Application of Raman Spectroscopy to Dynamic Binding Capacity Analysis.
Application of Raman Spectroscopy to Dynamic Binding Capacity Analysis.
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DOI：
10.1177/00037028231210293
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发表时间：
2023-12
影响因子：
3.5
通讯作者：
中科院分区：
文献类型：
--
作者：
蛋白A亲和层析是分离含有片段可结晶区域的生物治疗剂（BT）（包括单克隆和双特异性抗体）的关键步骤。动态结合容量（DBC）分析评估BT将与蛋白A柱结合的量。DBC随着色谱柱的使用而降低，随着时间的推移有效地减少了回收产物的量。药品监管机构通过测量包括DBC在内的参数来强制规定BT分离的色谱树脂寿命，因此在工业纯化管道中仔细监测该特征。高效亲和色谱法（HPAC）通常用于评估BT的浓度，当上样至色谱柱时，会导致BT在流通液中的显著突破。HPAC可以准确评估DBC及其随时间的变化，但仅报告蛋白质浓度，需要对每个新分析的BT进行校准，并且只能离线使用。在这里，我们利用拉曼光谱，并揭示，这种方法是至少一样有效的HPAC和紫外色谱法在监测蛋白A树脂的DBC。除了报告蛋白质浓度外，拉曼光谱中的化学信息还提供了关于聚集状态和蛋白质结构的信息，为工业生物加工管道提供了额外的质量控制。结合偏最小二乘（PLS）分析，拉曼光谱可用于确定BT的DBC而无需预先校准。在这里，我们在96孔板格式中离线进行拉曼分析，然而，在线进行该分析是可行的。这项研究证明了拉曼光谱作为一种显着改进的方法，在工业管道中的DBC监测的权力。这是抽象的视觉表现。
Protein A affinity chromatography is a key step in isolation of biotherapeutics (BTs) containing fragment crystallizable regions, including monoclonal and bispecific antibodies. Dynamic binding capacity (DBC) analysis assesses how much BT will bind to a protein A column. DBC reduces with column usage, effectively reducing the amount of recovered product over time. Drug regulatory bodies mandate chromatography resin lifetime for BT isolation, through measurement of parameters including DBC, so this feature is carefully monitored in industrial purification pipelines. High-performance affinity chromatography (HPAC) is typically used to assess the concentration of BT, which when loaded to the column results in significant breakthrough of BT in the flowthrough. HPAC gives an accurate assessment of DBC and how this changes over time but only reports on protein concentration, requires calibration for each new BT analyzed, and can only be used offline. Here we utilized Raman spectroscopy and revealed that this approach is at least as effective as both HPAC and ultraviolet chromatogram methods at monitoring DBC of protein A resins. In addition to reporting on protein concentration, the chemical information in the Raman spectra provides information on aggregation status and protein structure, providing extra quality controls to industrial bioprocessing pipelines. In combination with partial least square (PLS) analysis, Raman spectroscopy can be used to determine the DBC of a BT without prior calibration. Here we performed Raman analysis offline in a 96-well plate format, however, it is feasible to perform this inline. This study demonstrates the power of Raman spectroscopy as a significantly improved approach to DBC monitoring in industrial pipelines. This is a visual representation of the abstract.
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DOI：
10.1016/s1570-0232(03)00092-8
发表时间：
2003-06-25
影响因子：
3
作者：
Hahn, R;Schlegel, R;Jungbauer, A
通讯作者：
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DOI：
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影响因子：
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通讯作者：
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影响因子：
5.7
作者：
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通讯作者：
Mayolo-Deloisa K
DOI：
10.1039/d1an00985k
发表时间：
2021-08-09
期刊：
The Analyst
影响因子：
--
作者：
Beattie JW;Rowland-Jones RC;Farys M;Tran R;Kazarian SG;Byrne B
通讯作者：
Byrne B
影响因子：
2.9
作者：
MOORE, WH;KRIMM, S
通讯作者：
KRIMM, S