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Abstract: 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. [Copyright &y& Elsevier]

In order to purify lactoferrin from crude sweet wheyin one step,an expanded bed adsorption process was proposed with cation-exchangerFastline SP. A series of experiments were carried out to assess thepossibility of this process and optimize the operating conditions.First the adsorption equilibrium and kinetics of lactoferrin withFastline SP were measured. The results indicated that Fastline SPhad a fast adsorption of lactoferrin and was suitable for high-velocityoperation. Then the expansion properties, liquid mixing, and breakthroughbehaviors in the expanded bed were investigated, and the expansionfactor was set at 2.0. In addition, the elution condition was optimizedas 0.5 M NaCl in the phosphate buffer (pH 7.0). Finally, lactoferrinwas successfully recovered with a high purity of 88.5% and a reasonablerecovery of 77.1% in a single step. The purification factor reached553. [ABSTRACT FROM AUTHOR]

Abstract: The adsorption properties toward rutin of a cyclodextrin polymer adsorbent CroCD-TuC 3 have been studied. The adsorption capacity is reduced as temperature and pH of solution rises, but increases with the increase of solvent polarity. Compared with Sephadex™ G-15 dextran gel beads, CroCD-TuC 3 shows dramatically higher isosteric enthalpy due to a significant contribution of rutin/β-cyclodextrin inclusion complex formation in CroCD-TuC 3 skeleton. A highlight in our study is that the pore diffusion model has been employed to describe the mass transfer inside the adsorbent pores. It reveals that the diffusion inside the pores is the rate-restricting step in the whole adsorption process. The effective pore diffusivity of rutin in CroCD-TuC 3 calculated is much lower than the diffusivity in diluted solution. The pore diffusion model is an available tool to investigate the profile of mass transfer inside the pores, and provides an effective method to describe adsorption kinetics. [Copyright &y& Elsevier]

Abstract: Hydrophobic charge-induction chromatography (HCIC) is a novel bioseparation technology, especially for antibody purification. In order to better understand the molecular mechanism of HCIC, the typical ligand of 4-mercaptoethyl-pyridine (MEP) was coupled onto the cellulose matrix, and the binding and departing of IgG were studied with the molecular dynamics simulation. Based on the previous work with free MEP ligand (J. Phys. Chem. B, 116 (4) (2012) 1393–1400), the pocket around TYR319 and LEU309 on the CH2 domain of IgG was selected as the potential binding site for the Fc fragment of IgG (Fc-A), and the complex of matrix–ligand–Fc-A was formed for the molecular simulation. Both single ligand and ligand net were investigated in the present work. It was found that the MEP ligand immobilized on the cellulose matrix could capture the Fc-A at neutral pH during the simulation, and the Fc-A would depart quickly when pH was changed to 4.0. The hydrophobic interactions and hydrogen bonds controlled the binding of Fc-A on the immobilized ligands at neutral pH and the electrostatic repulsion caused the departing of Fc-A at acid condition. For the ligand net, multipoint binding was found, while one ligand dominated the binding of Fc-A and other ligands might enhance the adsorption of protein. In addition, the adsorption isotherm and the isothermal titration calorimetry (ITC) were used to evaluate the molecular interactions. The experimental results indicated that the hydrophobic interaction is the major driving force for the adsorption of IgG on the MEP resin, which was in good agreement with those findings of molecular simulation. The molecular simulation and thermodynamic results verified strongly the molecular mechanism of HCIC – the hydrophobic interactions for binding and the charge-induction repulsion for elution. Better understanding on the molecular interactions would be beneficial to design new HCIC ligands for improving the efficiency of antibody separation. [Copyright &y& Elsevier]

Immunoglobulin of egg yolk (IgY) was separated from fresh hen eggs by three steps, water dilution, salt-out precipitation, and hydrophobic charge induction chromatography (HCIC). In the purification step, HCIC was used as a novel technology and two chromatographic operation modes, packed bed and expanded bed, were compared to improve the separation efficiency. For packed bed mode, IgY were separated with the purity of 92% and the recovery of 58.4% at the operation condition of pH 7.5 and sample (total protein concentration of 11.6 mg/mL, IgY purity of 31%) loading of 0.2 mL/mL adsorbent. For expanded bed mode, a similar purity of 90% and the recovery of 59.5% were found at the fluid velocity of 500 cm/h and same sample loading of 0.24 mL/ mL adsorbent at pH 7.5. The results demonstrated that HCIC in expanded bed mode is a potential platform technology for antibody separation both in high efficiency and low cost. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Hydrophobic charge-induction chromatography (HCIC) with4-mercaptoethyl-pyridine(MEP) as the ligand is a novel technology for antibody purification.In the present work, the molecular simulation methods were used toinvestigate the interactions between MEP ligand and Fc fragment ofIgG (Fc-A). Six ligands with different structures of spacer arm werestudied with molecular docking and dynamics simulation at neutraland acidic pH. The binding modes and the interaction energies wereanalyzed. The results indicated that all ligands tested could bindinto the selected pocket on the CH2domain of Fc-A at neutralpH. The pyridine ring on the top of MEP ligands acts as a major roleto provide the hydrophobic association and hydrogen bond for the ligand–IgGbinding; meanwhile, the sulfone group on the spacer arm might formthe additional hydrogen bond and enhance the binding of ligand ontothe surface of IgG. The replacements of thioether sulfur atom on thespacer arm with either nitrogen or oxygen atom seem to have littleinfluence on the binding. The influences of pH on the ligand–IgGinteractions were also studied with the molecular dynamics simulation.It was found that MEP ligands would departed from the surface of Fc-Aat low pH due to the electrostatic repulsion. The ligands with a sulfonegroup on the spacer arm would weaken the electrostatic repulsion andneed more acidic conditions for the departing of ligand. The molecularsimulation results were in agreement with some experimental observations,which would be useful to elucidate the molecular mechanism of HCICand design a novel ligand to improve the efficiency of antibody separation. [ABSTRACT FROM AUTHOR]

