Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Engler-Bunte-Ring 3, 76131, Karlsruhe, Germany.
Biotechnol J. 2017 Sep;12(9). doi: 10.1002/biot.201700255. Epub 2017 Aug 14.
Recent studies highlighted the potential of PEGylated proteins to improve stabilities and pharmacokinetics of protein drugs. Ion-exchange chromatography (IEX) is among the most frequently used purification methods for PEGylated proteins. However, the underlying physical mechanisms allowing for a separation of different PEGamers (proteins with a varying number of attached PEG molecules) are not yet fully understood. In this work, mechanistic chromatography modeling is applied to gain a deeper understanding of the mass transfer and adsorption/desorption mechanisms of mono-PEGylated proteins in IEX. Using a combination of the general rate model (GRM) and the steric mass action (SMA) isotherm, simulation results in good agreement with the experimental data are achieved. During linear gradient elution of proteins attached with PEG of different molecular weight, similar peak heights, and peak shapes at constant gradient length are observed. A superimposed effect of increased desorption rate and reduced diffusion rate as a function of the hydrodynamic radius of PEGylated proteins is identified to be the reason of this anomaly. That is why the concept of the diffusion-desorption-compensation effect is proposed. In addition to the altered elution orders, PEGylation results in a considerable decrease of maximum binding capacity. By using the SMA model in a kinetic formulation, the adsorption behavior of PEGylated proteins in the highly concentrated state is described mechanistically. An exponential increase in the steric hindrance effect with increasing PEG molecular weight is observed. This suggests the formation of multiple PEG layers in the interstitial space between bound proteins and an associated shielding of ligands on the adsorber surface to be the cause of the reduced maximum binding capacity. The presented in silico approach thus complements the hitherto proposed theories on the binding mechanisms of PEGylated proteins in IEX.
最近的研究强调了聚乙二醇化蛋白质改善蛋白质药物稳定性和药代动力学的潜力。离子交换色谱（IEX）是最常用于分离聚乙二醇化蛋白质的纯化方法之一。然而，允许分离不同 PEGamers（具有不同数量附着的 PEG 分子的蛋白质）的基本物理机制尚未完全理解。在这项工作中，应用机制色谱模型来深入了解 IEX 中单聚乙二醇化蛋白质的传质和吸附/解吸机制。使用通用速率模型（GRM）和立体质量作用（SMA）等温线的组合，可以实现与实验数据非常吻合的模拟结果。在不同分子量的 PEG 修饰的蛋白质进行线性梯度洗脱时，观察到恒定梯度长度下相似的峰高和峰形。发现增加的解吸速率和降低的扩散速率作为聚乙二醇化蛋白质的流体力学半径的函数的叠加效应是这种异常的原因。因此，提出了扩散-解吸-补偿效应的概念。除了改变洗脱顺序外，PEG 化还导致最大结合容量显着降低。通过在动力学公式中使用 SMA 模型，可以从机理上描述聚乙二醇化蛋白质在高浓度状态下的吸附行为。观察到随着 PEG 分子量的增加，空间位阻效应呈指数增加。这表明在结合蛋白质之间的间隙空间中形成了多个 PEG 层，并且配体在吸附剂表面上的屏蔽是最大结合容量降低的原因。因此，提出的计算方法补充了迄今为止提出的 IEX 中聚乙二醇化蛋白质结合机制的理论。
