Chromatography process development aided by a dye-based assay
Chromatography process development aided by a dye-based assay
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DOI：
10.1002/jctb.6214
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发表时间：
2019-10-20
影响因子：
3.4
通讯作者：
Bracewell, Daniel G.
中科院分区：
文献类型：
--
作者：
Jasulaityte, Greta;Johansson, Hans J.;Bracewell, Daniel G.
背景：用于生产治疗性蛋白质的层析树脂的平均寿命通常在10到300个循环之间。为每个分离过程开发和建立该方法的稳健性是一项重大挑战，并受到广泛的监管监督。本文提出了一种新的基于荧光的残留聚集蛋白的分析方法，以帮助评估树脂再生的程度。结果用强阴、阳离子交换树脂Praesto Q和SP与牛血清白蛋白和单抗配合使用，证明了该方法的通用性。该分析需要将分子转子染料应用于游离树脂样品，并使用平板阅读器测量荧光强度，或在共聚焦激光扫描显微镜下进行可视化，以获得更详细的表征。在连续五次层析循环后，两种方法都显示荧光强度增加了10倍，同时动态结合能力成比例地降低。此外，分析表明，污垢与柱中微珠的空间位置、微珠通道结构和清洗条件有关。结论本工作提供了一种简便的测定方法，适用于树脂寿命研究，以加深对工艺的理解。(三)2019年化学工业学会
BACKGROUND The lifetime of chromatography resins typically averages between 10 and 300 cycles for the manufacture of a therapeutic protein. Developing and establishing the robustness of the method for each separation process represents a significant challenge and is subject to extensive regulatory oversight. This paper presents a novel fluorescence-based assay for residual aggregated proteins to aid the evaluation of the extent of resin regeneration. RESULTS The versatility of this method was demonstrated by using strong anion and cation exchange agarose resins Praesto Q and SP in conjunction with bovine serum albumin and monoclonal antibody feed materials. The assay entails applying a molecular rotor dye to a sample of free resin and measuring the fluorescence intensity using a plate reader or visualizing under a confocal laser scanning microscope to gain a more detailed characterization. Following five consecutive chromatography cycles, both methods revealed a 10-fold increase in fluorescence intensity along with a proportional reduction in dynamic binding capacity. Furthermore, the use of the assay suggested that fouling was dependent on spatial bead position in the column, bead channel structure and cleaning conditions. CONCLUSION This work presents a simple assay suitable for use in resin lifetime studies to enhance process understanding. (c) 2019 Society of Chemical Industry