The present work evaluated for the first time the use of continuous slug flow crystallizer (CSFC) in bulk seeded protein crystallization, using lysozyme (LYZ) as the model protein. LYZ crystals pre-prepared in batch crystallizer were used as seeds. The crystallization was performed at supersaturation level below the metastable-zone-limit. The resultant crystal size distribution (CSD), CSD’s reproducibility, LYZ’s bioactivity, and crystallization efficiency as characterized by the space-time-yield (STY, mg/h·L) were determined. The results showed that the CSFC’s performance was governed by the flowrate, where a trade-off existed between crystal quality and crystallization efficiency upon varying the flowrate. At low flowrates, which reduced the shear rate and prolonged the residence time, well-defined large tetragonal crystals were produced attributed to suppressed secondary nucleation and extended crystal growth, but low STY due to difficulty in transporting the seeds/products crystals. Higher flow rates led to higher STY, but increased production of small non-tetragonal crystals that formed agglomerates. Compared to batch crystallizer, the CSFC produced LYZ crystals of similar average size, morphology, and bioactivity, but with roughly 50% lower STY, as the batch’s high-shear environment promoted secondary nucleation, hence higher crystallization rate. The CSD’s width and reproducibility was nevertheless significantly improved in CSFC.

本工作首次评估了使用溶菌酶（LYZ）作为模型蛋白，在大量种子蛋白结晶中使用连续流式结晶器（CSFC）。在分批结晶器中预先制备的LYZ晶体用作种子。结晶在低于亚稳区极限的过饱和水平下进行。确定了以时空产率（STY，mg / h·L）为特征的所得晶体尺寸分布（CSD），CSD的再现性，LYZ的生物活性和结晶效率。结果表明，CSFC的性能受流速的影响，在流速变化时，晶体质量和结晶效率之间存在权衡。在低流量下会降低剪切速率并延长停留时间，产生了明确的大四方晶体，这归因于二次晶核的抑制和晶体生长的延长，但是由于难以运送晶种/产品晶体，STY较低。较高的流速导致较高的STY，但增加了形成团块的小的非四方晶体的产量。与分批结晶器相比，CSFC生产的LYZ晶体具有相似的平均尺寸，形态和生物活性，但STY降低了约50％，这是因为分批的高剪切环境促进了二次成核，从而提高了结晶速率。然而，CSFC中CSD的宽度和可重复性得到了显着改善。但增加了形成团聚体的小非四方晶体的产量。与分批结晶器相比，CSFC生产的LYZ晶体具有相似的平均尺寸，形态和生物活性，但STY降低了约50％，这是因为分批的高剪切环境促进了二次成核，从而提高了结晶速率。然而，CSFC中CSD的宽度和可重复性得到了显着改善。但增加了形成团聚体的小非四方晶体的产量。与分批结晶器相比，CSFC生产的LYZ晶体具有相似的平均尺寸，形态和生物活性，但STY降低了约50％，这是因为分批的高剪切环境促进了二次成核，从而提高了结晶速率。然而，CSFC中CSD的宽度和可重复性得到了显着改善。