Abstract: Hydrophobic charge-induction chromatography (HCIC) is a novel bioseparation technique, especially for antibody purification. In this study, HCIC was used for the purification of goose immunoglobulin IgY(ΔFc). IgY(ΔFc) is a kind of unique immunoglobulin existing in waterfowl, which natively lacks CH3 and CH4 domains of heavy chains and has some particular therapeutic applications. A new HCIC ligand, 2-mercapto-1-methyl-imidazole (MMI), was coupled to the divinyl-sulfone-activated agarose matrix to prepare the HCIC gel. The adsorption isotherms of IgY(ΔFc) were investigated at different pHs. The saturated capacity of IgY(ΔFc) at pH 5.0 could reach 187.5mg/ml gel, and the pH-dependent adsorption behaviors were found. After the goose plasma was pre-treated with caprylic acid to precipitate some impurities, the supernatant could be directly loaded onto the HCIC column for IgY(ΔFc) separation. The operation conditions were optimized, including the loading pH, elution pH and the loading volume. High separation efficiency was obtained with final purity of 98.6% and the yield of 85.0%. The results indicate that the new process with HCIC can be a promising technology for the cost-effective separation of IgY(ΔFc) from plasma. [Copyright &y& Elsevier]

Abstract: For the applications of polyelectrolyte complex (PEC) of chitosan and sodium cellulose sulfate (NaCS) as the controlled release drug carrier, the biodegradation characteristics of chitosan, NaCS and chitosan/NaCS PEC films were investigated with pepsin, trypsin, lipase, α-amylase, and cellulase. The results showed that pepsin, amylase, trypsin and lipase have the appreciable hydrolytic activity to chitosan, but cannot degrade NaCS. However, cellulase showed high cellulosic activity and low chitosanolytic activity. For the hydrolysis of chitosan/NaCS films, the degradation rates were greatly influenced by the molecular weights of chitosan and NaCS. In vitro tests showed that different formulations caused diverse disintegration time of the films through the gastrointestinal tract. The results indicated the PEC based on chitosan and NaCS showed good potential for the gastrointestinal delivery systems. [ABSTRACT FROM AUTHOR]

Abstract: The expansion and hydrodynamic properties of matrix are significant for expanded bed adsorption (EBA) processes. A series of new composite matrices CroCD-TuC are studied and estimated in an expanded bed. It is found that the heavier matrix is better suited for high operation fluid velocity than the lighters. Although the Richardson–Zaki equation can well correlate the bed voidage with fluid velocity for all CroCD-TuC matrices tested, the modifications are proposed to improve the accuracy of theoretical predictions of correlation parameters, including terminal settling velocity (U t ) and expansion index (n). Residence time distributions (RTDs) are determined, and the Bodenstein number (Bo) and axial dispersion coefficient (D ax ) are employed to analyze the liquid mixing in the expanded bed. It is found for CroCD-TuC matrices, both parameters notably changed with the variation of fluid velocity and viscosity. Furthermore, D ax is an intuitive parameter estimating the bed stability on various operating conditions, and also a restriction on developing the matrix for high operation fluid velocity. The comparison of the hydrodynamic properties on different matrices reveals that CroCD-TuC 3 and CroCD-TuC 4 seem superior to other matrices in hydrodynamic properties, making them promising matrices for further use. The correlations as the functions of fluid velocity and viscosity have been established which may provide beneficial information for practical applications of CroCD-TuC matrices in EBA processes. [Copyright &y& Elsevier]

Short peptide resin has a great advantage in the separation and purification of antibodies due to its high selectivity and stable ligand binding ability. In this study, a peptide library containing 480 tetrapeptides was established with six critical residues (Phe, Tyr, Leu, Glu, Ile, Lys) of Protein A binding to Fc part of the antibody and one Histidine. Then the library was screened by LibDock and five tetrapeptide ligands with high LibDock scores were selected. Subsequently, the electrostatic interaction energies and van der Waals interaction energies between the screened ligands and the Fc-A fragment of IgG were further calculated by molecular dynamics simulation to research the binding ability between ligands and target proteins. The simulation results showed that YEHF was the optimal ligand with higher van der Waals interaction energies. Then, four tetrapeptides (YFLH, FYHI, FYKH and YEHF) were selected as ligands for preparing adsorption resins, and the resins were used to bind hIgG and BSA by static adsorption experiments. The results showed that Ac-YEHF-4FF resin had higher hIgG binding ability and lower BSA binding ability, also indicating that YEHF was the optimal screened ligand. Therefore, molecular simulation was verified as a well use technology for antibody separation and purification. • A peptide library containing 480 tetrapeptides was established. • LibDock was used to screen the peptide library. • Molecular dynamics simulation was researched to get the binding ability between ligands and targets. • YEHF was identified as the optimal screened ligand. [ABSTRACT FROM AUTHOR]

The article examines a novel mixed-mode expanded bed adsorbent with anion-exchange properties using a benzylamine as the functional ligand. According to the authors, the prepared adsorbent had the ability to bind bovine serum albumin with a high adsorption capacity, while its tolerance of salt also improved. They reveal that the direct capture of target proteins from a biomass-containing feedstock without extra dilution steps is to be expected with a mixed-mode adsorbent prepared in this way.

Abstract: A novel TiO2-densified cellulose beads as the matrix used in expanded beds were exploited. The matrix was activated by epichlorohydrin and then coupled with diethylamine to function as an anion exchanger for the adsorption of proteins. Several factors influenced the efficiency of activation, such as the amount of epichlorohydrin, the concentration of NaOH, and the contents of TiO2 and cellulose in the matrix, were investigated. The optimal procedure for activation was found to be: mixing 20 g matrix beads in 40 mL 2.5 mol/L NaOH solution containing 8 mL epichlorohydrin for 8 h at 25 °C. The coupling efficiency of diethylamine with the activated matrix is close to 90% under the given conditions. The prepared adsorbent has favorable expanded bed characteristics and an accordant protein breakthrough behavior both in packed bed and in expanded bed, by possessing a dynamic adsorption capacity of 42.6 mg BSA/mL adsorbent at 10% breakthrough. The results demonstrated that the custom-made adsorbent can be applied to the expanded bed adsorption of proteins. [Copyright &y& Elsevier]

