Standardized approach for accurate and reliable model development of ion-exchange chromatography based on parameter-by-parameter method and consideration of extra-column effects.

Developing an accurate and reliable model for chromatographic separation that meets regulatory requirements and ensures consistency in model development remains challenging. In order to address this challenge, a standardized approach was proposed in this study with ion-exchange chromatography (IEC). The approach includes the following steps: liquid flow identification, system and column-specific parameters determination and validation, multi-component system identification, protein amount validation, steric mass action parameters determination and evaluation, and validation of the calibrated model's generalization ability. The parameter-by-parameter (PbP) calibration method and the consideration of extra-column effects were integrated to enhance the accuracy of the developed models. The experiments designed for implementing the PbP method (five gradient experiments for model calibration and one stepwise experiment for model validation) not only streamline the experimental workload but also ensure the extrapolation abilities of the model. The effectiveness of the standardized approach is successfully validated through an application about the IEC separation of industrial antibody variants, and satisfactory results were observed with R2 ≈ 0.9 for the majority of calibration and validation experiments. The standardized approach proposed in this work contributes significantly to improve the accuracy and reliability of the developed IEC models. Models developed using this standardized approach are ready to be applied to a broader range of industrial separation systems, and are likely find further applications in model-assisted decision-making of process development.

Multimodal charge-induction chromatography for antibody purification.

Hydrophobic charge-induction chromatography (HCIC) has advantages of high capacity, salt-tolerance and convenient pH-controlled elution. However, the binding specificity might be improved with multimodal molecular interactions. New ligand W-ABI that combining tryptophan and 5-amino-benzimidazole was designed with the concept of mutimodal charge-induction chromatography (MCIC). The indole and benzimidazole groups of the ligand could provide orientated mutimodal binding to target IgG under neutral pH, while the imidazole groups could induce the electrostatic repulsion forces for efficient elution under acidic pH. W-ABI ligand was coupled successfully onto agarose gel, and IgG adsorption behaviors were investigated. High affinity to IgG was found with the saturated adsorption capacity of 70.4 mg/ml at pH 7, and the flow rate of mobile phase showed little impact on the dynamic binding capacity. In addition, efficient elution could be achieved at mild acidic pH with high recovery. Two separation cases (IgG separation from albumin containing feedstock and monoclonal antibody purification from cell culture supernatant) were verified with high purity and recovery. In general, MCIC with the specially-designed ligand is an expanding of HCIC with improved adsorption selectivity, which would be a potential alternative to Protein A-based capture for the cost-effective purification of antibodies.

Molecular recognition of Fc-specific ligands binding onto the consensus binding site of IgG: insights from molecular simulation.

Immunoglobulin G (IgG) plays an important role in clinical diagnosis and therapeutics. Meanwhile, the consensus binding site (CBS) on the Fc domain of IgG is responsible for ligand recognition, especially for Fc-specific ligands. In this study, molecular simulation methods were used to investigate molecular interactions between the CBS of the Fc domain and seven natural Fc-specific ligands. The analysis on the binding energy of the Fc-ligand complex indicated that hydrophobic interactions provide the main driving force for the Fc-ligand binding processes. The hot spots on the ligands and Fc were identified with the computational alanine scanning approach. It was found that the residues of tryptophan and tyrosine on the ligands have significant contributions for the Fc-ligand binding, while Met252, Ile253, Asn434, His435, and Tyr436 are the key residues of Fc. Moreover, two binding modes based on tryptophan or tyrosine were summarized and constructed according to the pairwise interaction analysis. Guidelines for the rational design of CBS-specific ligands with high affinity and specificity were proposed.

Molecular insights into the binding selectivity of a synthetic ligand DAAG to Fc fragment of IgG.

Affinity chromatography with synthetic ligands has been focused as the potential alternative to protein A-based chromatography for antibody capture because of its comparable selectivity and efficiency. Better understanding on the molecular interactions between synthetic ligand and antibody is crucial for improving and designing novel ligands. In this work, the molecular interaction mechanism between Fc fragment of IgG and a synthetic ligand (DAAG) was studied with molecular docking and dynamics simulation. The docking results on the consensus binding site (CBS) indicated that DAAG could bind to the CBS with the favorable orientation like a tripod for the top-ranked binding complexes. The ligand-Fc fragment complexes were then tested by molecular dynamics simulation at neutral condition (pH 7.0) for 10 ns. The results indicated that the binding of DAAG on the CBS of Fc fragment was achieved by the multimodal interactions, combining the hydrophobic interaction, electrostatic interaction, hydrogen bond, and so on. It was also found that multiple secondary interactions endowed DAAG with an excellent selectivity to Fc fragment. In addition, molecular dynamics simulation conducted at acidic condition (pH 3.0) showed that the departure of DAAG ligand from the surface of Fc fragment was the result of reduced interaction energies. The binding modes between DAAG and CBS not only shed light on the molecular mechanisms of DAAG for antibody purification but also provide useful information for the improvement of ligand design.

Evaluation of mixed-mode chromatographic resins for separating IgG from serum albumin containing feedstock.

Mixed-mode chromatography has been focused as a cost-effective new technique for antibody purification. In this study, four mixed-mode resins with N-benzyl-N-methyl ethanol amine, 2-benzamido-4-mercaptobutanoic acide, 4-mercapto-ethyl-pyridine and phenylpropylamine as the ligands were tested and the multi-functional interactions between ligand and protein were discussed. Immunoglobulin G (IgG), bovine serum albumin (BSA) and the binary mixture of BSA and IgG were used as the model feedstock to compare the separation behaviors by pH gradient elution. The comparison analysis showed mixed-mode resin with N-benzyl-N-methyl ethanol amine as the ligand had the best ability to separate IgG and BSA. The results indicated that for four resins tested ionic interaction might play the dominant role in the separation of IgG and BSA while the hydrophobic interactions and hydrogen bonding have some subsidiary effects. The pH stepwise elution and sample loading were optimized to improve the IgG purification from serum albumin containing feedstock. High purity (92.3%) and high recovery (95.6%) of IgG were obtained. The results indicated that mixed-mode chromatography would be a potential option for antibody purification with the control of loading and elution conditions.