RESUMEN
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.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G , Ligandos , Humanos , Adsorción , Inmunoglobulina G/química , Inmunoglobulina G/sangre , Triptaminas/química , Cromatografía Liquida/métodos , Bencimidazoles/química , Concentración de Iones de HidrógenoRESUMEN
The combination of mixed-mode chromatography (MMC) and molecular imprinting technology (MIT) has been proven to be successful for protein separation, but suffered from cumbersome material preparation and limited performance. In this work, a new modification method marrying atom transfer radical polymerization (ATRP) and multicomponent reaction was proposed to simplify the preparation process. Using regenerated cellulose (RC) membrane as the substrate, immunoglobulin G (IgG) as the template protein and tryptamine as the ligand, a dual-recognition membrane adsorbers (MIM) was prepared by mild Ugi four-component reaction (Ugi-4CR) and surface initiated ATRP. Control the ATRP time is the key for surface imprinting. The static IgG uptake and selectivity of UGI membrane were 45 mg/mL and 1.8, respectively, while those of MIM-0.5 were 42.5 mg/mL and 14, indicating that the introduction of molecular imprinting technology significantly improved the selectivity of the membrane to IgG. The MIM-0.5 membrane retains the pH-dependent and salt-tolerant of HCIC. The dynamic flow-through results showed that the MIM-0.5 membrane could effectively separate IgG from IgG/BSA mixed solution with the purity of 88% and retained its bioactivity. This work demonstrated the feasibility of bonding HCIC and MIT to the membrane surface by Ugi-4CR and ATRP.
Asunto(s)
Cromatografía , Inmunoglobulina G , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/química , Ligandos , PolimerizacionRESUMEN
Antibodies are widely used as therapeutic drugs in the treatment of various diseases. Currently, Protein A affinity chromatography is still the most popular technique in antibody purification. However, it has some limitations such as high cost, leakage of ligands and requirement of harsh elution conditions. Hydrophobic charge-induction chromatography (HCIC) provides an alternative to Protein A affinity chromatography. The binding between HCIC resins and target proteins can be achieved via hydrophobic interactions at neutral pH, and proteins can be eluted via electrostatic repulsion between proteins and charged ligands under acidic conditions. HCIC is applied to the purification of antibodies and some specific proteins successfully, which is a promising technique with economic benefits and high efficiency. In this review, theoretical analysis and factors affecting HCIC adsorption are presented that provides functional mechanism of HCIC. HCIC ligands with different structures available on the market and reported in the literature are discussed and their recent applications in antibody purification are reviewed. Moreover, affecting factors such as ligand density, preparation approaches and additives of HCIC resins on their adsorption performance are summarized and discussed. In addition, future development of HCIC such as polymer-grafted HCIC resins, membrane chromatography with HCIC and continuous chromatography are proposed.
Asunto(s)
Cromatografía Liquida/métodos , Animales , Anticuerpos/aislamiento & purificación , Células CHO , Fenómenos Químicos , Cricetinae , Cricetulus , Humanos , Electricidad EstáticaRESUMEN
The aim of the present work was to develop a new scalable and cost-efficient process to isolate bovine immunoglobulin G from colostral whey with high purity and minimal loss of activity. The mixed mode material Mercapto-Ethyl-Pyridine-Hypercel™ was identified appropriate for direct capture of immunoglobulin G. The binding mechanism is primarily based on hydrophobic interactions at physiological conditions. As compared to immunoglobulin G, all other low molecular whey proteins such as α-Lactalbumin or ß-Lactoglobulin, except lactoperoxidase, are more hydrophilic and were therefore found in the flow-through fraction. In order to remove lactoperoxidase as an impurity the column was combined in series with a second mixed mode material (Capto™- with N-benzoyl-homocysteine as ligand) using the same binding conditions. At pH 7.5 the carboxyl group of this ligand is negatively charged and can hence bind the positively charged lactoperoxidase, whose isoelectric point is at pH 9.6. After sample application, the columns were eluted separately. By combining the two columns it was possible to obtain immunoglobulin G with a purity of >96.1% and yield of 65-80%. The process development was carried out using 1â¯mL columns and upscaling was performed in three steps up to a column volume of 8800â¯mL for the Hypercel™ column and 3000â¯mL for the Capto™- column. At this scale it is possible to obtain 130-150â¯g pure immunoglobulin G from 3â¯L colostrum within five hours, including the regeneration of both columns. Additionally, the impact of freeze-drying on the isolated immunoglobulin G was studied. The nativity of the freeze dried immunoglobulin was above 95%, which was proven by reversed phase liquid chromatography and validated by differential scanning calorimetry. The activity of immunoglobulin G was preserved over the isolation process and during drying as measured by enzyme-linked immunosorbent assay. In conclusion, by applying the proposed isolation process, it becomes feasible to obtain pure, active and stable imunnunoglobulin G at large scale.
