Faculty for Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia.
Technical development biosimilars, Global drug development, Novartis, Kolodvorska 27, 1234 Mengeš, Slovenia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jun 1;1174:122731. doi: 10.1016/j.jchromb.2021.122731. Epub 2021 Apr 25.
In-process monitoring of glycosylated protein concentration becomes very important with the introduction of perfusion bioprocesses. Affinity chromatography based on lectins allows selective monitoring when carbohydrates are accessible on the protein surface. In this work, we immobilized lectin on polyHIPE type of monoliths and implemented it for bioprocess monitoring. A spacer was introduced to lectin, which increased binding kinetics toward Fc-fusion protein, demonstrated by bio-layer interferometry. Furthermore, complete desorption using 0.25 M galactose was shown. Affinity column exhibited linearity in the range between 0.5 and 8 mg/ml and flow-unaffected binding for the flow-rates between 0.5 and 8 ml/min. Long-term stability over at least four months period was demonstrated. No unspecific binding of culture media components, including host cell proteins and DNA, was detected. Results obtained by affinity column matched concentration values obtained by a reference method.
随着灌注生物工艺的引入，糖基化蛋白浓度的过程监测变得非常重要。基于凝集素的亲和色谱法允许在蛋白质表面的碳水化合物可及时进行选择性监测。在这项工作中，我们将凝集素固定在多空高聚物类型的整体式柱上，并将其用于生物过程监测。引入了间隔物到凝集素中，这增加了与 Fc-融合蛋白的结合动力学，通过生物层干涉测量法证明。此外，使用 0.25 M 半乳糖可以完全洗脱。亲和柱在 0.5 至 8 mg/ml 范围内呈线性，在 0.5 至 8 ml/min 的流速范围内结合不受影响。至少四个月的长期稳定性得到了证明。未检测到培养基成分（包括宿主细胞蛋白和 DNA）的非特异性结合。通过亲和柱获得的结果与参考方法获得的浓度值相匹配。
