Hydrophobic Charge-induction Chromatography Resin Research Articles

Transforming waste into valuables: Preparation and evaluation of dual-ligand hydrophobic charge-induction chromatography using two poor performing ligands

In conventional chromatographic ligand screening, underperforming ligands are often dismissed. However, this practice may inadvertently overlook potential opportunities. This study aims to investigate whether these underperforming ligands can be repurposed as valuable assets. Hydrophobic charge-induction chromatography (HCIC) is chosen as the validation target for its potential as an innovative chromatographic mode. A novel dual-ligand approach is employed, combining two suboptimal ligands (5-Aminobenzimidazole and Tryptamine) to explore enhanced performance and optimization prospects. Various dual-ligand HCIC resins with different ligand densities were synthesized by adjusting the ligand ratio and concentration. The resins were characterized to assess appearance, functional groups, and pore features using SEM, FTIR, and ISEC techniques. Performance assessments were conducted using single-ligand mode resins as controls, evaluating the selectivity against human immunoglobulin G and human serum albumin. Static adsorption experiments were performed to understand pH and salt influence on adsorption. Breakthrough experiments were conducted to assess dynamic adsorption capacity of the novel resin. Finally, chromatographic separation using human serum was performed to evaluate the purity and yield of the resin. Results indicated that the dual-ligand HCIC resin designed for human antibodies demonstrates exceptional selectivity, surpassing not only single ligand states but also outperforming certain high-performing ligand types, particularly under specific salt and pH conditions. Ultimately, a high yield of 83.9 % and purity of 96.7 % were achieved in the separation of hIgG from human serum with the dual-ligand HCIC, significantly superior to the single-ligand resins. In conclusion, through rational design and proper operational conditions, the dual-ligand mode can revitalize underutilized ligands, potentially introducing novel and promising chromatographic modes.

A combined screening and in silico strategy for the rapid design of integrated downstream processes for process and product-related impurity removal.

Integrated designs of chromatographic processes for purification of biopharmaceuticals provides potential gains in operational efficiency and reductions of costs and material requirements. We describe a combined method using screening and in silico algorithms for ranking chromatographic steps to rapidly design orthogonally selective integrated processes for purifying protein therapeutics from both process- and product-related impurities. IFN-α2b produced in Pichia pastoris containing a significant product variant challenge was used as a case study. The product and product-related variants were screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography resins under various pH and salt linear gradient conditions. Data generated from reversed-phase chromatography of the fractions collected were used to generate a retention database for IFN-α2b and its variants. These data, in combination with a previously constructed process-related impurity database for P. pastoris, were input into an in silico process development tool that generated and ranked all possible integrated chromatographic sequences for their ability to remove both process and product-related impurities. Top-ranking outputs guided the experimental refinement of two successful three step purification processes, one comprising all bind-elute steps and the other having two bind-elute steps and a flowthrough operation. This approach suggests a new platform-like approach for rapidly designing purification processes for a range of proteins where separations of both process- and product-related impurities are needed.

Evaluation on adsorption selectivity of immunoglobulin G with 2-mercapto-1-methyl-imidazole-based hydrophobic charge-induction resins

Hydrophobic charge-induction chromatography (HCIC) with 2-mercapto-1-methyl-imidazole (MMI) ligands shows promising results in antibody purification. In this study, competitive adsorption processes between bovine immunoglobulin G (IgG) and bovine serum albumin (BSA) were investigated with MMI resins. Adsorption isotherms of IgG, BSA and IgG/BSA mixtures at various mass ratios were measured, and the Langmuir-Freundlich model was used to fit the adsorption experimental data. The results indicated that IgG could be preferentially adsorbed by MMI resins, and MMI-B-4FF-100 resin with the pore size of 43.2nm and the ligand density of 101μmol/g gel showed the best adsorption selectivity of IgG over BSA. In addition, binding behaviors of IgG and BSA in column were studied with MMI-B-4FF-100 at various pH values, and the optimized separation conditions were obtained as loading at pH 7.0 and elution at pH 4.0. Furthermore, IgG was effectively separated from IgG/BSA protein mixtures with MMI-B-4FF-100. The purities of IgG monomer reached above 95%, and the recovery kept about 90%. The results indicated that MMI-B-4FF-100 with high ligand density and moderate pore size would be a promising HCIC resin for IgG purification.

