Centro de Química Estrutural, Departamento de Engenharia Química, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Department of Chemical and Biomolecular Engineering, University of Delaware, 590 Avenue 1743, Newark, Delaware 19713, United States.
Mol Pharm. 2021 Dec 6;18(12):4415-4427. doi: 10.1021/acs.molpharmaceut.1c00604. Epub 2021 Oct 26.
Biopharmaceutical formulations may be compromised by freezing, which has been attributed to protein conformational changes at a low temperature, and adsorption to ice-liquid interfaces. However, direct measurements of unfolding/conformational changes in sub-0 °C environments are limited because at ambient pressure, freezing of water can occur, which limits the applicability of otherwise commonly used analytical techniques without specifically tailored instrumentation. In this report, small-angle neutron scattering (SANS) and intrinsic fluorescence (FL) were used to provide analysis of protein tertiary structure/folding at temperatures as low as -15 °C utilizing a high-pressure (HP) environment (up to 3 kbar) that prevents water from freezing. The results show that the α-chymotrypsinogen A (aCgn) structure is reasonably maintained under acidic pH (and corresponding pD) for all conditions of pressure and temperature tested. On the other hand, reversible structural changes and formation of oligomeric species were detected near -10 °C via HP-SANS for ovalbumin under neutral pD conditions. This was found to be related to the proximity of the temperature of cold denaturation of ovalbumin ( ∼ -17 °C; calculated via isothermal chemical denaturation and Gibbs-Helmholtz extrapolation) rather than a pressure effect. Significant structural changes were also observed for a monoclonal antibody, anti-streptavidin IgG1 (AS-IgG1), under acidic conditions near -5 °C and a pressure of ∼2 kbar. The conformational perturbation detected for AS-IgG1 is proposed to be consistent with the formation of unfolding intermediates such as molten globule states. Overall, the approaches described here offer a means to characterize the conformational stability of biopharmaceuticals and proteins more generally under cold-temperature stress by the assessment of structural alteration, self-association, and reversibility of each process. This offers an alternative to current methods that are based on higher temperatures and subsequent extrapolation of the data and interpretations to the cold-temperature regime.
生物制药制剂可能会因冷冻而受损，这归因于低温下的蛋白质构象变化，以及与冰-液相界面的吸附。然而，在亚零摄氏度环境下直接测量蛋白质的展开/构象变化受到限制，因为在环境压力下，水会发生冻结，这限制了通常使用的分析技术的适用性，除非使用专门设计的仪器。在本报告中，小角中子散射（SANS）和固有荧光（FL）被用于在高达 3 千巴的高压（HP）环境下，在低至-15°C 的温度下提供蛋白质三级结构/折叠的分析，该环境可防止水冻结。结果表明，在测试的所有压力和温度条件下，酸性 pH（和相应的 pD）下的α-糜蛋白酶原 A（aCgn）结构都能得到合理保持。另一方面，在中性 pD 条件下，卵清蛋白在接近-10°C 的温度下通过 HP-SANS 检测到可逆结构变化和寡聚体的形成。这被发现与卵清蛋白的冷变性温度（约-17°C；通过等温化学变性和吉布斯-亥姆霍兹外推法计算）接近有关，而不是与压力有关。在接近-5°C 和约 2 千巴的酸性条件下，抗生物素蛋白 IgG1（AS-IgG1）的单克隆抗体也观察到明显的结构变化。检测到的 AS-IgG1 构象扰动被认为与展开中间态的形成一致，如无定形球蛋白状态。总的来说，这里描述的方法通过评估结构改变、自组装和每个过程的可逆性，为在冷温应激下更一般地表征生物制药和蛋白质的构象稳定性提供了一种手段。这为基于更高温度和随后将数据和解释外推到低温区的现有方法提供了一种替代方法。