Aqueous two-phase extraction was used for efficient separation of biological particles with similar size, density, or other physical properties. The partitioning behavior of spores and parasporal crystals of Bacillus thuringiensi were studied in two types of aqueous two-phase systems: polymer/salt systems and polymer/polymer systems. The spores and crystals showed strong tendency towards the top polyethylene glycol (PEG)-rich phase and interface in PEG/salt systems, and were hardly influenced by several factors, such as concentration of PEG and salt, phase separation time, and phase ratio. Using the appropriate system (PEG 6000 2%/(NH4)2SO4 16%), collection of both spores and crystals directly from fermentation broth was carried out with a yield of 96% crystals on lab-scale. Separation of crystals from spores could be achieved using the polymer/polymer systems. It was found that phase-forming polymers, pH, and phase separation time showed significant influence on the partition of spores, and the optimum conditions were chosen as PEG 6000 6%/DEX60 7.5% system, pH 10.0, and 1.5∼2.0 hr for the separation of crystals from spores. The yield of crystals was 83%, and more than 90% of spores could be removed after four cycles of extractions of the bottom phase with fresh top PEG-rich phase. Finally, a process scheme was developed that included an initial extraction of both spores and crystals using PEG/salt system and integrated with subsequent removal of spores by extraction in PEG/dextran system. The feasibility of the integrated separation process was verified by lab-scale application. The total yield of crystals could reach 81.4%, and more than 90% of spores could be removed. [ABSTRACT FROM AUTHOR]

• Widely used modification to activity model deemed thermodynamically inconsistent. • Thermodynamically consistent multi-component activity model was proposed. • Proposed model considered the contributions of all components to activity. • Numerical experiments confirmed thermodynamical consistency of proposed model. • Adoption of thermodynamically consistent activity model was advocated. The single-component Mollerup model, with over 40 direct applications and 442 citations, is the most widely used activity model for chromatographic mechanistic modeling. Many researchers have extended this formula to multi-component systems by directly adding subscripts, a modification deemed thermodynamically inconsistent (referred to as the reference model). In this work, we rederived the asymmetric activity model for multi-component systems, using the van der Waals equation of state, and termed it the multi-component Mollerup model. In contrast to the reference model, our proposed model accounts for the contributions of all components to the activity. Three numerical experiments were performed to investigate the impact of the three different activity models on the chromatographic modeling. The results indicate that our proposed model represents a thermodynamically consistent generalization of the single-component Mollerup model to multi-component systems. This communication advocates adopting of the multi-component Mollerup model for activity modeling in multi-component chromatographic separation to enhance thermodynamic consistency. [ABSTRACT FROM AUTHOR]

• Model-assisted approaches for continuous chromatography were focused. • Modeling can improve the process development and find optimal operating points. • Model-assisted approaches can help to design a process with increasing feasibility and robustness. • Model predictive tools can achieve adaptive control for stable continuous process. • More model-based approaches need to be developed to support continuous manufacturing. Continuous bioprocessing is a promising trend in biopharmaceutical production, and multi-column continuous chromatography shows advantages of high productivity, high resin capacity utilization, small footprint, low buffer consumption and less waste. Due to the complexity and dynamic nature of continuous processing, traditional experiment-based approaches are often time-consuming and inefficient. In this review, model-assisted approaches were focused and their applications in continuous chromatography process development, validation and control were discussed. Chromatographic models are useful in describing particular process performances of continuous capture and polishing with multi-column chromatography. Model-assisted tools showed powerful ability in evaluating multiple operating parameters and identifying optimal points over the entire design space. The residence time distribution models, model-assisted process analytical technologies and model-predictive control strategies were also developed to reveal the propagation of disturbances, enhance real time monitor and achieve adaptive control to match the transient disturbances and deviations of continuous processes. Moreover, artificial neural networks and machine learning concepts were integrated into modeling approaches to improve data treatment. In general, further development in research and applications of model-assisted approaches for continuous chromatography are needed urgently to support the continuous manufacturing. [ABSTRACT FROM AUTHOR]

Catalytic properties of halophilic exoglucanase from a marine Aspergillus niger and the secondary structure change caused by high salinity was analyzed under high salinities condition. Exoglucanase activity in 12% NaCl solution (w/v) was 1.32 folds higher of that in NaCl free solution. Half-life time in 12% NaCl solution (w/v) was over 1.41 folds higher of that in NaCl free solution. The exoglucanase had not endoglucanase activity. Gibbs free energy change of exoglucanase denaturation, △ G , in 12% NaCl solution (w/v) was about 1.0 kJ/mol more than that in NaCl free solution at 45 °C, 50 °C, 55 °C and 60 °C. Melt temperature in 12% NaCl solution (w/v), 48.02 °C, was 3.32 °C higher than that in NaCl free solution, 44.70 °C. High salinity made contents of β-sheet and α-helix increased. The secondary structure change caused by high salinities improved exoglucanase thermostability and activity. The halostable exoglucanase from a marine Aspergillus niger was valuable for cellulose hydrolysis at high salinities. [ABSTRACT FROM AUTHOR]

• A simplified linear approximation (SLA) for estimating shielding steric factor σ. • σ was easily initialized by SLA and further determined by inverse method. • SLA yielded a reasonable σ compared to LA of the parameter-by-parameter method. • Time complexity was reduced from O (n 3) to O (n) without solving any linear system. • The new method outperformed LA in both numerical and real-world experiments. Mechanistic models play a crucial role in the process development and optimization of ion-exchange chromatography (IEC). Recent researches in steric mass action (SMA) model have heightened the need for better estimation of nonlinear parameter, steric shielding factor σ. In this work, a straightforward approach combination of simplified linear approximation (SLA) and inverse method (IM) was proposed to initialize and further determine σ , respectively. An existed, unique, and positive σ can be derived from SLA. Compared with linear approximation (LA) developed in our previous study, σ of the multi-component system can be calculated easily without solving the complex system of linear equations, leading to a time complexity reduction from O (n 3) to O (n). The proposed method was verified first in numerical experiments about the separation of three charge variants. The calculated σ was more reasonable than that of LA, and the error of elution profiles with the parameters estimated by SLA+IM was only one-sixth of that by LA in numerical experiments. Moreover, the error accumulation effect could also be reduced. The proposed method was further confirmed in real-world experiments about the separation of monomer-dimer mixtures of monoclonal antibody. The results gave a lower error and better physical understanding compared to LA. In conclusion, SLA+IM developed in the present work provides a novel and straightforward way to determine σ. This simplification would help to save the effort of calibration experiments and accelerate the process development for the multi-component IEC separation. [ABSTRACT FROM AUTHOR]

