KTH Royal Institute of Technology, Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, Solna, Sweden.
Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden.
Methods Mol Biol. 2024;2804:127-138. doi: 10.1007/978-1-0716-3850-7_8.
Within the vast field of medical biotechnology, the biopharmaceutical industry is particularly fast-growing and highly competitive, so reducing time and costs associated to process optimization becomes instrumental to ensure speed to market and, consequently, profitability. The manufacturing of biopharmaceutical products, namely, monoclonal antibodies (mAbs), relies mostly on mammalian cell culture processes, which are highly dynamic and, consequently, difficult to optimize. In this context, there is currently an unmet need of analytical methods that can be integrated at-line in a bioreactor, for systematic monitoring and quantification of key metabolites and proteins. Microfluidic-based assays have been extensively and successfully applied in the field of molecular diagnostics; however, this technology remains largely unexplored for Process Analytical Technology (PAT), despite holding great potential for the at-line measurement of different analytes in bioreactor processes, combining low reagent/molecule consumption with assay sensitivity and rapid turnaround times.Here, the fabrication and handling of a microfluidic cartridge for protein quantification using bead-based affinity assays is described. The device allows geometrical multiplexed immunodetection of specific protein analytes directly from bioreactor samples within 2.5 h and minimal hands-on time. As a proof-of-concept, quantification of Chinese hamster ovary (CHO) host cell proteins (HCP) as key impurities, IgG as product of interest, and lactate dehydrogenase (LDH) as cell viability marker was demonstrated with limits of detection (LoD) in the low ng/mL range. Negligible matrix interference and no cross-reactivity between the different immunoassays on chip were found. The results highlight the potential of the miniaturized analytical method for PAT at reduced cost and complexity in comparison with sophisticated instruments that are currently the state-of-the-art in this context.
在医学生物技术的广阔领域中，生物制药行业的发展尤为迅速且竞争激烈，因此缩短与工艺优化相关的时间和成本对于确保快速上市并最终实现盈利至关重要。生物制药产品（即单克隆抗体（mAbs））的制造主要依赖于哺乳动物细胞培养工艺，这些工艺非常动态，因此难以优化。在这种情况下，目前需要一种分析方法，可以在线集成到生物反应器中，用于系统监测和定量关键代谢物和蛋白质。基于微流控的分析方法已在分子诊断领域得到广泛而成功的应用；然而，尽管该技术在工艺分析技术（PAT）中具有很大的潜力，可以在线测量生物反应器过程中的不同分析物，具有试剂/分子消耗低、分析灵敏度高和周转时间短等优点，但在 PAT 中，这种技术仍然在很大程度上未得到探索。这里，描述了一种使用基于珠的亲和测定法进行蛋白质定量的微流控芯片的制造和处理。该设备允许在 2.5 小时内从生物反应器样品中直接进行几何多路复用免疫检测特定蛋白质分析物，并且需要的人工操作时间最少。作为概念验证，使用微流控芯片定量检测了中国仓鼠卵巢（CHO）宿主细胞蛋白（HCP）作为关键杂质、作为感兴趣产品的 IgG 以及乳酸脱氢酶（LDH）作为细胞活力标志物，检测限（LoD）在低 ng/mL 范围内。发现芯片上的不同免疫分析之间几乎没有基质干扰和交叉反应。结果突出了这种小型化分析方法在降低成本和复杂性方面的潜力，与目前在这方面处于最先进水平的复杂仪器相比。
