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回收有价值的物质:使用新型抗脂多糖抗体微球缀合物从 LPS 污染制剂中去除内毒素后回收高蛋白。

Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates.

机构信息

Chemical Biology Department, R&D Reagents, Miltenyi Biotec B.V. & Co. KG, 51429 Bergisch Gladbach, Germany.

Division of Allergy & Immunology, Department of Biosciences & Medical Biology, Paris Lodron University of Salzburg, 5020 Salzburg, Austria.

出版信息

Int J Mol Sci. 2023 Sep 12;24(18):13971. doi: 10.3390/ijms241813971.


DOI:10.3390/ijms241813971
PMID:37762274
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10531372/
Abstract

Endotoxins or lipopolysaccharides (LPS), found in the outer membrane of Gram-negative bacterial cell walls, can stimulate the human innate immune system, leading to life-threatening symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 ×10 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield.

摘要

内毒素或脂多糖(LPS)存在于革兰氏阴性细菌细胞壁的外膜中,能够刺激人体先天免疫系统,导致危及生命的症状。因此,内毒素含量的监管限制适用于注射用药物,在商业化之前必须去除多余的 LPS。大多数可用的内毒素去除系统基于 LPS 与带电和/或疏水性表面的非特异性吸附。尽管这些方法可以有效地去除内毒素,但缺乏特异性可能导致药物配方中必需蛋白质的不必要损失。在这项工作中,我们开发了与抗脂多糖 A 抗体偶联的微颗粒,用于选择性去除内毒素。使用流式细胞术和显微镜技术对抗脂多糖 A 颗粒进行了表征。这些颗粒在水中的耗竭容量超过 6×10 内毒素单位/mg 颗粒,这是通过两种独立的方法(鲎变形细胞溶解物试验和纳米颗粒跟踪分析)确定的。此外,我们在一系列越来越复杂的制剂中比较了这些颗粒与非特异性内毒素去除系统:水中的牛血清白蛋白<缓冲液中的胰岛素<桦树花粉提取物。我们证明了特异性抗脂多糖 A 颗粒在不影响其去除内毒素能力的情况下,具有更高的蛋白质回收率。因此,我们认为抗脂多糖 A 抗体的特异性层可能有利于提高产品产量。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/600f/10531372/f1f60fc02a0b/ijms-24-13971-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/600f/10531372/8fa0d544f652/ijms-24-13971-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/600f/10531372/29f7335c1072/ijms-24-13971-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/600f/10531372/f2c59c4da87e/ijms-24-13971-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/600f/10531372/745fd2886b94/ijms-24-13971-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/600f/10531372/f1f60fc02a0b/ijms-24-13971-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/600f/10531372/8fa0d544f652/ijms-24-13971-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/600f/10531372/29f7335c1072/ijms-24-13971-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/600f/10531372/f2c59c4da87e/ijms-24-13971-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/600f/10531372/745fd2886b94/ijms-24-13971-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/600f/10531372/f1f60fc02a0b/ijms-24-13971-g005.jpg

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Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates.

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[6]
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[10]
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引用本文的文献

[1]
Preparation of Polymyxin B-Functionalized Cryogels for Efficient Endotoxin Removal from Protein Solutions.

Gels. 2025-5-28

[2]
Editorial of Special Issue "Current Trends in Chemistry Towards Biology".

Int J Mol Sci. 2024-7-3

[3]
Generation of Endotoxin-Specific Monoclonal Antibodies by Phage and Yeast Display for Capturing Endotoxin.

Int J Mol Sci. 2024-2-15

本文引用的文献

[1]
Broad-Spectrum Clearance of Lipopolysaccharides from Blood Based on a Hemocompatible Dihistidine Polymer.

ACS Appl Mater Interfaces. 2023-7-12

[2]
Specific Clearance of Lipopolysaccharide from Blood Based on Peptide Bottlebrush Polymer for Sepsis Therapy.

Adv Mater. 2023-8

[3]
Heterogeneity of Lipopolysaccharide as Source of Variability in Bioassays and LPS-Binding Proteins as Remedy.

Int J Mol Sci. 2023-5-7

[4]
Mechanistic insights into silica nanoparticle-allergen interactions on antigen presenting cell function in the context of allergic reactions.

Nanoscale. 2023-2-2

[5]
Single-Particle Functionality Imaging of Antibody-Conjugated Nanoparticles in Complex Media.

ACS Appl Bio Mater. 2023-1-16

[6]
Bacterial Species Associated with Highly Allergenic Plant Pollen Yield a High Level of Endotoxins and Induce Chemokine and Cytokine Release from Human A549 Cells.

Inflammation. 2022-12

[7]
A Journey from Structure to Function of Bacterial Lipopolysaccharides.

Chem Rev. 2022-10-26

[8]
Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles.

J Vis Exp. 2021-3-28

[9]
Initiating pollen sensitization - complex source, complex mechanisms.

Clin Transl Allergy. 2020-8-31

[10]
Biologic excipients: Importance of clinical awareness of inactive ingredients.

PLoS One. 2020-6-25

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