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An efficient mathematical tool for the design and scaling up of protein chromatography is suggested, in which the model parameters can be determined quickly over a wide operating space without large material investments. The design method is based on mathematical modelling of column dynamics and moment analysis. The accuracy of the dynamic models that are most frequently used for simulations of chromatographic processes is analyzed, and possible errors that can be generated using the moment analysis are indicated. The so-called transport dispersive model was eventually employed for the process simulations. The model was modified to account for the protein dispersion in void volumes of chromatographic systems. The manner of the model calibration was suggested, which was based on a few chromatographic runs and verified over a wide space of the operating parameters, including composition and flow rate of the mobile phase, column dimensions, residence time, and mass loading. The model system for the study was ion-exchange chromatography. The analysis was performed based on the elution profiles of basic fibroblast growth factor 2 and lysozyme, on two different IEX media.

A model-based approach for scaling up chromatographic capture step was developed. The purification of human basic fibroblast growth factor protein 2 (FGF2) from an E. coli homogenate on a cation exchange resin was selected as a case study. Non-ideal effects accompanying the capture operation were examined, including: reduction in the protein diffusivity in the presence of the homogenate, competitive adsorption between FGF2 and undefined impurities, and flow behavior in external column volumes. The viscosity of the homogenate was measured as a function of dilution degree and shear stress, and its contribution to the diffusivity reduction was quantified. A dynamic model was formulated which accounted for underlying kinetic and thermodynamic dependencies. The model parameters were determined for a lab scale system using a small 2-mL column. The model was successfully used to scale up the capture operation from the lab scale column to a preparative bench scale column of about 1 L volume.

Basic fibroblast growth factor (FGF-2) is a labile protein which degrades over time or forms aggregates. A purification process combining a cation ion exchange chromatography (CIEX) capture step with a polishing step by hydrophobic interaction chromatography (HIC) efficiently depleted process-related impurities such as host cell proteins (HCPs), dsDNA and endotoxins from the clarified E. coli homogenate containing the overexpressed FGF-2. Fresh homogenates did not contain verifiable amounts of process-related impurities such as degraded product variants. However, the storage of the capture eluates for longer than two weeks at 4 °C led to product degradation up to 49%. Mass spectrometry showed that the truncated FGF-2 was devoid of the nine N-terminal amino acids. Removal of such product-related impurities is included in state of the art purifications because these byproducts bear the risk of having different biological activities. Therefore, the established purification process has been evaluated for its potential to deplete such truncated FGF-2 variants. The HIC polishing step using Toyopearl® Hexyl-650C with a linear salt gradient over 4 column volumes from 1.65 M trisodium citrate to 0 M separated this variant from the full-length FGF-2. The truncated form eluted before the full length product due to the loss of 9 hydrophobic amino acids. The developed ion exchange HPLC method enabled us to quantify the truncated variant and full-length FGF-2. A purity of > 98% full-length FGF-2 with a step yield of up 86% is achieved. As long as the degree of truncation does not exceed 25% of the stored eluates, the purification process delivers the product in the desired quality of > 98% full-length FGF-2, HCP content below 10 ppm/dose, dsDNA content below 10 ng/mL/dose and endotoxin level below 400 EU/dose. This purification sequence allowed the production of FGF-2 which meets distinctive quality standards defined by regulatory bodies with respect to the depletion of both - process- and product-related impurities.