation in densely packed cellular environments. We expect electrostatic interactions are a significant factor in determining the stabilizing ability of ATP towards maintaining proteins in non-dispersed states in vivo.
提高抗聚集性的一种常见策略是通过合理诱变对蛋白质进行超荷化，这会带来高胶体稳定性，但往往会产生降低构象稳定性的不良影响。我们表明，通过使用小的多价聚磷酸离子、三磷酸腺苷（ATP）和三聚磷酸（TPP）作为辅料，可以克服这种权衡。通过动态和静态光散射监测，这些离子在热应激下抑制卵清蛋白和牛血清白蛋白（BSA）聚集方面同样有效。单体损失动力学研究，结合对天然状态蛋白质-蛋白质相互作用和ζ电位的测量，表明这些离子通过结合并使蛋白质过度带电来提高蛋白质胶体稳定性，从而减少聚集体的生长。在研究的另外三种蛋白质核糖核酸酶A（RNaseA）、α-胰凝乳蛋白酶原（α-Cgn）和溶菌酶中，我们只观察到RNaseA的聚集体生长减少，尽管溶菌酶和α-Cgn也会发生多价磷酸离子的过度带电。由于这些盐不会改变蛋白质的构象稳定性，一旦更好地阐明决定多价离子结合是否会增加胶体稳定性的蛋白质结构因素，将它们用作辅料可能是稳定生物药物的一种有前景的策略。我们的发现也具有生物学意义。最近，有人提出ATP在维持细胞内生物凝聚物和防止蛋白质在密集的细胞环境中聚集方面也起着重要作用。我们预计静电相互作用是决定ATP在体内维持蛋白质非分散状态的稳定能力的一个重要因素。
