Column-free optical deconvolution of intrinsic fluorescence for a monoclonal antibody and its product-related impurities
Column-free optical deconvolution of intrinsic fluorescence for a monoclonal antibody and its product-related impurities
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单克隆抗体及其产品相关杂质的内在荧光的无柱光学解卷积
DOI：
10.1016/j.chroma.2023.464463
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发表时间：
2023
影响因子：
4.1
通讯作者：
Uçan D
中科院分区：
文献类型：
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作者：
Uçan D
用高压液相色谱体积排阻色谱(HPLC-SEC)对单抗聚集物和片段进行定量通常需要耗时的离线测量，因此与实时监测不兼容。然而，它对制造和工艺开发至关重要，并且仍然是评估与产品相关的杂质的行业标准。在这里，我们证明了我们以前建立的固有的时间分辨荧光(TRF)方法可以用于在典型下游过程的不同阶段对抗体产品纯度的生物过程关键质量属性(CQA)进行量化，并有可能被开发用于在线生物过程监测。这是直接以行业标准的高效液相色谱-SEC为基准的。对于单体含量和聚集体片段含量，TRF光谱和高效液相色谱-SEC的输出之间存在很强的线性相关性，R2系数分别为0.99和0.69。当蛋白质总浓度高于1.41 mg/mL时，高效液相-SEC UV-Vis色谱图显示检测器饱和的迹象，这降低了蛋白质定量的准确性，因此需要额外的样品稀释步骤。相比之下，由于更高的信噪比，TRF光谱在这些浓度下的准确度提高了。我们的方法有可能将验证mAb生物过程阶段聚集含量所需的时间和劳动力从高效液相色谱-SEC所需的几个小时减少到每个样品几分钟。
The quantification of monoclonal antibody (mAb) aggregates and fragments using high pressure liquid chromatography-size exclusion chromatography (HPLC-SEC) typically requires off-line measurements that are time-consuming and therefore not compatible with real-time monitoring. However, it has been crucial to manufacturing and process development, and remains the industrial standard in the assessment of product-related impurities. Here we demonstrate that our previously established intrinsic time-resolved fluorescence (TRF) approach can be used to quantify the bioprocess critical quality attribute (CQA) of antibody product purity at various stages of a typical downstream process, with the potential to be developed for in-line bioprocess monitoring. This was directly benchmarked against industry-standard HPLC-SEC. Strong linear correlations were observed between outputs from TRF spectroscopy and HPLC-SEC, for the monomer and aggregate-fragment content, withR2coefficients of 0.99 and 0.69, respectively. At total protein concentrations above 1.41 mg/mL, HPLC-SEC UV-Vis chromatograms displayed signs of detector saturation which reduced the accuracy of protein quantification, thus requiring additional sample dilution steps. By contrast, TRF spectroscopy increased in accuracy at these concentrations due to higher signal-to-noise ratios. Our approach opens the potential for reducing the time and labour required for validating aggregate content in mAb bioprocess stages from the several hours required for HPLC-SEC to a few minutes per sample.