Institute for Biochemistry, University of Applied Sciences, Mannheim, Germany.
CMC Microbial Platform, Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany.
J Sep Sci. 2022 Jun;45(12):2008-2023. doi: 10.1002/jssc.202200098. Epub 2022 Apr 6.
The mechanistic modeling of preparative liquid chromatography is still a challenging task. Nonideal thermodynamic conditions may require activity coefficients for the mechanistic description of preparative chromatography. In this work, a chromatographic cation exchange step with a polypeptide having a complex elution behavior in low and high loading situations is modeled. Model calibration in the linear range of the isotherm is done by applying counterion-induced linear gradient elution experiments between pH 3.3 and 4.3. Inverse fitting with column loads up to 25 mg/mL is performed for parameter estimation in the nonlinear range. The polypeptide elution peak shows an anti-Langmuirian behavior with fronting under low loading conditions and a switch to a Langmuirian behavior with increasing load. This unusual elution behavior could be described with an extended version of the sigmoidal Self-Association isotherm including two activity coefficients for the polypeptide and counterion in solution. The activity coefficient of the solute polypeptide shows a strong influence on the model parameters and is crucial in the linear and nonlinear range of the isotherm. The modeling procedure results in a unique and robust model parameter set that is sufficient to describe the complex elution behavior and allows modeling over the full isotherm range.
制备液相色谱的机理建模仍然是一项具有挑战性的任务。非理想热力学条件可能需要活度系数来对制备色谱进行机理描述。在这项工作中，对具有在低和高负载情况下具有复杂洗脱行为的多肽进行了色谱阳离子交换步骤的建模。通过在 pH 3.3 和 4.3 之间进行抗衡离子诱导的线性梯度洗脱实验，在等温线的线性范围内进行模型校准。在非线性范围内，对柱载量高达 25mg/mL 进行逆拟合，以进行参数估计。多肽洗脱峰表现出反 Langmuir 行为，在低负载条件下前伸，随着负载增加转变为 Langmuir 行为。这种异常的洗脱行为可以用扩展的 Sigmoidal 自缔合等温线来描述，其中包括溶液中多肽和抗衡离子的两个活度系数。溶质多肽的活度系数对模型参数有很大影响，在等温线的线性和非线性范围内都是至关重要的。建模过程产生了一组独特且稳健的模型参数集，足以描述复杂的洗脱行为，并允许在整个等温线范围内进行建模。
