Mapping the Aggregation Kinetics of a Therapeutic Antibody Fragment
Mapping the Aggregation Kinetics of a Therapeutic Antibody Fragment
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DOI：
10.1021/acs.molpharmaceut.5b00387
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发表时间：
2016-02-01
影响因子：
4.9
通讯作者：
Dalby, Paul A.
中科院分区：
文献类型：
--
作者：
Chakroun, Nesrine;Hilton, David;Dalby, Paul A.
生物药物的分析表征是开发和预测其在生物过程中的行为的早期阶段的基本步骤。蛋白质聚集是一个特别常见的问题，因为它影响到产品开发的所有阶段。在目前的工作中，我们研究了A33Fab的稳定性和聚集动力学，A33Fab是一种具有治疗意义的人源化抗体片段，在广泛的pH、离子强度和温度范围内。我们发现，A33Fab在热加速条件下的聚集倾向与pH和离子强度有关，低pH时离子强度的不稳定作用更强。在没有添加盐的情况下，A33Fab分子似乎受到保护，不会由于低pH下的静电胶体排斥而聚集。透射电子显微镜分析表明，在低pH和高pH条件下形成的聚集体种类明显不同。据报道，热转变的表观中点(T-m，T-APP值)或未折叠摩尔分数与聚集率之间的相关性仅在65摄氏度的高温下才显著，其中来自未折叠状态的聚集占主导地位。在所有其他条件下，特别是在4-45℃，A33Fab主要以类天然状态聚集，动力学服从Arrhenius行为。尽管如此，在45摄氏度、23摄氏度和4摄氏度观察到的聚集率的等级顺序仍然没有很好地相互关联，这表明在高温下强制降解不是预测低温行为的好方法。
The analytical characterization of biopharmaceuticals is a fundamental step in the early stages of development and prediction of their behavior in bioprocesses. Protein aggregation in particular is a common issue as it affects all stages of product development. In the present work, we investigate the stability and the aggregation kinetics of A33Fab, a therapeutically relevant humanized antibody fragment at a wide range of pH, ionic strength, and temperature. We show that the propensity of A33Fab to aggregate under thermally accelerated conditions is pH and ionic-strength dependent with a stronger destabilizing effect of ionic strength at low pH. In the absence of added salts, A33Fab molecules appear to be protected from aggregation due to electrostatic colloidal repulsion at low pH. Analysis by transmission electron microscopy identified significantly different aggregate species formed at low and high pH. The correlations between apparent midpoints of thermal transitions (T-m,T-app values), or unfolded mole fractions, and aggregation rates are reported here to be significant only at the elevated incubation temperature of 65 degrees C, where aggregation from the unfolded state predominates. At all other conditions, particularly at 4-45 degrees C, aggregation of A33 Fab was predominantly from a native-like state, and the kinetics obeyed Arrhenius behavior. Despite this, the rank order of aggregation rates observed at 45 degrees C, 23 and 4 degrees C still did not correlate well to each other, indicating that forced degradation at elevated temperatures was not a good screen for predicting behavior at low temperature.