Quantitative modeling and in-depth analysis of multi-state binding and buffer equilibria in chromatography

• Quantitative Modellierung und Tiefenanalyse von Mehrzustandsbindungen und Puffergleichgewichten in der Chromatographie

Diedrich, Juliane Dorothea; Wiechert, Wolfgang (Thesis advisor); Jupke, Andreas (Thesis advisor)

Aachen (2019, 2020)
Dissertation / PhD Thesis

Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2019

Abstract

The industrial chromatographic purification of biotechnological products, such as milk powder or insulin, is an important part of their production process. The understanding of basic mechanisms in chromatography, such as physical adsorption processes in chromatography columns for example, is key for control and approval of such biotechnological products. Mechanistic models can can contribute to this understanding by a great extent. In this thesis models from two perspectives are introduced. In the first part of this work, unusual peak shapes of an industrial purification of monoclonal antibodies by ion exchange chromatography are observed. With standard binding models this behavior can not be explained. Therefore, a hypothesis of the binding mechanism is formalized. Based on this hypothesis a binding model is developed that describes adsorption of molecules into multiple binding states. By means of the industrial data binding parameters are estimated. The model explains four data sets with one set of parameters. Subsequently, the model serves for a quantitative analysis of the binding mechanism with two binding states. By the analysis, a binding mechanism is revealed that explains the unusual peak shapes at the column outlet and strongly supports the hypothesis formalized in the first place. The introduced approach demonstrates how unusual elution behavior at the column outlet can be explained by detailed description of binding mechanisms inside chromatography columns. The local pH is an important factor influencing the binding behavior of proteins, in particular the adsorption behavior of antibodies is very sensitive towards pH. Therefore, defined buffer conditions are important for chromatographic protein separations. Often, factors impacting the separations, such as pH impacting adsorption behavior, are simulated separate from each other. Therefore, in the second part of this thesis a systematic modeling approach is developed that enables mechanistic description of chromatography including molecule-molecule interactions. Based on this modeling framework a variety of physical and chemical processes and their influence on chromatographic processes can be described. For example, it enables the modeling of buffer reactions, protein aggregation, or enzymatic conversions in fixed-bed reactors. The framework is introduced by means of a pH gradient example with a simple Tris buffer. By assistance of a more complex buffer formulation, which contains eight different buffers, the abilities of the framework are demonstrated predicting linear and non-linear pH elution profiles. The buffer model is used for an in-depth analysis of the interaction of the buffer components with the adsorbent and the influence on the pH at the column outlet. A result of this analysis is that by adsorption of buffer components the equilibrium concentrations of the buffer components shift and influence each other. The developed modeling framework allows to quantitatively predict influences of multiple physical processes, such as pH and adsorption mechanisms, on protein adsporption and elution.