BMC Biotechnol. 2013 May 20;13:44. doi: 10.1186/1472-6750-13-44.
In situ magnetic separation (ISMS) has emerged as a powerful tool to overcome process constraints such as product degradation or inhibition of target production. In the present work, an integrated ISMS process was established for the production of his-tagged single chain fragment variable (scFv) D1.3 antibodies ("D1.3") produced by E. coli in complex media. This study investigates the impact of ISMS on the overall product yield as well as its biocompatibility with the bioprocess when metal-chelate and triazine-functionalized magnetic beads were used.
Both particle systems are well suited for separation of D1.3 during cultivation. While the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium. After the ISMS step, elevated copper concentrations above 120 mg/L in the medium negatively influenced D1.3 production. Due to the stable nature of the model protein scFv D1.3 in the biosuspension, the application of ISMS could not increase the overall D1.3 yield as was shown by simulation and experiments.
We could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb D1.3 fragments, and measured maximum loads of 0.08 g D1.3 per g of beads. Although copper-loaded metal-chelate beads did adsorb his-tagged D1.3 well during cultivation, this particle system must be optimized by minimizing metal leakage from the beads in order to avoid negative inhibitory effects on growth of the microorganisms and target production. Hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility. Such optimized particle systems can be regarded as ISMS platform technology, especially for the production of antibodies and their fragments with low stability in the medium. The proposed model can be applied to design future ISMS experiments in order to maximize the overall product yield while the amount of particles being used is minimized as well as the number of required ISMS steps.
原位磁分离（ISMS）已成为克服产品降解或目标产物抑制等工艺限制的有力工具。本工作建立了一种集成的 ISMS 工艺，用于生产复杂介质中大肠杆菌产生的带有 His 标签的单链片段可变（scFv）D1.3 抗体（“D1.3”）。本研究考察了在使用金属螯合和三嗪功能化磁珠时，ISMS 对整体产物收率的影响及其与生物过程的生物相容性。
两种粒子体系都非常适合在培养过程中分离 D1.3。虽然三嗪珠对生物过程没有负面影响，但金属螯合珠的应用导致介质中二价铜离子泄漏。ISMS 步骤后，介质中铜浓度升高至 120mg/L 以上，对 D1.3 生产产生负面影响。由于生物悬浮液中模型蛋白 scFv D1.3 的稳定性质，如模拟和实验所示，应用 ISMS 并不能提高整体 D1.3 收率。
我们可以证明三嗪功能化珠是一种选择性吸附 D1.3 片段的合适低成本替代品，最大负载量为 0.08g D1.3/g 珠。虽然负载铜的金属螯合珠在培养过程中很好地吸附了带有 His 标签的 D1.3，但该颗粒体系必须通过最小化颗粒中金属的泄漏来优化，以避免对微生物生长和目标产物产生负面影响。在此，应测试其他类型的金属螯合络合物以证明其生物相容性。这种优化的颗粒体系可作为 ISMS 平台技术，特别是用于生产在介质中稳定性低的抗体及其片段。所提出的模型可用于设计未来的 ISMS 实验，以在最小化使用的颗粒量和所需的 ISMS 步骤数的同时最大化整体产物收率。