• Ac-YEHF was confirmed as the optimal tetrapeptide for antibody purification. • Ac-YEHF-4FF resin could be well used to separate hIgG from human serum with high purity and yield. • Ac-YEHF-4FF resin could biomimetic the structure and function of Protein A. Short peptide biomimetic chromatography technology as a developing protein separation technology has huge potential for antibody purification. In this study, four tetrapeptide ligands (Ac-FYKH, Ac-YEHF, Ac-YFLH and Ac-FYHI) with high potential binding ability to antibody were selected for the optimal ligand to antibody purification. The results showed that Ac-YEHF-4FF resin had higher binding capacity and selectivity for hIgG among the four resins. And at pH 7.0 and 0.3 ml/min, the highest Q 10%-hIgG of Ac-YEHF-4FF resin was 26.2 mg/ml resin while its Q 10%-BSA was just 2.2 mg/ml resin. Further, Ac-YEHF-4FF resin was used to purify protein mixtures. By binding at pH 7.0 and being eluted at pH 5.0 and pH 4.0, Ac-YEHF-4FF resin was well used to separate hIgG from BSA containing feedstock, HSA containing feedstock and human serum with the purity and yield both more than 95 %. And the screened resin could also separate mAb from CHO cell culture supernatant with purity 94.3 % and yield 97.5 %. The adsorption and separation results of Ac-YEHF-4FF resin indicated that the goal of getting the efficacy of critical residues from protein A to biomimetic its structure and function could be achieved, which had great significance to the establishment and improvement of tetrapeptide biomimetic chromatography, and also provided a new method for the field of antibody separation and purification. [ABSTRACT FROM AUTHOR]

The effect mechanisms of sodium chloride (NaCl) addition on the partitioning and solubility of immunoglobulin G (IgG) in polyethylene glycol (PEG)/hydroxypropyl starch (HPS) aqueous two-phase system (ATPS) were studied. The influences of NaCl on PEG/HPS phase diagram and the hydrophobicity of IgG were investigated. The results indicated that the hydrophobicity difference between two phases and the IgG hydrophobicity were both enhanced with NaCl addition, so IgG preferred to partition into the top phase with more hydrophobicity. The solubilities of IgG in PEG and HPS solutions with or without NaCl were measured. It was found that NaCl addition could significantly reduce IgG precipitation in PEG solution. The effects of NaCl on the interaction between IgG and PEG was studied by Isothermal Titration Calorimetry (ITC). It was found that the main interactions between IgG and PEG changed from electrostatic force to hydrophobic interaction with NaCl addition. IgG would be wrapped by PEG molecules due to hydrophobic interaction so that the solubility of IgG in PEG solution was enhanced. The results reveal the mechanisms of NaCl addition on the partitioning and solubility of IgG in PEG/HPS systems, and PEG/HPS ATPS might be a promising alternative for IgG separation with good purity and yield. [Display omitted] • Mechanisms of NaCl on partition and solubility of IgG in PEG/HPS system were revealed. • Hydrophobicity of IgG with NaCl addition was determined by fluorescence probe method. • Solubility of IgG in PEG solution was significantly improved with NaCl addition. • Interactions between IgG and PEG were measured by Isothermal Titration Calorimetry. [ABSTRACT FROM AUTHOR]

Magnetic particles modified with 5-amino-benzimidazole (ABI), a ligand for hydrophobic charge-induction chromatography, were prepared and used for antibody capture. In this study, with IgG as the model target, and bovine serum albumin (BSA) as the model impurity, the separation mechanism and process of IgG was investigated. The adsorption isotherms of IgG and BSA were measured, and the effects of pH were investigated in the range of pH 4.0–8.0. The maximum adsorption capacity of IgG on the particles was 180 mg/ml at pH 7.0, while low adsorption capacity of BSA (64 mg/ml) was found at pH 7.0, resulting in good selectivity. The protein-ligand interactions were elucidated by adding NaCl and glycerol. The results indicated the hydrophobic interactions were the main forces for IgG-ligand association. Moreover, the batch uptake and desorption experiments demonstrated the fast adsorption and desorption processes for IgG separation. The purity of IgG separated from mimetic serum could reach 98.6%, and the purity of monoclonal antibody (mAb) from a cell culture supernatant was 97.1%. Magnetic particles with hydrophobic charge-induction ligands showed a robust performance and could purify antibody directly from the complicated feedstock without clarification, which would improve the efficiency of antibody purification. [ABSTRACT FROM AUTHOR]

• Modeling multi-component hydrophobic interaction chromatography was focused. • Parameter-by-parameter estimation method for multi-component system was developed. • A simplified linear approximation was proposed for more efficient estimation. • New method was verified through two-component calibration experiments. • Process optimization was performed to demonstrate the application potentials. Mechanistic models are powerful tools for chromatographic process development and optimization. However, hydrophobic interaction chromatography (HIC) mechanistic models lack an effective and logical parameter estimation method, especially for multi-component system. In this study, a parameter-by-parameter method for multi-component system (called as mPbP-HIC) was derived based on the retention mechanism to estimate the six parameters of the Mollerup isotherm for HIC. The linear parameters (k s , i and k eq , i) and nonlinear parameters (n i and q max , i) of the isotherm can be estimated by the linear regression (LR) and the linear approximation (LA) steps, respectively. The remaining two parameters (k p , i and k kin , i) are obtained by the inverse method (IM). The proposed method was verified with a two-component model system. The results showed that the model could accurately predict the protein elution at a loading of 10 g/L. However, the elution curve fitting was unsatisfactory for high loadings (12 g/L and 14 g/L), which is mainly attributed to the demanding experimental conditions of the LA step and the potential large estimation error of the parameter q max. Therefore, the inverse method was introduced to further calibrate the parameter q max , thereby reducing the estimation error and improving the curve fitting. Moreover, the simplified linear approximation (SLA) was proposed by reasonable assumption, which provides the initial guess of q max without solving any complex matrix and avoids the problem of matrix unsolvable. In the improved mPbP-HIC method, q max would be initialized by the SLA and finally determined by the inverse method, and this strategy was named as SLA+IM. The experimental validation showed that the improved mPbP-HIC method has a better curve fitting, and the use of SLA+IM reduces the error accumulation effect. In process optimization, the parameters estimated by the improved mPbP-HIC method provided the model with excellent predictive ability and reasonable extrapolation. In conclusion, the SLA+IM strategy makes the improved mPbP-HIC method more rational and can be easily applied to the practical separation of protein mixture, which would accelerate the process development for HIC in downstream of biopharmaceuticals. [ABSTRACT FROM AUTHOR]

