Department of Applied Science and Technology, Politecnico di Torino, 24 corso Duca degli Abruzzi, Torino, 10129 Italy.
Dipartimento di Scienze del Farmaco, Università degli Studi del Piemonte Orientale "A. Avogadro", Largo Donegani 2/3, 28100 Novara, Italy.
J Pharm Sci. 2019 Jan;108(1):431-438. doi: 10.1016/j.xphs.2018.09.002. Epub 2018 Sep 14.
Biopharmaceuticals are often stored in a lyophilized form. However, stresses due to both the freezing and the drying steps of the lyophilization process can be harmful to protein stability, and appropriate excipients must be added to minimize detrimental effects. In this work, molecular dynamics was used to provide insight into the mechanisms of protein stabilization by different osmolytes, using lactate dehydrogenase as model protein. Our simulations indicate that good cryoprotectants are not always equally good as lyoprotectants, suggesting that synergistic effects may arise when different excipients are combined. This observation is in accordance with the experimental results. In fact, the enzymatic activity of lactate dehydrogenase after freeze-drying was investigated for various formulations, and the trend predicted by molecular dynamics was confirmed. More specifically, we found that the most effective stabilization of the protein structure is achieved when a good cryoprotectant is coupled with an efficient lyoprotectant. Ultimately, we propose a new approach to the design of formulations for protein-based therapeutics to be lyophilized, which combines simulations and experiments. In this new concept, the computational investigation allows a more knowledge-driven and targeted experimental campaign for the selection of the optimal excipients, making the whole process extremely time- and cost-effective.
生物制药通常以冻干形式储存。然而，冻干过程中的冷冻和干燥步骤所产生的压力会对蛋白质稳定性造成损害，因此必须添加适当的赋形剂以将有害影响最小化。在这项工作中，使用分子动力学模拟深入了解了不同渗透剂稳定蛋白质的机制，以乳酸脱氢酶作为模型蛋白。我们的模拟结果表明，良好的冷冻保护剂并不总是同样适合作为冻干保护剂，这表明当不同赋形剂结合使用时可能会产生协同作用。这一观察结果与实验结果一致。事实上，我们针对不同配方研究了乳酸脱氢酶在冷冻干燥后的酶活性，实验结果证实了分子动力学的预测趋势。更具体地说，我们发现当将良好的冷冻保护剂与有效的冻干保护剂结合使用时，可以实现对蛋白质结构的最有效稳定。最终，我们提出了一种新的方法来设计要冻干的蛋白质治疗药物制剂，该方法结合了模拟和实验。在这个新概念中，计算研究可以更有针对性地进行实验，以选择最佳赋形剂，从而使整个过程极具时间和成本效益。
