Predicting multimodal chromatography of therapeutic antibodies using multiscale modeling.

Multimodal chromatography has emerged as a powerful method for the purification of therapeutic antibodies. However, process development of this separation technique remains challenging because of an intricate and molecule-specific interaction towards multimodal ligands, leading to time-consuming and costly experimental optimization. This study presents a multiscale modeling approach to predict the multimodal chromatographic behavior of therapeutic antibodies based on their sequence information. Linear gradient elution (LGE) experiments were performed on an anionic multimodal resin for 59 full-length antibodies, including five different antibody formats at pH 5.0, 6.0, and 7.0 that were used for parameter determination of a linear adsorption model at low loading density conditions. Quantitative structure-property relationship (QSPR) modeling was utilized to correlate the adsorption parameters with up to 1374 global and local physicochemical descriptors calculated from antibody homology models. The final QSPR models employed less than eight descriptors per model and demonstrated high training accuracy (R² > 0.93) and reasonable test set prediction accuracy (Q² > 0.83) for the adsorption parameters. Model evaluation revealed the significance of electrostatic interaction and hydrophobicity in determining the chromatographic behavior of antibodies, as well as the importance of the HFR3 region in antibody binding to the multimodal resin. Chromatographic simulations using the predicted adsorption parameters showed good agreement with the experimental data for the vast majority of antibodies not employed during the model training. The results of this study demonstrate the potential of sequence-based prediction for determining chromatographic behavior in therapeutic antibody purification. This approach leads to more efficient and cost-effective process development, providing a valuable tool for the biopharmaceutical industry.

Antibody sequence-based prediction of pH gradient elution in multimodal chromatography.

Multimodal chromatography has emerged as a promising technique for antibody purification, owing to its capacity to selectively capture and separate target molecules. However, the optimization of chromatography parameters remains a challenge due to the intricate nature of protein-ligand interactions. To tackle this issue, efficient predictive tools are essential for the development and optimization of multimodal chromatography processes. In this study, we introduce a methodology that predicts the elution behavior of antibodies in multimodal chromatography based on their amino acid sequences. We analyzed a total of 64 full-length antibodies, including IgG1, IgG4, and IgG-like multispecific formats, which were eluted using linear pH gradients from pH 9.0 to 4.0 on the anionic mixed-mode resin Capto adhere. Homology models were constructed, and 1312 antibody-specific physicochemical descriptors were calculated for each molecule. Our analysis identified six key structural features of the multimodal antibody interaction, which were correlated with the elution behavior, emphasizing the antibody variable region. The results show that our methodology can predict pH gradient elution for a diverse range of antibodies and antibody formats, with a test set R² of 0.898. The developed model can inform process development by predicting initial conditions for multimodal elution, thereby reducing trial and error during process optimization. Furthermore, the model holds the potential to enable an in silico manufacturability assessment by screening target antibodies that adhere to standardized purification conditions. In conclusion, this study highlights the feasibility of using structure-based prediction to enhance antibody purification in the biopharmaceutical industry. This approach can lead to more efficient and cost-effective process development while increasing process understanding.

Standardized method for mechanistic modeling of multimodal anion exchange chromatography in flow through operation.

Multimodal chromatography offers an increased selectivity compared to unimodal chromatographic methods and is often employed for challenging separation tasks in industrial downstream processing (DSP). Unfortunately, the implementation of multimodal polishing into a generic downstream platform can be hampered by non-robust platform conditions leading to a time and cost intensive process development. Mechanistic modeling can assist experimental process development but readily applicable and easy to calibrate multimodal chromatography models are lacking. In this work, we present a mechanistic modeling aided approach that paves the way for an accelerated development of anionic mixed-mode chromatography (MMC) for biopharmaceutical purification. A modified multimodal isotherm model was calibrated using only three chromatographic experiments and was employed in the retention prediction of four antibody formats including a Fab, a bispecific, as well as an IgG1 and IgG4 antibody subtype at pH 5.0 and 6.0. The chromatographic experiments were conducted using the anionic mixed-mode resin Capto adhere at industrial relevant process conditions to enable flow through purification. An existing multimodal isotherm model was reduced to hydrophobic interactions in the linear range of the adsorption isotherm and successfully employed in the simulation of six chromatographic experiments per molecule in concert with the transport dispersive model (TDM). The model reduction to only three parameters did prevent structural parameter non-identifiability and enabled an analytical isotherm parameter determination that was further refined by incorporation of size exclusion effects of the selected multimodal resin. During the model calibration, three linear salt gradient elution experiments were performed for each molecule followed by an isotherm parameter uncertainty assessment. Lastly, each model was validated with a set of step and isocratic elution experiments. This standardized modeling approach facilitates the implementation of multimodal chromatography as a key unit operation for the biopharmaceutical downstream platform, while increasing the mechanistic insight to the multimodal adsorption behavior of complex biologics.