• 20 model protein adsorption by four mixed-mode resins was investigated. • QSAR models were developed with random forest and gradient boosting methods. • The QSAR models had high predictive performance of protein adsorption capacities. • The SHAP theory was applied to evaluate the key descriptors obtained. Mixed-mode chromatography is promising for protein separation, but the structural diversities of proteins result in distinct adsorption behaviors. This study used machine learning methods to establish the quantitative structure–activity relationships (QSAR) between the protein adsorption capacity on mixed-mode resins and the molecule properties of target proteins and mixed-mode ligands. Four mixed-mode resins and twenty proteins were tested at different pHs and salt concentrations. Two machine learning models, random forest and gradient boosting, were developed successfully to predict protein adsorption capacities. The determination coefficients (R2) of the training dataset, validation dataset, and test dataset ranged around 0.94–0.97, 0.79–0.82, and 0.90–0.93, respectively. Several key descriptors that have significant impacts on adsorption capacities were identified by a two-step descriptor elimination method. Moreover, the SHapley Additive exPlanations (SHAP) method was used to reveal the mechanism of target protein adsorption on mixed-mode resins. The results provided a valuable guidance for the design and selection mixed-mode resins for the separation and purification of target proteins. [ABSTRACT FROM AUTHOR]

• A novel method was developed for one-by-one estimation of SMA parameters. • A retention model was suggested for direct prediction of protein retention of IEC. • Superior performances in the parameter estimation and retention prediction. • Effects of protein loadings on the developed method were investigated. • A five-experiment strategy for better implementation of the developed method. Ion exchange chromatography (IEC) is one of the most widely-used techniques for protein separation and has been characterized by mechanistic models. However, the time-consuming and cumbersome model calibration hinders the application of mechanistic models for process development. A new methodology called "parameter-by-parameter method (PbP)" was proposed with mechanistic derivations of the steric mass action (SMA) model of IEC. The protocol includes four steps: (1) first linear regression (LR1) for characteristic charge; (2) second linear regression (LR2) for equilibrium coefficient; (3) linear approximation (LA) for shielding factor; (4) inverse method (IM) for kinetic coefficient. Four SMA parameters could be one-by-one determined in sequence, reducing the number of unknown parameters per species from four to one, and predicting almost consistent retention. Numerical single-component experiments were investigated firstly, and the PbP method showed excellent agreement between experiments and simulations. The effects of loadings on the PbP and Yamamoto methods were compared. It was found that the PbP method had higher accuracy and robustness than the Yamamoto method. Moreover, a five-experiment strategy was suggested to implement the PbP method, which is straightforward to reduce the cost of calibration experiments. Finally, a real-world multi-component separation was challenged and further confirmed the feasibility of the PbP method. In general, the proposed method can not only reliably estimate the SMA parameters with comprehensive physical understanding but also accurately predict retention over a wide range of loading conditions. [ABSTRACT FROM AUTHOR]

The advancement of recombinant virus‐like particle‐based vaccines has attracted global attention owing to substantially safety and high efficacy in provoking a protective immunity against various chronic and infectious diseases in humans and animals. A robust, low‐cost, and scalability separation and purification technology is of utmost importance in the downstream processing of recombinant virus‐like particles to produce affordable and safe vaccines. Being a relatively simple, environmentally friendly, and efficient biomolecules recovery approach, aqueous two‐phase systems have received great attention from researchers worldwide. This review aims to highlight the challenges and outlook in addition to the current applications of aqueous two‐phase systems in downstream processing of virus‐like particles. The efforts will confidently reinforce scholars' knowledge and fill in the valuable research gap in the aspect of concerning recombinant virus‐like particle‐based vaccines development, particularly related to the virus‐like particles downstream production processes. [ABSTRACT FROM AUTHOR]

Abstract: Novel capsules loaded with lactoferrin (LF) were prepared using polyelectrolyte complexes that were formed by water soluble chitosan (WSC), sodium cellulose sulfate (NaCS) and sodium polyphosphate (PPS). Normal chitosan (soluble in acidic conditions) was chosen as a control to prepare similar capsules with NaCS and PPS. 1H NMR and FTIR spectra analysis showed that WSC was in a form of chitosan hydrochloride which can be directly dissolved and protonated in acid-free water. SEM results showed that the capsules had a typical wall-capsule structure with a regular spherical shape and an average diameter of 1.97mm. TGA studies revealed that the thermal stability of the capsules were enhanced and the moisture content of the drug-free/loaded capsules were 6.3% and 3.2%. SDS-PAGE results showed that the primary structures of the processed LF in the capsules were unchanged. Drug loading (LE%) and encapsulation efficiency (EE%) analysis showed that the capsules had a higher LE% (45.6%) and EE% (70.7%) than that of the control. In vitro release studies showed that the capsules had a regular and sustainable release profiles in simulated colonic fluid. All of these results indicated that the capsules prepared could be used as a candidate protein drug carrier for colon. [Copyright &y& Elsevier]

Highlights: [•] Antibody aggregates removal using three mixed mode chromatography were evaluated. [•] Aggregates binding more strongly to the mixed-mode resins. [•] Cooperativity of different molecular interactions is important for removal. [•] Removal efficiency is dependent on ligand structure with different interactions. [Copyright &y& Elsevier]

