Hospira Zagreb, Zagreb, Croatia.
Eur J Pharm Biopharm. 2010 Nov;76(3):357-65. doi: 10.1016/j.ejpb.2010.09.006. Epub 2010 Sep 18.
Severe immunogenic and other debilitating human disorders potentially induced by protein aggregates have brought this phenomenon into the focus of biopharmaceutical science over the past decade. Depending on its driving forces, the process induced in the model protein rHuG-CSF may be either reversible or irreversible, resulting in the assembly of self-associated protein species or irreversible aggregates of various final morphologies. The aim of our work was to investigate the correlation between irreversible and reversible aggregation and the protective effect of non-specific formulation stabilisers, selected from the group of carbohydrates and polyols including trehalose, xylitol, cellobiitol, turanose, cellobiose, leucrose, lactitol, lyxose, and sorbitol, against both irreversible protein aggregation and reversible self-association processes of the rHuG-CSF. The formation of irreversible aggregates was thermally induced and evaluated using differential scanning calorimetry and size-exclusion chromatography. As opposed to the irreversible aggregation process, the process of self-association was induced by the agitation experiment by directly augmenting the protein solution contact surfaces. Absence of statistical connectivity between different stabilisers' ability to inhibit self-association or aggregation reactions indicates that these are two distinct physicochemical processes with different formulation stabilizing outcomes. Reaction mechanism of thermally induced aggregation observed in the study was in line with published literature data, while the reaction mechanism for self-association process was postulated. The postulate has been verified experimentally by isothermal calorimetry and agitation set of experiments conducted after size-exclusion chromatography and asymmetrical flow field-flow fractionation separation of monomeric, dimeric, trimeric, oligomeric, and large self-associated forms detected on multi-angle light scattering, fluorescence, UV, and refractive index detectors. Besides defining the mechanism and kinetic of self-association in stabilized rHuG-CSF formulations, special attention was also paid to the shifts and ranks of the free energy of the aggregation or self-association transition states.
在过去的十年中，蛋白质聚集物潜在引发的严重免疫和其他衰弱性人类疾病使这一现象成为生物制药科学的焦点。取决于其驱动力，模型蛋白 rHuG-CSF 中诱导的过程可能是可逆的，也可能是不可逆的，导致自身相关的蛋白质物种的组装或各种最终形态的不可逆聚集体。我们的工作旨在研究不可逆和可逆聚集之间的相关性，以及从碳水化合物和多元醇组中选择的非特异性制剂稳定剂对 rHuG-CSF 的不可逆蛋白质聚集和可逆自身聚集过程的保护作用，所选稳定剂包括海藻糖、木糖醇、纤维二糖醇、曲拉通、纤维二糖、莱苏糖、乳糖醇、木酮糖和山梨糖醇。不可逆聚集体的形成是通过差示扫描量热法和尺寸排阻色谱法热诱导和评估的。与不可逆聚集过程相反，自身聚集过程是通过直接增加蛋白质溶液接触表面的搅拌实验诱导的。不同稳定剂抑制自身聚集或聚集反应能力之间不存在统计学相关性，这表明这是两个具有不同制剂稳定效果的不同物理化学过程。研究中观察到的热诱导聚集反应的反应机制与已发表的文献数据一致，而自身聚集过程的反应机制则被假设。该假设通过等温量热法和在尺寸排阻色谱和不对称流场流分离后进行的搅拌实验得到了实验验证，在多角光散射、荧光、紫外和折射率检测器上检测到单体、二聚体、三聚体、寡聚体和大自身相关形式的分离。除了确定稳定的 rHuG-CSF 制剂中自身聚集的机制和动力学外，还特别关注聚集或自身聚集过渡态的自由能的位移和等级。
