Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold.
Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold.
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DOI：
10.1016/j.chroma.2015.08.068
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发表时间：
2015-10-09
期刊：
影响因子：
--
通讯作者：
Bracewell DG
中科院分区：
文献类型：
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作者：
Mazzer AR;Perraud X;Halley J;O'Hara J;Bracewell DG
IgG在低pH下的聚集遵循pH依赖的指数衰减动力学。IgG的聚集行为受到蛋白a层析步骤的影响。分离过程似乎加速了聚合机制。蛋白A层析是一种在生物过程中几乎普遍存在的单抗捕获方法。已知使用低pH缓冲液从蛋白A中洗脱有助于产物聚集。然而，一组更有限的证据表明，低pH值可能不是蛋白a层析中聚集的唯一原因，相反，该过程的其他方面可能起着重要作用。本文提出了一种定义良好的方法来研究这个问题。蛋白A层析（典型的病毒灭活舱）后，在洗脱缓冲液中孵育IgG4，中和样品中单体的数量通过尺寸排除层析测定；使用不同pH值的洗脱缓冲液来诱导IgG4的聚集。通过将指数衰减函数拟合到数据中，确定了单体随时间衰减的速率常数。在没有层析步骤的情况下进行了类似的实验，即在低pH下IgG4聚集。蛋白a层析后的聚集速率常数明显高于低pH单独暴露的速率常数；在测试的pH范围内，聚集率的明显变化是明显的。
IgG aggregation at low pH follows pH-dependent exponential decay kinetics. IgG aggregation behaviour is influenced by a protein A chromatography step. The mechanism of aggregation appears to be accelerated by the separation process. Protein A chromatography is a near-ubiquitous method of mAb capture in bioprocesses. The use of low pH buffer for elution from protein A is known to contribute to product aggregation. Yet, a more limited set of evidence suggests that low pH may not be the sole cause of aggregation in protein A chromatography, rather, other facets of the process may contribute significantly. This paper presents a well-defined method for investigating this problem. An IgG4 was incubated in elution buffer after protein A chromatography (typical of the viral inactivation hold) and the quantity of monomer in neutralised samples was determined by size exclusion chromatography; elution buffers of different pH values predetermined to induce aggregation of the IgG4 were used. Rate constants for monomer decay over time were determined by fitting exponential decay functions to the data. Similar experiments were implemented in the absence of a chromatography step, i.e. IgG4 aggregation at low pH. Rate constants for aggregation after protein A chromatography were considerably higher than those from low pH exposure alone; a distinct shift in aggregation rates was apparent across the pH range tested.