Neun Barry W., Dobrovolskaia Marina A.
Nanotechnology Characterization Lab, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, MD 21702
This document describes a protocol for the quantitative detection of β-(1,3)-D-glucans in nanoparticle preparations using a chromogenic endpoint Glucatell assay. β-(1,3)-D-glucans are products of cell walls of some microorganisms, including yeast and fungi. The most common source of β-(1,3)-D-glucans is fungi and . B-(1,3)-D-glucans are also found at low levels ( < 60 pg/mL) in the blood of healthy humans where they are believed to distribute from the gastrointestinal tract following the consumption of β-(1,3)-D-glucan-containing foods (plants, grains, cereal, mushrooms etc.,). In patients with an invasive fungal infection, these levels increase at or above 80 pg/mL [–5]. β-(1,3)-D-glucans may become undesirable contaminants in pharmaceutical products, where they are introduced during manufacturing through filtration processes utilizing cellulose-based filters or contaminated starting materials or common excipients such as sucrose. β-(1,3)-D-glucans are not as immunologically potent as bacterial endotoxins. However, they are pro-inflammatory and can also activate the immune system. There is an increasing amount of information suggesting that β-(1,3)-D-glucans may exaggerate endotoxin-mediated toxicities as well as synergize with other immunologically active impurities introduced into pharmaceutical products during manufacturing and, therefore, lead to adverse immune effects [–8]. Moreover, FDA immunogenicity guidance for industry suggests minimizing the levels of β-(1,3)-D-glucans in therapeutic protein formulations to decrease the immunogenicity of these products [9]. Unlike bacterial endotoxins, β-(1,3)-D-glucans are currently not regulated; there is no compendial standard for their detection and no harmonized approach to acceptable levels. Nevertheless, there is a growing trend in industry and among regulatory authorities worldwide to detect β-(1-3)-D-glucans and understand their safety levels [6, 8]. Since many nanotechnology platforms are not immunologically inert, understanding the presence of immunologically reactive contaminants besides endotoxin becomes an essential step in understanding the safety margins for formulations containing such materials [10]. This becomes especially important when such nanomaterials are intended for immunotherapy involving the intentional application of immune checkpoint inhibitors to enhance the immune response. The assay used in this protocol is based on the commercial kit approved for the detection of β-(1,3)-D-glucans in human serum to diagnose fungal infection. We adapted an R&D version of this assay for the screening of nanomaterial formulations not to disqualify nanoparticles, but to inform the formulation of immunologically safe nanomaterials.
本文描述了一种使用显色终点Glucatell测定法对纳米颗粒制剂中β-(1,3)-D-葡聚糖进行定量检测的方案。β-(1,3)-D-葡聚糖是包括酵母和真菌在内的一些微生物细胞壁的产物。β-(1,3)-D-葡聚糖最常见的来源是真菌。在健康人的血液中也发现了低水平（<60 pg/mL）的β-(1,3)-D-葡聚糖，据信它们是在食用含β-(1,3)-D-葡聚糖的食物（植物、谷物、麦片、蘑菇等）后从胃肠道分布而来的。在侵袭性真菌感染患者中，这些水平会升高至80 pg/mL及以上[–5]。β-(1,3)-D-葡聚糖可能成为药品中不受欢迎的污染物，它们在生产过程中通过使用纤维素基过滤器的过滤工艺、受污染的起始原料或常见辅料（如蔗糖）而被引入。β-(1,3)-D-葡聚糖的免疫活性不如细菌内毒素强。然而，它们具有促炎作用，还能激活免疫系统。越来越多的信息表明，β-(1,3)-D-葡聚糖可能会加剧内毒素介导的毒性，以及与生产过程中引入药品的其他免疫活性杂质协同作用，从而导致不良免疫反应[–8]。此外，FDA针对行业的免疫原性指南建议尽量降低治疗性蛋白质制剂中β-(1,3)-D-葡聚糖的水平，以降低这些产品的免疫原性[9]。与细菌内毒素不同，β-(1,3)-D-葡聚糖目前不受监管；没有检测它们的药典标准，也没有关于可接受水平的统一方法。然而，全球行业和监管机构中检测β-(1-3)-D-葡聚糖并了解其安全水平的趋势正在增加[6, 8]。由于许多纳米技术平台并非免疫惰性，了解除内毒素外具有免疫反应性的污染物的存在成为了解含此类材料制剂安全边际的重要一步[10]。当此类纳米材料用于免疫治疗，即有意应用免疫检查点抑制剂以增强免疫反应时，这一点尤为重要。本方案中使用的测定法基于已批准用于检测人血清中β-(1,3)-D-葡聚糖以诊断真菌感染的商业试剂盒。我们对该测定法的研发版本进行了调整，用于筛选纳米材料制剂并非为了排除纳米颗粒，而是为了为免疫安全的纳米材料配方提供信息。
