Institute for Bioengineering, The School of Engineering, The University of Edinburgh, Edinburgh, EH9 3DW, UK.
Fujifilm Diosynth Biotechnologies, Teesside, TS23 1LH, UK.
J Chromatogr A. 2024 May 10;1722:464873. doi: 10.1016/j.chroma.2024.464873. Epub 2024 Apr 4.
3D printing offers the unprecedented ability to fabricate chromatography stationary phases with bespoke 3D morphology as opposed to traditional packed beds of spherical beads. The restricted range of printable materials compatible with chromatography is considered a setback for its industrial implementation. Recently, we proposed a novel ink that exhibits favourable printing performance (printing time ∼100 mL/h, resolution ∼200 µm) and broadens the possibilities for a range of chromatography applications thanks to its customisable surface chemistry. In this work, this ink was used to fabricate 3D printed ordered columns with 300 µm channels for the capture and polishing of therapeutic monoclonal antibodies. The columns were initially assessed for leachables and extractables, revealing no material propensity for leaching. Columns were then functionalised with protein A and SO ligands to obtain affinity and strong cation exchangers, respectively. 3D printed protein A columns showed >85 % IgG recovery from harvested cell culture fluid with purities above 98 %. Column reusability was evaluated over 20 cycles showing unaffected performance. Eluate samples were analysed for co-eluted protein A fragments, host cell protein and aggregates. Results demonstrate excellent HCP clearance (logarithmic reduction value of > 2.5) and protein A leakage in the range of commercial affinity resins (<100 ng/mg). SO functionalised columns employed for polishing achieved removal of leaked Protein A (down to 10 ng/mg) to meet regulatory expectations of product purity. This work is the first implementation of 3D printed columns for mAb purification and provides strong evidence for their potential in industrial bioseparations.
3D 打印技术提供了前所未有的能力，可以制造具有定制 3D 形态的色谱固定相，而不是传统的球形珠粒填充床。与色谱兼容的可打印材料范围有限，被认为是其工业实施的一个障碍。最近，我们提出了一种新型墨水，由于其可定制的表面化学性质，它具有良好的打印性能（打印时间约为 100 毫升/小时，分辨率约为 200 微米），并拓宽了一系列色谱应用的可能性。在这项工作中，这种墨水被用于制造具有 300 微米通道的 3D 打印有序柱，用于捕获和抛光治疗性单克隆抗体。这些柱子最初评估了浸出物和提取物，结果表明没有材料浸出的倾向。然后，柱子用蛋白 A 和 SO 配体进行功能化，分别获得亲和和强阳离子交换剂。3D 打印的蛋白 A 柱从收获的细胞培养液中回收超过 85%的 IgG，纯度超过 98%。评估了柱子的可重复使用性，超过 20 个循环，性能未受影响。洗脱样品用于分析共洗脱的蛋白 A 片段、宿主细胞蛋白和聚集体。结果表明，HCP 清除率（对数减少值大于 2.5）和蛋白 A 泄漏率在商业亲和树脂范围内（<100ng/mg）均表现良好。用于抛光的 SO 功能化柱实现了泄漏蛋白 A 的去除（降至 10ng/mg），以满足产品纯度的监管要求。这项工作是首次将 3D 打印柱用于 mAb 纯化，并为其在工业生物分离中的潜力提供了有力证据。