Asunto(s)
Técnicas de Química Analítica/métodos , Cromatografía , Calostro/química , Inmunoglobulina G/aislamiento & purificación , Leche/química , Suero Lácteo/química , Animales , Bovinos , Ensayo de Inmunoadsorción Enzimática , Proteínas de la Leche/análisisRESUMEN
The structural and functional properties of a series of dextran-grafted and non-grafted hydrophobic charge-induction chromatographic (HCIC) agarose resins were characterized by macroscopic and microscopic techniques. The effects of dextran grafting and mobile phase conditions on the pore dimensions of the resins were investigated with inverse size exclusion chromatography (ISEC). A significantly lower pore radius (17.6nm) was found for dextran-grafted than non-grafted resins (29.5nm), but increased salt concentration would narrow the gap between the respective pore radii. Two proteins, human immunoglobulin G (hIgG) and bovine serum albumin (BSA), were used to examine the effect of protein characteristics. The results of adsorption isotherms showed that the dextran-grafted resin with high ligand density had substantially higher adsorption capacity and enhanced the salt-tolerance property for hIgG, but displayed a significantly smaller benefit for BSA adsorption. Confocal laser scanning microscopy (CLSM) showed that hIgG presented more diffuse and slower moving adsorption front compared to BSA during uptake into the resins because of the selective binding of multiple species from polyclonal IgG; polymer-grafting with high ligand density could enhance the rate of hIgG transport in the dextran-grafted resins without salt addition, but not for the case with high salt and BSA. The results indicate that microscopic analysis using ISEC and CLSM is useful to improve the mechanistic understanding of resin structure and of critical functional parameters involving protein adsorption and transport, which would guide the rational design of new resins and processes.
Asunto(s)
Técnicas de Química Analítica/instrumentación , Dextranos/química , Adsorción , Animales , Cromatografía en Gel , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Ligandos , Porosidad , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Cloruro de Sodio/químicaRESUMEN
Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the functional ligand has been developed as a new technology for antibody purification. In the present work, molecular simulation methods were developed to investigate the interactions between the Fc fragment of IgG and a MEP ligand net. The MM/PBSA method was used to evaluate the binding energy for the MEP ligand net at different densities. It was found that ligand density had significant influence on the binding of Fc. Potential binding conformations were further analyzed by molecular dynamics simulation. It was found that the interaction between Fc and MEP ligand net is driven by self-adaptive conformation adjustment and multiple-site binding. Hydrophobic forces dominate the binding interaction, which appeared as the results of synergistic actions of binding sites located on CH2, CH3, and the consensus binding site (CBS) of the Fc fragment. At acidic pH, the electrostatic repulsion between the basic residues and the protonated pyridine ring group on MEP ligands is the main driving force for the detachment of the Fc fragment from the MEP net.
