Subvisible Particle Counting Provides a Sensitive Method of Detecting and Quantifying Aggregation of Monoclonal Antibody Caused by Freeze-Thawing: Insights Into the Roles of Particles in the Protein Aggregation Pathway
Subvisible Particle Counting Provides a Sensitive Method of Detecting and Quantifying Aggregation of Monoclonal Antibody Caused by Freeze-Thawing: Insights Into the Roles of Particles in the Protein Aggregation Pathway
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DOI：
10.1002/jps.22305
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发表时间：
2011-02-01
影响因子：
3.8
通讯作者：
Carpenter, John F.
中科院分区：
文献类型：
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作者：
Barnard, James G.;Singh, Satish;Carpenter, John F.
本研究的目的是评价微流成像（MFI）作为检测和定量IgG（2）单克隆抗体（mAb）冻融过程中不溶性微粒形成的灵敏工具。将在20 mM组氨酸缓冲液（pH 5.5）中配制的蛋白质溶液进行三次冻融循环，并通过MFI和尺寸排阻色谱法（SEC）进行分析。MFI显示每个冻融循环后颗粒数量增加，而SEC未检测到聚集体。对形成的颗粒的总质量的估计显示，对颗粒形成的监测允许检测仅占总蛋白质质量百分之一的蛋白质聚集体。此外，解决了由于不同制剂或不同冻融方案引起的蛋白质聚集水平差异，即使蛋白质聚集不能通过SEC检测到。为了检查基于SEC和MFI的总聚集体质量估计值是否定量一致，将mAb在磷酸盐缓冲盐水中冻融。该过程产生了足够水平的不溶性聚集体，通过SEC检测，作为色谱图中单体峰面积的减少。SEC检测到的单体损失与MFI检测到的不溶性微粒总质量之间具有良好的一致性。(C)2010 Wiley-Liss，Inc.和American Pharmacologist Association J Pharm Sci 100：492-503，2011
The objective of this study was to evaluate microflow imaging (MFI) as a sensitive tool to detect and quantify subvisible particle formation during freeze-thawing of an IgG(2) monoclonal antibody (mAb). Solutions of the protein formulated in 20 mM of histidine buffer (pH 5.5) were subjected to three freeze thaw cycles and analyzed by MFI and size-exclusion chromatography (SEC). MFI showed increased particle numbers after each freeze-thaw cycle, whereas aggregates were not detected by SEC. Estimates of the total mass of particles formed revealed that monitoring of particle formation allows for the detection of protein aggregates comprising only hundredths of a percent of the total protein mass. Furthermore, differences in protein aggregation levels due to different formulations or different freeze-thawing protocols were resolved, even though protein aggregation could not be detected by SEC. To examine whether SEC and MFI-based estimations of total aggregate mass were in quantitative agreement, mAb was freeze-thawed in phosphate-buffered saline. This process created sufficient level of insoluble aggregates to be detected by SEC as a reduction in the monomer peak area in the chromatogram. There was good agreement between the loss of monomer detected by SEC and the total mass of subvisible particles detected by MFI. (C) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:492-503, 2011