Department of Chemical and Biochemical Engineering Missouri University of Science and Technology, Rolla, MO, 65409, USA.
Sci Rep. 2019 Jun 20;9(1):8867. doi: 10.1038/s41598-019-45402-w.
The presence of endotoxin, also known as lipopolysaccharides (LPS), as a side product appears to be a major drawback for the production of certain biomolecules that are essential for research, pharmaceutical, and industrial applications. In the biotechnology industry, gram-negative bacteria (e.g., Escherichia coli) are widely used to produce recombinant products such as proteins, plasmid DNAs and vaccines. These products are contaminated with LPS, which may cause side effects when administered to animals or humans. Purification of LPS often suffers from product loss. For this reason, special attention must be paid when purifying proteins aiming a product as free as possible of LPS with high product recovery. Although there are a number of methods for removing LPS, the question about how LPS removal can be carried out in an efficient and economical way is still one of the most intriguing issues and has no satisfactory solution yet. In this work, polymeric poly-ε-caprolactone (PCL) nanoparticles (NPs) (d = 780 ± 285 nm) were synthesized at a relatively low cost and demonstrated to possess sufficient binding sites for LPS adsorption and removal with 100% protein recovery. The PCL NPs removed greater than 90% LPS from protein solutions suspended in water using only one milligram (mg) of NPs, which was equivalent to ~1.5 × 10 endotoxin units (EU) per mg of particle. The LPS removal efficacy increased to a higher level (100%) when phosphate buffered saline (PBS containing 137 mM NaCl) was used as a protein suspending medium in place of water, reflecting positive effects of increasing ionic strength on LPS binding interactions and adsorption. The results further showed that the PCL NPs not only achieved 100% LPS removal but also ~100% protein recovery for a wide concentration range from 20-1000 μg/ml of protein solutions. The NPs were highly effective in different buffers and pHs. To scale up the process further, PCL NPs were incorporated into a supporting cellulose membrane which promoted LPS adsorption further up to ~100% just by running the LPS-containing water through the membrane under gravity. Its adsorption capacity was 2.8 × 10 mg of PCL NPs, approximately 2 -fold higher than that of NPs alone. This is the first demonstration of endotoxin separation with high protein recovery using polymer NPs and the NP-based portable filters, which provide strong adsorptive interactions for LPS removal from protein solutions. Additional features of these NPs and membranes are biocompatible (environment friendly) recyclable after repeated elution and adsorption with no significant changes in LPS removal efficiencies. The results indicate that PCL NPs are an effective LPS adsorbent in powder and membrane forms, which have great potential to be employed in large-scale applications.
内毒素（也称为脂多糖[LPS]）作为副产物的存在似乎是生产某些对研究、制药和工业应用至关重要的生物分子的主要缺点。在生物技术行业中，革兰氏阴性菌（例如大肠杆菌）被广泛用于生产重组产品，如蛋白质、质粒 DNA 和疫苗。这些产品被 LPS 污染，当施用于动物或人类时可能会引起副作用。LPS 的纯化通常会导致产物损失。因此，在纯化蛋白质时，必须特别注意，以使产品尽可能不含 LPS，并具有较高的产物回收率。尽管有许多去除 LPS 的方法，但如何以高效和经济的方式进行 LPS 去除仍然是最引人关注的问题之一，目前尚无令人满意的解决方案。在这项工作中，以相对较低的成本合成了聚合的聚己内酯（PCL）纳米颗粒（NPs）（d=780±285nm），并证明其具有足够的 LPS 吸附和去除结合位点，蛋白回收率约为 100%。PCL NPs 仅使用 1 毫克（mg）的 NPs 即可从悬浮在水中的蛋白质溶液中去除超过 90%的 LPS，这相当于每毫克颗粒约 1.5×10 个内毒素单位（EU）。当磷酸盐缓冲盐水（PBS 中含有 137mM NaCl）代替水作为蛋白质悬浮介质时，LPS 去除效果提高到更高水平（~100%），这反映了增加离子强度对内毒素结合相互作用和吸附的积极影响。结果进一步表明，PCL NPs 不仅可以去除 100%的 LPS，还可以在 20-1000μg/ml 的蛋白质溶液的宽浓度范围内实现约 100%的蛋白质回收率。NPs 在不同的缓冲液和 pH 值下均具有高效性。为了进一步扩大该过程，将 PCL NPs 掺入支撑纤维素膜中，通过重力使含有 LPS 的水通过膜，进一步促进 LPS 吸附，达到约 100%的 LPS 去除率。其吸附能力为 2.8×10mg PCL NPs，大约是 NPs 单独使用时的 2 倍。这是首次使用聚合物 NPs 和基于 NP 的便携式过滤器进行内毒素分离并实现高蛋白质回收率的证明，这些过滤器为从蛋白质溶液中去除 LPS 提供了强大的吸附相互作用。这些 NPs 和膜的其他特点是生物相容性（环保），可在重复洗脱和吸附后回收，而 LPS 去除效率没有明显变化。结果表明，PCL NPs 是一种有效的 LPS 吸附剂，无论是粉末形式还是膜形式，都具有在大规模应用中应用的巨大潜力。