Asunto(s)
Anticuerpos/química , Cromatografía/instrumentación , Resinas Sintéticas/química , Anticuerpos/aislamiento & purificación , Sitios de Unión , Cromatografía/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Simulación de Dinámica Molecular , Unión Proteica , Piridinas/química , Electricidad EstáticaRESUMEN
Hydrophobic charge-induction chromatography is a new technology for antibody purification. To improve antibody adsorption capacity of hydrophobic charge-induction resins, new poly(glycidyl methacrylate)-grafted hydrophobic charge-induction resins with 5-aminobenzimidazole as a functional ligand were prepared. Adsorption isotherms, kinetics, and dynamic binding behaviors of the poly(glycidyl methacrylate)-grafted resins prepared were investigated using human immunoglobulin G as a model protein, and the effects of ligand density were discussed. At the moderate ligand density of 330 µmol/g, the saturated adsorption capacity and equilibrium constant reached the maximum of 140 mg/g and 25 mL/mg, respectively, which were both much higher than that of non-grafted resin with same ligand. In addition, effective pore diffusivity and dynamic binding capacity of human immunoglobulin G onto the poly(glycidyl methacrylate)-grafted resins also reached the maximum at the moderate ligand density of 330 µmol/g. Dynamic binding capacity at 10% breakthrough was as high as 76.3 mg/g when the linear velocity was 300 cm/h. The results indicated that the suitable polymer grafting combined with the control of ligand density would be a powerful tool to improve protein adsorption of resins, and new poly(glycidyl methacrylate)-grafted hydrophobic charge-induction resins have a promising potential for antibody purification applications.
Asunto(s)
Cromatografía/instrumentación , Inmunoglobulina G/aislamiento & purificación , Ácidos Polimetacrílicos/química , Sefarosa/química , Adsorción , Bencimidazoles/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/química , Cinética , Ligandos , Resinas Sintéticas/químicaRESUMEN
Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the ligand is a novel technology for antibody purification. In this study, isothermal titration calorimetry (ITC) was used to evaluate the molecular interactions between MEP ligand and immunoglobulin G (IgG). Three types of IgG molecules including human IgG (hIgG), bovine IgG (bIgG) and a monoclonal antibody (mAb) were investigated with human serum albumins (HSA) and bovine serum albumin (BSA) as the comparison. The thermodynamic parameters obtained from ITC were compared with the adsorption data. The results indicated that MEP binding to protein at neutral pH was entropy driven and induced by multimodal molecular interactions that was dominated by hydrophobic forces. The interactions between MEP and IgGs were stronger than that of albumins, which resulted in high binding affinity of IgGs. Moreover, the effects of pH and salt addition on MEP-hIgG binding were studied. The change of enthalpy increased obviously with the decrease of pH, which revealed that the electrostatic forces dominated the MEP-hIgG interactions at acidic condition and caused typical charge-induced elution of HCIC. Salt addition influenced both hydrophobic and electrostatic interactions. With the increase of salt concentration, the hydrophobic interactions decreased first and then increased, while the electrostatic interactions showed the opposite trend. This resulted in trade-off between the multimodal interactions, which caused the salt-tolerant property of MEP resin. In general, ITC studies revealed the molecular mechanism of three critical characteristics of HCIC, multimodal interactions, pH-dependent and salt-tolerant properties.
Asunto(s)
Calorimetría , Cromatografía , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/metabolismo , Ligandos , Adsorción , Anticuerpos/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Entropía , Humanos , Concentración de Iones de Hidrógeno , Unión Proteica , Piridinas/química , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Cloruro de Sodio/química , Electricidad Estática , TermodinámicaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Técnicas de Química Analítica/métodos , Cromatografía , Inmunoglobulina G/aislamiento & purificación , Adsorción , Bencimidazoles/química , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , LigandosRESUMEN
Expanded bed adsorption (EBA) can capture target proteins directly from unclarified feedstock without prior solid-liquid separation. Hydrophobic charge-induction chromatography (HCIC) is a promising technology for biomolecule separation with high capacity, good selectivity and relatively low cost without the pretreatment of dilution or salt addition. In this work, EBA and HCIC were combined to develop a new separation technology, hydrophobic charge-induction EBA. Two HCIC ligands, 4-mercapto-ethyl-pyridine (MEP) and 5-aminobenzimidazole (ABI), were coupled onto agarose beads containing tungsten carbide to prepare the resins for EBA, named T-MEP and T-ABI, respectively. The static adsorption and dynamic binding behaviors of bovine IgG (bIgG) were investigated. Two resins had similar saturated adsorption capacities and salt-tolerant properties, but T-ABI showed higher dynamic binding capacity than T-MEP, indicating that ABI ligand was more suitable for EBA. The performances in expanded bed were verified. With the protein mixture (2mg/ml bIgG and 10mg/ml bovine serum albumin) as the model feedstock, the effects of loading and elution pH, expansion factor and loading volume on the separation performance of bIgG were evaluated. Finally, T-ABI EBA was used to separate bIgG directly from bovine whey with optimized operation conditions. The purity and recovery of bIgG reached 90.6% and 78.2%, respectively. The purification factor was about 19.3. The results demonstrated that the combination of HCIC and EBA would be a potential platform for antibody capture with less feedstock pretreatments, high efficiency and relatively low cost.