A novel polymer-grafted hydrophobic charge-induction chromatographic resin for enhancing protein adsorption capacity

Hydrophobic charge-induction chromatography (HCIC) is a developing technology for antibody purification. To enhance the protein adsorption capacity, a novel polymer-grafted HCIC resin was developed, in which the surface-initiated activator generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) was explored as a controlled polymerization technique to reconstruct matrix structure and ligand distribution. Using poly(glycidyl methacrylate, GMA) as grafting polymer and 2-mercapto-1-methyl-imidazole (MMI) as functional ligand, poly(GMA)-grafted HCIC resins were prepared with series of grafting and ligand densities. Adsorption behaviors of human immunoglobulin G (hIgG) on the prepared resins demonstrated the necessity of controlling grafting and ligand density. Saturated adsorption capacity (Qm) and effective pore diffusivity (De) reached the maximum under medium ligand density when the grafting density was kept constant. The highest Qm and De values were found under the highest grafting density, which were 73% and 7.17times higher than the non-grafted resin, respectively. Column breakthrough tests indicated that the dynamic binding capacity of the resin with optimized grafting density and ligand density was up to 34.6mg/g when linear velocity was 300cm/h, which was 86.3% higher than dextran-grafted resin. The resin was then used to separate hIgG from a protein mixture (hIgG/human serum albumin=1:4), high purity (>99%) and recovery (>90%) of hIgG were found with 50-cycle reuses, which verified the selectivity and robustness of G-MMI resin prepared. In general, the surface-initiated AGET ATRP provides a controlled grafting strategy to improve protein binding capacity for chromatographic separation, and new resins developed have great potential in large-scale protein purification applications.

Hydrophobic charge-induction resin with 5-aminobenzimidazol as the functional ligand: preparation, protein adsorption and immunoglobulin G purification.

A new hydrophobic charge-induction chromatography resin was prepared with 5-aminobenzimidazol as functional ligand and polyacrylic ester beads as matrix. Adsorption isotherms and adsorption in columns were investigated using human immunoglobulin G and bovine serum albumin as model proteins, and the influence of pH and NaCl concentration was discussed. Results showed that the ligand density was 195 μmol/mL gel, and protein selectivity can be improved by controlling pH and salt addition. An optimized purification process (sample loading at pH 8.0 with 0.2 M NaCl and elution at pH 5.0) was performed to purify human immunoglobulin G from bovine serum albumin containing feedstock, which resulted in human immunoglobulin G purity of 99.7% and recovery of 94.6%. A similar process was applied for the purification of monoclonal antibody from cell culture supernatant, which showed antibody purity of 94.9% and recovery of 92.5%. The results indicated that the new resin developed had comparable performance as Protein A chromatography and would be suitable for antibody purification from complex feedstock.

Characterization of immunoglobulin adsorption on dextran-grafted hydrophobic charge-induction resins: Cross-effects of ligand density and pH/salt concentration

Hydrophobic charge-induction chromatography (HCIC) is a promising technology for antibody purification. New HCIC resins MMI-B-XL with dextran-grafted agarose gel as the matrix and 2-mercapto-1-methyl-imidazole (MMI) as the functional ligand were prepared with different ligand densities. The adsorption behaviors (static adsorption equilibrium and adsorption kinetics) of human immunoglobulin G (hIgG) on series of MMI-B-XL resins at varying pHs and salt concentrations were investigated. The cross-effects of solid phase property (ligand density) and liquid phase conditions (pH and salt concentration) were focused. The results showed that the new resins had typical pH-dependent and salt-tolerant characteristics for hIgG adsorption, but differences were found for the resins with different ligand densities. For MMI-B-XL resins with higher ligand density, an obvious higher saturated adsorption capacity (Qm) and effective pore diffusivity (De) could be obtained, which were less affected at pH 7.0∼8.9 but dropped drastically at pH 5.0. Salt addition had less influence on protein adsorption onto MMI-B-XL with higher ligand density. Qm and De both reached minimum values at 0.2mol/L NaCl for all MMI-B-XL resins tested. The results of dynamic binding in the column demonstrated that MMI-B-XL with higher ligand density had better performance for hIgG adsorption, especially under high linear velocities. The mechanism of the cross-effects of ligand density and pH/salt concentration on IgG adsorption was discussed, which provides new insights into protein adsorption and mass transport for dextran-grafted HCIC resins.

Preparation and evaluation of dextran-grafted agarose resin for hydrophobic charge-induction chromatography

Hydrophobic charge-induction chromatography (HCIC) is a new and effective technology for antibody separation. In the present work, HCIC resin MMI-B-XL was prepared with dextran-grafted agarose gel as the matrix and 2-mercapto-1-methyl-imidazole (MMI) as the functional ligand. The preparation procedures were optimized, and the maximum ligand density could reach as high as 200μmol/g gel. The adsorption isotherms and kinetics on new resins were investigated with human immunoglobulin G (hIgG) as the model protein, which were compared with non-grafted HCIC resin MMI-B-6FF. It was found that the saturated adsorption capacity (Qm) increased with the increase of ligand density for MMI-B-XL. Moreover, the effective diffusivity (De) could be dramatically enhanced with the increase of ligand density for MMI-B-XL, and the De for MMI-B-XL with the ligand density of 200μmol/g gel was 18–40 times higher than that for MMI-B-6FF. The breakthrough experiments indicated that new resins with the ligand density of 200μmol/g gel could be used for high superficial velocity and high dynamic adsorption could be obtained. The results indicated that dextran-grafted layer on the resin could increase the ligand density, enhance the mass transport in the pore, and improve the dynamic adsorption at high velocity, which showed a potential application for large-scale antibody purification.

