Survey of chemical unfolding complexity as a unique stability assessment assay for monoclonal antibodies.

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry Pub Date : 2025-03-01 Epub Date: 2024-11-29 DOI:10.1016/j.ab.2024.115729

J Alaina Floyd, Jeremy M Shaver

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引用次数: 0

Abstract

Seventy-two intentionally sequence-diverse antibody variable regions were selected, expressed as IgG1 antibodies, and evaluated by chemical unfolding to survey the complexities of denaturant induced unfolding behavior. A two-transition fit well described the curves and uncovered a wide range of sensitivities to denaturant. Four general types of unfolding curves were observed: balanced traces (each transition responsible for half of the total unfolding curve), low-unfolding traces (first transition is a majority of the unfolding curve), high-unfolding traces (the second transition is the majority of the unfolding curve), and coincident traces (the two transitions are found close to each other, approximating a single transition). The complexity of the data from this survey indicates that focusing on the first inflection point or fitting a single transition model is likely an over-simplistic method for measuring stability by the chemical unfolding assay. Additionally, other conformational assays, such as thermal and low pH unfolding, showed no correlation with the chemical unfolding results, indicating that each of these assays provide alternate information on the different pathways of antibody conformational stability. These results provide a basis for beginning to better connect unfolding behavior to other physical phenotypic behaviors and production process behaviors.

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化学展开复杂性作为单克隆抗体独特稳定性评估方法的研究进展。

选择72个序列不同的抗体可变区，表达为IgG1抗体，并通过化学展开来评估变性剂诱导展开行为的复杂性。两个过渡拟合很好地描述了曲线，并揭示了对变性剂的广泛敏感性。观察到四种一般类型的展开曲线：平衡轨迹（每个转变占整个展开曲线的一半），低展开轨迹（第一次转变是展开曲线的大部分），高展开轨迹（第二次转变是展开曲线的大部分）和重合轨迹（两个转变彼此接近，近似于单个转变）。该调查数据的复杂性表明，专注于第一个拐点或拟合单一过渡模型可能是通过化学展开试验测量稳定性的过于简单的方法。此外，其他构象分析，如热展开和低pH展开，显示与化学展开结果没有相关性，这表明这些分析提供了抗体构象稳定性不同途径的替代信息。这些结果为开始更好地将展开行为与其他物理表型行为和生产过程行为联系起来提供了基础。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Analytical biochemistry 生物-分析化学

CiteScore

5.70

自引率

0.00%

发文量

283

审稿时长

44 days

期刊介绍： The journal''s title Analytical Biochemistry: Methods in the Biological Sciences declares its broad scope: methods for the basic biological sciences that include biochemistry, molecular genetics, cell biology, proteomics, immunology, bioinformatics and wherever the frontiers of research take the field. The emphasis is on methods from the strictly analytical to the more preparative that would include novel approaches to protein purification as well as improvements in cell and organ culture. The actual techniques are equally inclusive ranging from aptamers to zymology. The journal has been particularly active in: -Analytical techniques for biological molecules- Aptamer selection and utilization- Biosensors- Chromatography- Cloning, sequencing and mutagenesis- Electrochemical methods- Electrophoresis- Enzyme characterization methods- Immunological approaches- Mass spectrometry of proteins and nucleic acids- Metabolomics- Nano level techniques- Optical spectroscopy in all its forms. The journal is reluctant to include most drug and strictly clinical studies as there are more suitable publication platforms for these types of papers.