Asunto(s)
Bencimidazoles/química , Técnicas de Química Analítica/métodos , Cromatografía , Inmunoglobulina G/aislamiento & purificación , Adsorción , Animales , Bovinos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/metabolismo , Ligandos , Piridinas/química , Sefarosa/química , Albúmina Sérica Bovina/química , Cloruro de Sodio/químicaRESUMEN
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.
Asunto(s)
Bencimidazoles/análisis , Bencimidazoles/química , Cromatografía/instrumentación , Inmunoglobulina G/química , Adsorción , Animales , Anticuerpos Monoclonales/química , Células CHO , Bovinos , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , Electroforesis en Gel de Poliacrilamida , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/aislamiento & purificación , Ligandos , Sales (Química) , Albúmina Sérica Bovina/química , Cloruro de Sodio/químicaRESUMEN
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 7.0â¼8.9 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.
Asunto(s)
Dextranos/química , Imidazoles/química , Inmunoglobulina G/química , Sefarosa/química , Cloruro de Sodio , Adsorción , Cromatografía Liquida , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , LigandosRESUMEN
Hydrophobic charge-induction chromatography, a novel chromatographic technique for bioseparation, was developed to isolate and purify bovine IgG with high purity. In this work, the raw IgG solution, a precipitate from bovine colostrum powder solution with 40% (wt/vol) ammonium sulfate, was dissolved in 50mM phosphate buffer and used as loading solution for investigating chromatographic conditions on a mercapto-ethyl-pyridine (MEP) HyperCel (Pall Corp., Port Washington, NY) sorbent. The initial IgG concentration had no effect on the dynamic binding capacity of MEP HyperCel resin, but the linear velocity of loading solution had an obvious effect on the dynamic IgG binding capacity and IgG recovery. The maximum linear velocity was optimized as 0.4cm/min of loading solution, and 90% recovery of IgG was achieved. Under these optimized binding conditions, the pH and ionic strength for the elution process were selected as pH 4.5 and 0.5 M NaCl, respectively. Subsequently, hydrophobic charge-induction chromatography was performed on a MEP HyperCel sorbent to isolate IgG using bovine colostrum whey as the loading solution. Under the optimized operation conditions, a remarkable process improvement in IgG purification was received, which includes a yield of 91.5%, a purity of 93.9% (wt/wt), and a purification factor of 6.8. The results indicated that MEP HyperCel chromatography offers an efficient means to purify IgG from bovine colostrums.
Asunto(s)
Bovinos , Cromatografía Liquida/métodos , Calostro/química , Inmunoglobulina G/química , Animales , Femenino , Interacciones Hidrofóbicas e Hidrofílicas , Embarazo , Unión ProteicaRESUMEN
The chromatographic separation of two proteins into a displacement train of two adjoined rectangular bands was accomplished using a novel method for hydrophobic charge induction chromatography (HCIC) which employs a self-sharpening pH front as the displacer. This method exploits the fact that protein elution in HCIC is promoted by a pH change, but is relatively independent of salt effects, so that a retained pH front can be used in place of a traditional displacer in displacement chromatography. The retained pH front was produced using the two adsorbed buffering species tricine and acetic acid. The separation of lysozyme and α-chymotrypsinogen A into adjoined, rectangular bands was accomplished with overall recoveries based on the total mass injected greater than 90 and 70%, respectively. The addition of urea to the buffer system increased the sharpness of the pH front by 36% while the yields of lysozyme and α-chymotrypsinogen A based on the total mass eluted increased from 76% to 99% and from 37% to 85%, respectively, when the purities of both proteins in their product fractions were fixed at 85%. The results demonstrate that the method developed in this study is a useful variant of HCIC and is also a useful alternative to other displacement chromatography methods.
