Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
Research and Development, Bilfinger Industrietechnik Salzburg GmbH, Salzburg, Austria.
Biotechnol Prog. 2019 Sep;35(5):e2864. doi: 10.1002/btpr.2864. Epub 2019 Jun 22.
Glycosylation, as the most prominent posttranslational modification, is recognized as an important quality attribute of monoclonal antibodies affected by various bioprocess parameters and cellular physiology. A method of lectin-based bio-layer interferometry (LBLI) to relatively rank galactosylation and fucosylation levels was developed. For this purpose, Fc-glycosylated immunoglobulin G (IgG) was recombinantly produced with varying bioprocess conditions in 15 L bioreactor and accumulated IgG was harvested. The reliability, the robustness and the applicability of LBLI to different samples has been proven. Data obtained from LC-MS analysis served as reference and were compared to the LBLI results. The introduced method is based on non-fluidic bio-layer interferometry (BLI), which becomes recently a standard tool for determining biomolecular interactions in a label-free, real-time and high-throughput manner. For the intended purpose, biotinylated lectins were immobilized on disposable optical fiber streptavidin (SA) biosensor tips. Aleuria aurantia lectin (AAL) was used to detect the core fucose and Ricinus communis agglutinin 120 (RCA120) to determine galactosylation levels. In our case study it could be shown that fucosylation was not affected by variations in glucose feed concentration and cultivation temperature. However, the galactosylation could be correlated with the ratio of mean specific productivity (q ) and ammonium (q ) but was unrelated to the ratio of mean q and the specific glucose consumption (q ). This presented method strengthens the applicability of the BLI platform, which already enables measurement of several product related characteristics, such as product quantity as well as kinetic rates (k ,k ) and affinity constants (k ) analysis.
糖基化是最显著的翻译后修饰之一，被认为是单克隆抗体的一个重要质量属性，受各种生物工艺参数和细胞生理学的影响。本研究开发了一种基于凝集素的生物层干涉（LBLI）方法，用于相对评估半乳糖基化和岩藻糖基化水平。为此，使用不同的生物工艺条件在 15L 生物反应器中重组生产 Fc 糖基化免疫球蛋白 G（IgG），并收获积累的 IgG。已经证明 LBLI 对不同样品的可靠性、稳健性和适用性。从 LC-MS 分析获得的数据作为参考，并与 LBLI 结果进行比较。所介绍的方法基于非流动生物层干涉（BLI），该技术最近已成为一种标准工具，可用于以无标记、实时和高通量的方式测定生物分子相互作用。为此，生物素化凝集素被固定在一次性光纤链霉亲和素（SA）生物传感器尖端上。Aleuria aurantia 凝集素（AAL）用于检测核心岩藻糖，蓖麻凝集素 120（RCA120）用于确定半乳糖基化水平。在我们的案例研究中，结果表明，糖基化不受葡萄糖进料浓度和培养温度变化的影响。然而，半乳糖基化可以与平均比生产力（q ）和铵（q ）的比值相关，但与平均 q 与特定葡萄糖消耗（q ）的比值无关。该方法增强了 BLI 平台的适用性，该平台已经能够测量几个与产品相关的特性，例如产品数量以及动力学速率（k,k ）和亲和常数（k ）分析。
