Detection and Quantification of β-(1,3)-D-Glucan Contamination in Nanoparticle Formulations by Factor C Depleted LAL (Glucatell®) Assay: Version 1

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.