Abstract

Lipopolysaccharide (LPS), as a stubborn contamination, should be monitored and kept in an acceptable level during the pharmaceutical production process. Recombinant hepatitis B surface antigen (r-HBsAg) is one of the recombinant biological products, which is probable to suffer from extrinsic endotoxin due to its long and complex production process. This research aims to assess the potential interaction between LPS and r-HBsAg by recruiting immunoaffinity chromatography (IAC) as a novel tool to quantify the interaction. Molecular modeling was performed on the HBsAg molecule to theoretically predict its potential binding and interaction sites. Then dynamic light scattering (DLS) analysis was implemented on HBsAg, LPS, and mixtures of them to reveal the interaction. The virus-like particle (VLP) structure of HBsAg and the ribbon-like structure of LPS were visualized by transmission electron microscopy (TEM). Finally, the interaction was quantified by applying various LPS/HBsAg ratios ranging from 1.67 to 120 EU/dose in the IAC. Consequently, the LPS/HBsAg ratios in the eluate were measured from 1.67 to a maximum of 92.5 EU/dose. The results indicated that 77 to 100% of total LPS interacted with HBsAg by an inverse relationship to the incubated LPS concentration. The findings implied that the introduced procedure is remarkably practical in the quantification of LPS interaction with a target recombinant protein.

摘要

脂多糖（LPS）作为一种顽固污染物，在药品生产过程中应进行监测并保持在可接受的水平。重组乙型肝炎表面抗原（r-HBsAg）是重组生物制品之一，由于其生产过程漫长而复杂，很可能受到外源性内毒素的影响。本研究旨在通过采用免疫亲和层析 (IAC) 作为量化相互作用的新工具来评估 LPS 和 r-HBsAg 之间的潜在相互作用。对 HBsAg 分子进行分子建模，从理论上预测其潜在的结合和相互作用位点。然后对 HBsAg、LPS 及其混合物进行动态光散射 (DLS) 分析，以揭示相互作用。通过透射电子显微镜 (TEM) 观察 HBsAg 的病毒样颗粒 (VLP) 结构和 LPS 的带状结构。最后，通过在 IAC 中应用 1.67 至 120 EU/剂量的各种 LPS/HBsAg 比率来量化相互作用。因此，测得洗出液中的 LPS/HBsAg 比率从 1.67 到最大 92.5 EU/剂量。结果表明，77% 至 100% 的总 LPS 与 HBsAg 相互作用，与孵育的 LPS 浓度呈反比关系。研究结果表明，所引入的程序在定量 LPS 与目标重组蛋白的相互作用方面非常实用。测得洗出液中的 LPS/HBsAg 比率为 1.67 至最大 92.5 EU/剂量。结果表明，77% 至 100% 的总 LPS 与 HBsAg 相互作用，与孵育的 LPS 浓度呈反比关系。研究结果表明，所引入的程序在定量 LPS 与目标重组蛋白的相互作用方面非常实用。测得洗出液中的 LPS/HBsAg 比率为 1.67 至最大 92.5 EU/剂量。结果表明，77% 至 100% 的总 LPS 与 HBsAg 相互作用，与孵育的 LPS 浓度呈反比关系。研究结果表明，所引入的程序在定量 LPS 与目标重组蛋白的相互作用方面非常实用。