Abstract: Polymeric cryogels are sponge-like materials with supermacroporous structure, allowing them to be of interest as new chromatographic supports, cell scaffolds and drug carriers in biological and biomedical areas. The matrices of cryogels are always prepared in the form of monoliths by cryo-polymerization under frozen conditions. However, there are limited investigations on the production of cryogels in the form of adsorbent beads suitable for bioseparation. In this work, we provide a new approach by combining the microchannel liquid-flow focusing with cryo-polymerization for the preparation of polyacrylamide-based supermacroporous cryogel beads with a narrow particle size distribution. The present method was achieved by introducing the aqueous phase solution containing monomer, cross-linker and redox initiators, and the water-immiscible organic oil phase containing surfactant simultaneously into a microchannel with a cross-shaped junction, where the aqueous drops with uniform sizes were generated by the liquid shearing and the segmentation due to the steady flow focusing of the immiscible phase streams. These liquid drops were in situ suspended into the freezing bulk oil phase for cryo-polymerization and the cryogel matrix beads were obtained by thawing after the achievement of polymerization. By grafting the polymer chains containing sulfo binding groups onto these matrix beads, the cation-exchange cryogel beads for protein separation were produced. The results showed that at the aqueous phase velocities from 0.5 to 2.0cm/s and the total velocities of the water-immiscible phase from 2.0 to 6.0cm/s, the obtained cryogel beads by the present method have narrow size distributions with most of the bead diameters in the range from 800 to 1500μm with supermacropores in sizes of about 3–50μm. These beads also have high porosities with the averaged maximum porosity of 96.9% and the mean effective porosity of 86.2%, which are close to those of the polyacrylamide-based cryogel monoliths. The packed bed using the cryogel beads with mean diameter of 1248μm, as an example, has reasonable and acceptable liquid dispersion, but high water permeability (4.29×10−10 m2) and high bed voidage (90.2%) owing to the supermacropores within the beads, enhanced the rapid binding and separation of protein from the feedstock even at high flow velocities. The purity of the obtained lysozyme from chicken egg white by one-step chromatography using the packed bed was in the range of about 78–92% at the flow velocities of 0.5–15cm/min, indicating that the present cryogel beads could be an effective chromatographic adsorbent for primary bioseparation. [Copyright &y& Elsevier]

The enormous potential of mRNA-based therapeutics in vaccines and medicine has led to the consideration of various mRNA sizes, ranging from ∼1000 to 10,000 nucleotides (nt). However, the commonly used Oligo-deoxythymidine (Oligo dT) affinity resins are not initially designed for such a wide range of sizes, and present challenges in achieving optimal performance for the separation of a given mRNA. In this paper, two factors of the resin beads, pore size and grafting were focused. The effects of varying pore sizes, ranging from 150 to 850 nm, on both static and dynamic adsorption for varying-length mRNA (1000–8500 nt) were investigated. It was found that static adsorption capacity and uptake kinetics were correlated with the mRNA length. Interestingly, dynamic binding capacities (DBCs) showed an increase-then-decrease trend with increasing pore size. An optimal pore size of about 350 nm could lead to the highest DBCs for all tested mRNA. Additionally, the effects of polymer grafting on the adsorption performance were studied. The results revealed that polymer-grafted resin exhibited 24–55% higher DBCs than non-grafted resin. The enhanced DBCs could be attributed to the improved uptake rate facilitated by the polymer grafted. These investigations emphasize the importance of optimizing pore size and grafting for Oligo dT affinity resins, providing valuable guidance for selecting and designing new resins for different mRNA products. • Adsorption behaviors of Oligo dT affinity resins were studied for varying mRNA. • Effects of pore size and grafting on the adsorption were focused. • Static adsorption capacity and uptake kinetics were correlated with mRNA length. • Highest dynamic binding capacities were found with the pore size of 350 nm. • Grafted resin showed 24-55% higher dynamic binding capacity than non-grafted resin. [ABSTRACT FROM AUTHOR]

Mixed-mode chromatography (MMC) with the specially-designed ligands shows good adsorption selectivity for protein separation. In this study MMC was explored to purify recombinant human serum albumin (rHSA) from Pichia pastoris culture broth, which combined bind-elute and flow-through modes. Four mixed-mode resins were evaluated for their adsorption capacities of HSA. It was found that two home-made MMC resins showed special performances. IC-B-6FF resin with indole-3-acetic acid-cysteine ligand had high adsorption and selectivity towards HSA, and SFA-B-6FF resin with sulfapyridine ligand showed minimal binding to HSA. Therefore, a two-step MMC separation strategy was designed that IC-B-6FF was used to capture rHSA directly from yeast culture broth and then SFA-B-6FF resin was used to polish the remaining impurities by flow-through mode. The results revealed that the purity of rHSA reached 95.1% with a yield of 81.2% after the two-step separation. The removal rate of host cell proteins (HCPs) was 99.8% with final HCP level as low as 50 ppm. The results demonstrate that MMC combining bind-elute and flow-through modes has a promising potential to separate protein with high selectivity and process efficiency. • Human serum albumin (HSA) adsorption by mixed-mode resins was studied. • Mixed-mode resins with high-HSA-adsorption and low-HSA-adsorption were tested. • Two-step separation strategy for rHSA purification was proposed. • The rHSA was purified from the fermentation broth of Pichia pastoris. • The purity of rHSA reached 95.1% with the recovery of 81.2%. [ABSTRACT FROM AUTHOR]

• Parameter estimation was studied for hydrophobic interaction chromatography. • A step-by-step method was developed for isotherm parameters estimation. • Effects of operating conditions on the method developed was investigated. • New method was validated through numerical experiments. • A six-experiment strategy was proposed for enabling the method developed. Hydrophobic interaction chromatography (HIC) is used as a critical polishing step in the downstream processing of biopharmaceuticals. Normally the process development of HIC is a cumbersome and time-consuming task, and the mechanical models can provide a powerful tool to characterize the process, assist process design and accelerate process development. However, the current estimation of model parameters relies on the inverse method, which lacks an efficient and logical parameter estimation strategy. In this study, a parameter-by-parameter (PbP) method based on the theoretical derivation and simplifying assumptions was proposed to estimate the Mollerup isotherm parameters for HIC. The method involves three key steps: (1) linear regression (LR) to estimate the salt-protein interaction parameter and the equilibrium constant; (2) linear approximation (LA) to estimate the stoichiometric parameter and the maximum binding capacity; and (3) inverse method to estimate the protein-protein interaction parameter and the kinetic coefficient. The results indicated that the LR step should be used for dilution condition (loading factor below 5%), while the LA step should be conducted when the isotherm is in the transition or nonlinear regions. Six numerical experiments were conducted to implement the PbP method. The results demonstrated that the PbP method developed allows for the systematic estimation of HIC parameters one-by-one, effectively reducing the number of parameters required for inverse method estimation from six to two. This helps prevent non-identifiability of structural parameters. The feasibility of the PbP-HIC method was further validated by real-world experiments. Moreover, the PbP method enhances the mechanistic understanding of adsorption behavior of HIC and shows a promising application to other stoichiometric displacement model-derived isotherms. [ABSTRACT FROM AUTHOR]

