Purification of therapeutic monoclonal antibodies usually involves a protein A affinity capture step. Because column breakthrough of antibody in complex, UV-absorbing culture fluid cannot be readily detected in real time, processes are designed so conservatively that column capacity is usually underutilized, wasting adsorbent and reducing productivity. We have developed a fluorescence-based monitoring technology which allows real-time mAb monitoring and used it to detect IgG in column breakthrough. The column effluent was continuously contacted with soluble fluorescein-labeled Fc-binding ligands to produce an immediately-detectable shift in both fluorescence polarization and intensity. To extend the upper limit of inlet flow rate, a 14:1 split-ratio flow splitter was tested with an inlet flow of 15 mL/min (0.9 L/h), producing a sampling stream at 1 mL/min while still enabling continuous detection functionality. We observed significant shifts in fluorescence intensity in CHO cell culture fluid spiked with human IgG, and detected 0.02–0.1 g/L human IgG in protein A column breakthrough at a flow velocity of 80 cm/h. The increase in fluorescence intensity upon 0.1% breakthrough of an 1 g/L feed was used to trigger column switching using Python-enabled two-way communication with the standard Unicorn OPC process control protocol. The technology allows rapid, continuous and reliable monitoring of IgG in a flowing process stream, without elaborate sample preparation.

治疗性单克隆抗体的纯化通常涉及蛋白A亲和力捕获步骤。由于无法轻松，实时地检测抗体在复杂的吸收紫外线的培养液中的柱穿透作用，因此设计过程过于保守，通常无法充分利用柱容量，从而浪费了吸附剂并降低了生产率。我们已经开发了一种基于荧光的监测技术，该技术可以实时监测mAb，并将其用于检测色谱柱穿透中的IgG。使柱流出物连续与可溶性荧光素标记的Fc结合配体接触，以立即检测到荧光偏振和强度的变化。为了扩展入口流量的上限，测试了14：1的分流比分流器，其入口流量为15 mL / min（0.9 L / h），产生1 mL / min的采样流，同时仍具有连续检测功能。我们观察到掺有人IgG的CHO细胞培养液中荧光强度发生了显着变化，并以80 cm / h的流速检测到蛋白A柱穿透中的0.02–0.1 g / L人IgG。1 g / L进料突破0.1％时，荧光强度的增加被用来触发色谱柱切换，该切换使用了支持Python的双向通讯与标准Unicorn OPC过程控制协议。该技术可以快速，连续和可靠地监测流动过程流中的IgG，而无需进行复杂的样品制备。蛋白质A柱中的1 g / L人IgG以80 cm / h的流速穿透。1 g / L进料突破0.1％时，荧光强度的增加被用来触发色谱柱切换，该切换使用了支持Python的双向通讯与标准Unicorn OPC过程控制协议。该技术可以快速，连续和可靠地监测流动过程流中的IgG，而无需进行复杂的样品制备。蛋白质A柱中的1 g / L人IgG以80 cm / h的流速穿透。1 g / L进料突破0.1％时，荧光强度的增加被用来触发色谱柱切换，该切换使用了支持Python的双向通讯与标准Unicorn OPC过程控制协议。该技术可以快速，连续和可靠地监测流动过程流中的IgG，而无需进行复杂的样品制备。