Bal Vivekananda, Hong Moo Sun, Wolfrum Jacqueline M, Barone Paul W, Springs Stacy L, Sinskey Anthony J, Kotin Robert M, Braatz Richard D
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, United States.
School of Chemical and Biological Engineering, Seoul National University, Seoul 08826, Republic of Korea.
Cryst Growth Des. 2025 May 27;25(11):3687-3696. doi: 10.1021/acs.cgd.4c01720. eCollection 2025 Jun 4.
Crystallization of proteins, specifically proteins of medical relevance, is performed for various reasons, such as to understand the protein structure and to design therapies. Obtaining kinetic constants in rate laws for nucleation and growth of advanced biotherapeutics such as capsids, an assembly of macromolecules, is challenging and essential to the design of crystallization processes. In this work, coupled population balance and species balance equations are developed to extract nucleation and growth kinetics for the crystallization of recombinant adeno-associated virus (rAAV) capsids. A comparison of model results with that of experimental data for capsid crystallization in a hanging-drop vapor diffusion system shows that the slow rate of vapor diffusion from the droplet controls the initial nucleation and growth processes, and the capsid nucleation occurs via heterogeneous nucleation in the microdroplet. Results also show that the capsids, which are of very high molecular weight (∼3.6 MDa), have a similar tendency to nucleate as small organic molecules such as glycine (∼75 Da), low-molecular-weight proteins, and small-molecule active pharmaceutical ingredients due to their ball-shaped outer structure/shape. Capsids also show a prolonged nucleation period as for proteins and other macromolecules but have a slow growth rate with a growth rate prefactor seven orders of magnitude smaller than that of lysozyme. The capsid crystal growth rate is weakly sensitive to supersaturation compared to lysozyme and is limited by the transport of capsids due to slow Brownian motion resulting from the very high molecular weight.
蛋白质的结晶，特别是具有医学相关性的蛋白质结晶，出于各种原因而进行，例如了解蛋白质结构和设计治疗方法。对于诸如衣壳（一种大分子组装体）等先进生物治疗剂的成核和生长速率定律中获取动力学常数，既具有挑战性，又对结晶过程的设计至关重要。在这项工作中，开发了耦合的种群平衡和物种平衡方程，以提取重组腺相关病毒（rAAV）衣壳结晶的成核和生长动力学。将模型结果与悬滴气相扩散系统中衣壳结晶的实验数据进行比较表明，液滴中缓慢的气相扩散速率控制着初始成核和生长过程，并且衣壳成核是通过微滴中的异相成核发生的。结果还表明，分子量非常高（约3.6 MDa）的衣壳，由于其球形的外部结构/形状，与诸如甘氨酸（约75 Da）、低分子量蛋白质和小分子活性药物成分等小有机分子具有相似的成核倾向。衣壳也像蛋白质和其他大分子一样表现出较长的成核期，但生长速率较慢，其生长速率前置因子比溶菌酶小七个数量级。与溶菌酶相比，衣壳晶体生长速率对过饱和度的敏感性较弱，并且由于极高分子量导致的缓慢布朗运动，衣壳的传输限制了其生长速率。
