Abstract

Lipopolysaccharide (LPS) is one of the most challenging contaminants in biopharmaceutical industries. Cationic amphiphilic peptides (CAPs) -based affinity matrices can be potent tools for LPS removal in such situations. In this study, two newly designed CAPs derived from the LPS binding site of factor C of horseshoe crab S3E3 and S3E3A were immobilized chemo-selectively on diaminodipropylamine (DADPA) and iodoacetyl functionalized Sepharose beads. Both peptides were immobilized via their carboxyl or sulfhydryl (thiol) groups by amide or thioether bonds, respectively. The generated four affinity matrices were used to remove LPS from bovine serum albumin (BSA). The effects of different influential factors including pH, NaCl, Ethylenediaminetetraacetic acid (EDTA), and LPS concentrations on LPS removal efficiency and protein recovery were investigated by Plackett Burman (PB) method.

Statistical analysis revealed that immobilized S3E3 removed LPS more effectively than immobilized S3E3A. Increasing pH and LPS concentration had negative effects on LPS removal efficiency and protein recovery. Increasing NaCl concentration improved protein recovery but reduced LPS removal efficiency. Other factors such as EDTA and type of buffer had no significant effect on the measured responses.

摘要

脂多糖（LPS）是生物制药行业最具挑战性的污染物之一。在这种情况下，基于阳离子两亲肽（CAP）的亲和基质可以成为去除LPS的有效工具。在这项研究中，两个新设计的从马蹄蟹S3E3和S3E3A的C因子LPS结合位点获得的CAPs被化学选择性地固定在二氨基二丙胺（DADPA）和碘乙酰基官能化的琼脂糖凝胶珠上。两种肽分别通过其羧基或巯基（硫醇）基团通过酰胺或硫醚键固定。生成的四个亲和矩阵用于从牛血清白蛋白（BSA）中去除LPS。pH，NaCl，乙二胺四乙酸（EDTA），

统计分析表明，固定的S3E3比固定的S3E3A更有效地去除LPS。pH和LPS浓度的增加对LPS去除效率和蛋白质回收率产生负面影响。增加NaCl浓度可提高蛋白质回收率，但降低LPS去除效率。诸如EDTA和缓冲液类型等其他因素对测得的响应没有显着影响。