• A continuous capture process under ΔUV control with varied feedstockwas evaluated. • The effects of target protein and impurity changes from −10 to 10 mAU/min were analyzed. • A reduced concentration of protein or impurity increases the risk of protein loss. • Proposed contour map depicting the comprehensive impacts of feedstock variations. Multi-column periodic counter-current chromatography is a promising technology for continuous antibody capture. However, dynamic changes due to disturbances and drifts pose some potential risks for continuous processes during long-term operation. In this study, a model-based approach was used to describe the changes in breakthrough curves with feedstock variations in target proteins and impurities. The performances of continuous capture of three-column periodic counter-current chromatography under ΔUV dynamic control were systematically evaluated with modeling to assess the risks under different feedstock variations. As the concentration of target protein decreased rapidly, the protein might not breakthrough from the first column, resulting in the failure of ΔUV control. Small reductions in the concentrations of target proteins or impurities would cause protein losses, which could be predicted by the modeling. The combination of target protein and impurity variations showed complicated effects on the process performance of continuous capture. A contour map was proposed to describe the comprehensive impacts under different situations, and nonoperation areas could be identified due to control failure or protein loss. With the model-based approach, after the model parameters are estimated from the breakthrough curves, it can rapidly predict the process stability under dynamic control and assess the risks under feedstock variations or UV signal drifts. In conclusion, the model-based approach is a powerful tool for continuous process evaluation under dynamic changes and would be useful for establishing a new real-time dynamic control strategy. [ABSTRACT FROM AUTHOR]

• Ac-FYHE-4FF resin as the novel resin was prepared for antibody purification. • High binding capacity and selectivity of hIgG could be achieved by novel resin. • Novel resin could purify mAb or hIgG from feed liquid with high purity and yield. Biomimetic affinity chromatography with short peptide ligands is a developing technology, which has great potential for antibody separation and purification. In this study, a tetrapeptide library with critical residues of natural ligands to hIgG was constructed and a novel tetrapeptide ligand (Ac-FYHE) with high LibDock scores was selected by molecular docking. Then, Ac-FYHE ligand was linked to agarose bead to prepare a new chromatography resin. The properties of antibody adsorption were measured and evaluated by static/dynamic adsorption. It was found that the resin with ligand Ac-FYHE has high binding capacity and selectivity for hIgG. The results showed the Q m-hIgG of Ac-FYHE-4FF resin was 87.9 mg/g resin while the Q m-BSA of this resin was only 16.5 mg/g resin at pH 7.0. Moreover, at pH 7.0, Q 10% of Ac-FYHE-4FF resin was 24.1 mg/mL for hIgG but just 2.1 mg/mL for BSA, which presented high selectivity of the screened resin at pH 7.0. Subsequently, the adsorption and separation properties of the Ac-FYHE-4FF resin were further investigated. As a result, with the addition of 0.5 M NaCl, Q m decreased by less than 20% but Q m decreased by 70% with the addition of 50% (v/v) ethylene glycol, which indicated that hydrophobic interaction would be the driving force for the binding between resin and hIgG. Besides, pH 7.5 and pH 4.5 could be the optimal loading and elution condition for hIgG, respectively. Finally, the Ac-FYHE-4FF resin was applied to separate mAb or/and hIgG from BSA containing feedstock, CHO cell culture supernatant and human serum, and the purity and recovery were both more than 90% with only one-step separation. The results indicate that the Ac-FYHE-4FF resin developed in this work would be promising for antibody separation and purification. [ABSTRACT FROM AUTHOR]

The adeno-associated virus (AAV) has been widely employed as a popular gene therapy vector. However, current methods for its separation and purification exhibit numerous limitations. In this study, polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS) was utilized as an efficient and environmentally-benign approach to separate AAV. The separation efficiency of AAV particles from other cellular components was investigated using various ATPS compositions. The effects of PEG molecular weight, types of phase-forming salts, and concentrations of different components were tested and compared. The optimal separation conditions were determined to be 17 % (w/w) PEG 600, 16 % (w/w) sodium citrate, and 15 % (w/w) crude cell lysate. The AAV particles were predominantly enriched in the interphase, while the impurities were observed in the top and bottom phases. High AAV recovery (>99 %) and efficient impurity removal (>95 %) were achieved. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transmission electron microscopy were used to characterize and verify the separated AAV particles. The results indicated that aqueous two-phase extraction possesses a robust capability for the enrichment and separation of AAV particles directly from crude cell lysates, and it shows promising potential for the separation of other viral particles. [Display omitted] • New method was developed to enrich and separate of adeno-associated virus (AAV). • Polyethylene glycol/salt aqueous two-phase systems were evaluated and optimized. • AAV was almost enriched in the interphase and impurities in top or bottom phases. • High AAV yield and high impurity removal were obtained. [ABSTRACT FROM AUTHOR]

• Physics-informed neural network (PINN) was used to describe the breakthrough curve. • Designed PINN model structure for lumped kinetic model (LKM). • Optimized the training procedure of PINN to improve performance. • Built a PINN model with all LKM parameters and used in chromatography process. • Better performance than numerical methods in breakthrough curve simulation. Numerical method is widely used for solving the mechanistic models of chromatography process, but it is time-consuming and hard to response in real-time. Physics-informed neural network (PINN) as an emerging technology combines the structure of neural network with physics laws, and is getting noticed for solving physics problems with a balanced accuracy and calculation speed. In this research, a proof-of-concept study was carried out to apply PINN to chromatography process simulation. The PINN model structure was designed for the lumped kinetic model (LKM) with all LKM parameters. The PINN structure, training data and model complexity were optimized, and an optimal mode was obtained by adopting an in-series structure with a nonuniform training data set focusing on the breakthrough transition region. A PINN for LKM (LKM-PINN) consisting of four neural networks, 12 layers and 606 neurons was then used for the simulation of breakthrough curves of chromatography processes. The LKM parameters were estimated with two breakthrough curves and used to infer the breakthrough curves at different residence times, loading concentrations and column sizes. The results were comparable to that obtained with numerical methods. With the same raw data and constraints, the average fitting error for LKM-PINN model was 0.075, which was 0.081 for numerical method. With the same initial guess, the LKM-PINN model took 160 s to complete the fitting, while the numerical method took 7 to 72 min, depending on the fitting settings. The fitting speed of LKM-PINN model was further improved to 30 s with random initial guess. Thus, the LKM-PINN model developed in this study is capable to be applied to real-time simulation for digital twin. [ABSTRACT FROM AUTHOR]