Caprylate as the albumin-selective modifier to improve IgG purification with hydrophobic charge-induction chromatography

Hydrophobic charge-induction chromatography (HCIC) is a novel downstream bioprocessing technology for antibody purification and it has several advantages over traditional purification processes. However, its separation selectivity still needs to be improved. In this work, sodium caprylate (NaCA) was used as the selective modifier to improve IgG purification from serum albumin containing feedstock with a typical HCIC resin, MEP HyperCel. The effects of NaCA on the adsorption equilibrium of bovine serum immunoglobulin G (IgG) and bovine serum albumin (BSA), as well as the dynamic binding and displacement behaviors were investigated. The binding and elution behaviors of these two proteins in the column were studied. It was found that adding 50–75mM NaCA in the liquid phase could effectively reduce the adsorption of BSA on the MEP resin, but the same treatment has little influence on the adsorption of IgG. Moreover, the mechanism of the competitive binding between caprylate and MEP ligands on the surface of BSA is discussed. It was found that by controlling NaCA addition in the loading or washing buffer, the process efficiency of IgG purification from BSA containing feedstock can be improved, and the purity of IgG could reach to over 98%. The results indicated that caprylate could be a promising albumin-selective modifier to improve the separation efficiency of antibodies with the HCIC process.

Evaluation of immunoglobulin adsorption on the hydrophobic charge-induction resins with different ligand densities and pore sizes

Hydrophobic charge-induction chromatography (HCIC) is a novel technology for antibody purification. The ligand densities and pore properties of HCIC resins have significant effects on the separation behavior of protein, however, the understandings are quite limited. In the present work, new HCIC ligand, 2-mercapto-1-methylimidazole (MMI) was coupled to three agarose matrices with different pore sizes. A series of MMI resins with different ligand density and pore size was prepared by the control of ligand coupling. The adsorption isotherms and kinetics on the series of MMI resins were investigated with bovine serum immunoglobulin as the model IgG, and the effects of salt addition were studied. The Langmuir equation and pore diffusion model were used to fit the experimental data, and the influences of ligand density, pore size and salt addition on the saturated adsorption capacity, the dissociation constant and the effective diffusivity were discussed. It was found that the adsorption capacities and the effective pore diffusion coefficient increased with the increase of ligand density and pore size. The effects of salt addition on the adsorption behaviors were dependent on the ligand density. For low ligand density the IgG adsorption was salt-promoted, while the resins with high ligand density showed a salt-independent property. The results indicated that for a given protein the ligand density and pore size of HCIC resins should be optimized for improving the protein adsorption.

Enhancing IgG purification from serum albumin containing feedstock with hydrophobic charge-induction chromatography

Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the ligand is a novel technology for antibody purification, however, the separation selectivity still needs to be improved for the applications, especially for the impurity of serum albumin. In this study, with bovine serum immunoglobulin G (IgG) as the model, the purification of IgG from the serum albumin containing feedstock was developed with the commercial HCIC resin MEP HyperCel, focusing on the optimization of operation pH and salt addition. The adsorption isotherms of IgG and bovine serum albumin (BSA) were investigated at different pHs, and the binding and elution behaviors of two proteins in the column were also studied at varying pHs. In addition, the protein–ligand interactions were investigated with some additives in the buffer. It was found that the conditions of pH 6 with 0.1M NaCl or pH 8 could be used to effectively remove BSA from the MEP resin without the influence on IgG adsorption. Two modes with control of loading or washing buffer were tested to enhance the purification of IgG from BSA containing feedstock, and the purity of IgG was improved to about 95% compared with 62.9% for the control. The results demonstrated that the control of loading pH or the addition of NaCl in the buffer might be an effective method to improve the purification of antibody with the HCIC process.

Initial purification of recombinant botulinum neurotoxin fragments for pharmaceutical production using hydrophobic charge induction chromatography

Initial purification of two serotypic variants of recombinant botulinum neurotoxin toxin heavy chain fragment [rBoNT(H c)], produced intracellularly in the yeast Pichia pastoris, using hydrophobic charge induction chromatography (HCIC) is reported. HCIC employs a matrix containing a weakly ionizable ligand that binds proteins through hydrophobic interactions at neutral pH and elutes the proteins by charge repulsion at acidic pH. HCIC optimization led to different purification conditions for each of the proteins even though they have 58% sequence similarity. The HCIC resin has a higher affinity for the fragment of serotype A than that of serotype B. The 10% dynamic breakthrough capacity for the serotype A fragment is >12.5 mg per ml of resin and is ∼3.5 mg or the serotype B fragment per ml of resin. Stable elution conditions are also different for the two serotypes. The serotype A fragment is unstable when citrate is used to elute the product. However the serotype B fragment is stable when eluted with citrate buffer, and it is further purified by a overnight precipitation caused by the citrate buffer. This paper reports the development strategy, dynamic capacity breakthrough curves, resin and separation reproducibility, and preliminary scale-up data. The summation of the data demonstrates that HCIC is a scaleable process step for biopharmaceutical production of rBoNT(H c) proteins.