Asunto(s)
Cromatografía Liquida , Interacciones Hidrofóbicas e Hidrofílicas , Extracción Líquido-Líquido/métodos , Proteínas/química , Adsorción , Quimotripsinógeno/análisis , Quimotripsinógeno/aislamiento & purificación , Concentración de Iones de Hidrógeno , Muramidasa/análisis , Muramidasa/aislamiento & purificación , Proteínas/aislamiento & purificación , Fuerza Protón-MotrizRESUMEN
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 200 µmol/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 200 µmol/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 200 µmol/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.
Asunto(s)
Cromatografía Liquida/instrumentación , Dextranos/química , Interacciones Hidrofóbicas e Hidrofílicas , Sefarosa/química , Adsorción , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/aislamiento & purificación , Cinética , LigandosRESUMEN
The manufacture of human serum albumin by chromatographic procedures involves gel filtration chromatography as a final polishing step. Despite this step being essential to remove high molecular weight impurity proteins and thus ensure a stable and safe final product, it is relatively inefficient. This paper explores the use of hydrophobic charge induction chromatographic media, MEP HyperCel as an alternative to Sephacryl S200HR gel filtration for the polishing of human serum albumin derived by ion exchange chromatographic purification of Cohn Supernatant I. The use of MEP HyperCel results in a product with a higher purity than achieved with gel filtration and in a less time consuming manner and with potential resource savings. MEP HyperCel appears to have great potential for incorporation into downstream processes in the plasma fractionation industry as an efficient means of achieving polishing of intermediates or capture of proteins of interest.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Albúmina Sérica/aislamiento & purificación , Cromatografía por Intercambio Iónico/instrumentación , Contaminación de Medicamentos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Albúmina Sérica/análisis , Albúmina Sérica/química , Albúmina Sérica/normasRESUMEN
Hydrophobic charge-induction chromatography (HCIC) has emerged as a useful addition to Protein A chromatography for antibody purification due to its remarkable merits in cost and stability. However, the instability of antibody during acidic elution, which may cause inactivation and aggregation, is still a major concern for the efficiency of this method. The aim of this study is to develop a new strategy of competitive elution with inclusion complexes in HCIC, and to apply it to antibody elution under neutral pH conditions. Interactions between 4-mercaptoethylpyridine (MEP), a typical ligand of HCIC, and four different types of cyclodextrins (CDs) were investigated by molecular docking; immunoglobulin G (IgG) elution capacities of CDs were characterized on MEP-based HCIC mediums. The results demonstrated the general effectiveness of CD-based eluents for HCIC. This type of displacement eluents could allow an efficient elution of bound antibody over a broad range of pH and ion strength. With 15 mM ß-CD, elution of human IgG was achieved at physiological pH, with an average IgG recovery of 87%. When this elution strategy was used to separate antibody directly from human serum, substantial elution of bound IgG could be obtained at pH 7.4, with product purity comparable to traditional method with an acidic buffer. We expect such method can be of special interest in developing HCIC elution strategy for the proteins like antibody that are sensitive to acidic conditions.
Asunto(s)
Cromatografía , Ciclodextrinas/química , Inmunoglobulina G/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Piridinas/químicaRESUMEN
Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pHAsunto(s)
Productos Biológicos/aislamiento & purificación
, Cromatografía en Gel/métodos
, Cromatografía Líquida de Alta Presión/métodos
, Escherichia coli/genética
, Factor Estimulante de Colonias de Granulocitos/aislamiento & purificación
, Dicroismo Circular
, Electroforesis en Gel de Poliacrilamida
, Ensayo de Inmunoadsorción Enzimática
, Factor Estimulante de Colonias de Granulocitos/genética
RESUMEN
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.