Department of Chemistry, Biosystems Research Complex, Clemson University, Clemson, South Carolina 29634-0973, United States.
Anal Chem. 2023 Dec 5;95(48):17886-17893. doi: 10.1021/acs.analchem.3c04044. Epub 2023 Nov 23.
Cultured cell lines are very commonly used for the mass production of therapeutic proteins, such as monoclonal antibodies (mAbs). In particular, Chinese hamster ovary (CHO) cell lines are widely employed due to their high tolerance to variations in experimental conditions and their ability to grow in suspension or serum free media. CHO cell lines are known for their ability to produce high titers of biotherapeutic products such as immunoglobulin G (IgG). An emergent alternative means of treating diseases, such as cancer, is the use of gene therapies, wherein genetic cargo is "packaged" in nanosized vesicular structures, referred to as "vectors". One particularly attractive vector option is extracellular vesicles (EVs), of which exosomes are of greatest interest. While exosomes can be harvested from virtually any human body fluid, bovine milk, or even plants, their production in cell cultures is an attractive commercial approach. In fact, the same CHO cell types employed for mAb production also produce exosomes as a natural byproduct. Here, we describe a single integrated 2D liquid chromatography (2DLC) method for the quantitative recovery of both exosomes and antibodies from a singular sample aliquot. At the heart of the method is the use of polyester capillary-channeled polymer (C-CP) fibers as the first dimension column, wherein exosomes/EVs are captured from the supernatant sample and subsequently determined by multiangle light scattering (MALS), while the mAbs are captured, eluted, and quantified using a protein A-modified C-CP fiber column in the second dimension, all in a 10 min workflow. These efforts demonstrate the versatility of the C-CP fiber phases with the capacity to harvest both forms of therapeutics from a single bioreactor, suggesting an appreciable potential impact in the field of biotherapeutics production.
细胞培养物系通常用于大规模生产治疗性蛋白，如单克隆抗体（mAbs）。特别是，由于对实验条件变化的高耐受性以及在悬浮或无血清培养基中生长的能力，中国仓鼠卵巢（CHO）细胞系得到了广泛应用。CHO 细胞系以能够产生高滴度的生物治疗产品（如免疫球蛋白 G（IgG））而闻名。另一种新兴的治疗疾病的方法是使用基因疗法，其中遗传货物被“包装”在纳米级囊泡结构中，称为“载体”。一种特别有吸引力的载体选择是细胞外囊泡（EVs），其中外泌体最受关注。虽然外泌体可以从几乎任何人体体液、牛牛奶甚至植物中提取，但在细胞培养物中生产它们是一种有吸引力的商业方法。事实上，用于 mAb 生产的相同 CHO 细胞类型也会产生外泌体作为天然副产物。在这里，我们描述了一种从单一样品等分试样中定量回收外泌体和抗体的单一集成二维液相色谱（2DLC）方法。该方法的核心是使用聚酯毛细管通道聚合物（C-CP）纤维作为第一维柱，其中外泌体/ EVs 从上清液样品中捕获，随后通过多角度光散射（MALS）进行测定，而 mAbs 则通过第二维中的蛋白质 A 修饰的 C-CP 纤维柱进行捕获、洗脱和定量，所有这些都在 10 分钟的工作流程内完成。这些努力证明了 C-CP 纤维相的多功能性，能够从单个生物反应器中收获这两种形式的治疗药物，这表明在生物治疗药物生产领域具有相当大的潜在影响。