Highlights • Directed expression of two β-glucosidases (BGLs) was achieved in marine Aspergillus niger. • Two BGLs were relatively acidophilic and BGL2 was halophilic. • Protein expression was improved by preliminary medium optimization. Abstract The hydrolysis step by β-glucosidase (BGL) is generally recognized as the major limiting step in cellulose degradation and the BGLs with prominent enzymatic properties are of great importance for efficient utilization of lignocellulosic biomass. In order to identify some salt-tolerant BGLs, two BGL genes were cloned from marine Aspergillus niger ZJUBE-1 genome. Then two bgl expression cassettes driven by gpdA promoter were respectively transformed into marine A. niger for homologous constitutive expression. Directed expression was achieved for the domination of target BGLs in fermentation broth. Conveniently, two BGLs were purified to homogeneity by two separation steps, ultrafiltration and anion exchange chromatography. The purified BGL1 and BGL2 showed maximum activity at pH 3.0–4.0 and 3.5–4.5, respectively, suggesting these two BGLs were relatively acidophilic, especially for BGL1. Besides, BGL1 was stable to most of metal ions, while BGL2 was sensitive to Cu2+, Fe3+ and Ag+. Most specially, BGL2 activity increased by 44% in the presence of 4 M NaCl, suggesting BGL2 was halophilic. Homology modeling revealed that longer loops and linkers as well as polymerous Glu492 may contribute to the halophilism of BGL2. At last, the medium for directed expression was optimized and the content as well as the purity of target protein was improved. [ABSTRACT FROM AUTHOR]

• Model-assisted approaches for continuous chromatograph were reviewed. • Model-assisted methods for process development were focused and discussed. • Strategies for model-assisted process characterization was proposed. • Current model-assisted process design approaches were reviewed. • Perspectives of model-assisted tools for the future digitalized manufacturing were discussed. Continuous manufacturing in monoclonal antibody production has generated increased interest due to its consistent quality, high productivity, high equipment utilization, and low cost. One of the major challenges in realizing continuous biological manufacturing lies in implementing continuous chromatography. Given the complex operation mode and various operation parameters, it is challenging to develop a continuous process. Due to the process parameters being mainly determined by the breakthrough curves and elution behaviors, chromatographic modeling has gradually been used to assist in process development and characterization. Model-assisted approaches could realize multi-parameter interaction investigation and multi-objective optimization by integrating continuous process models. These approaches could reduce time and resource consumption while achieving a comprehensive and systematic understanding of the process. This paper reviews the application of modeling tools in continuous chromatography process development, characterization and design. Model-assisted process development approaches for continuous capture and polishing steps are introduced and summarized. The challenges and potential of model-assisted process characterization are discussed, emphasizing the need for further research on the design space determination strategy and parameter robustness analysis method. Additionally, some model applications for process design were highlighted to promote the establishment of the process optimization and process simulation platform. [ABSTRACT FROM AUTHOR]

• Process design was studied for multicolumn counter-current solvent gradient purification. • The trade-off between purity and recovery was focused. • Both model protein mixture and mAb sample with charge variants were tested. • The mismatching was found between normally designed window and elution profiles. • An improved process design strategy was proposed to improve the process performances. The multicolumn counter-current solvent gradient purification (MCSGP) method has proven effective in addressing the issue of elution profile overlap for difficult-to-separate proteins, leading to improved purity and recovery. However, during the MCSGP process, the flow rate and proportion of loaded proteins undergo changes, causing a significant discrepancy between the elution profiles of batch process design and the actual MCSGP process. This mismatch negatively impacts the purity and recovery of the target protein. To address this challenge, an improved process design (reDesign) was proposed with the first-run MCSGP to mimic the actual continuous process and obtain elution profiles that closely resemble the real ones. The reDesign was demonstrated with both a model protein mixture and a sample of monoclonal antibody (mAb) with charge variants. For model protein mixture, the reDesign-based MCSGP process (reMCSGP) showed a remarkable improvement in recovery, increasing from 83.6% to 97.8% while maintaining a purity of more than 95%. For mAb sample, the recovery of reMCSGP improved significantly to 93.9%, surpassing the performance of normal MCSGP processes at a given purity level of more than 84%. In general, the new process design strategy developed in this work could generate a more representative elution profile that closely mirrors actual conditions in continuous processes, which enhances the separation performance of MCSGP. [ABSTRACT FROM AUTHOR]

This study investigated adsorption selectivity of immunoglobulin M (IgM), immunoglobulin A (IgA) and immunoglobulin (IgG) on four mixed-mode resins with the functional ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. IgM purification processes with mixed-mode resins were also proposed. All resins showed typical pH-dependent adsorption, and high adsorption capacity was found at pH 5.0–8.0 with low adsorption capacity under acidic conditions. Meanwhile, high selectivity of IgM/IgA and IgM/IgG was obtained with ABI-4FF and MMI-4FF resins at pH 4.0–5.0, which was used to develop a method for IgM, IgA and IgG separation by controlling loading and elution pH. Capture of monoclonal IgM from cell culture supernatant with ABI-4FF resins was studied and high purity (∼99%) and good recovery (80.8%) were obtained. Moreover, IgM direct separation from human serum with combined two-step chromatography (ABI-4FF and MMI-4FF) was investigated, and IgM purity of 65.2% and a purification factor of 28.3 were obtained after optimization. The antibody activity of IgM was maintained after purification. The results demonstrated that mixed-mode chromatography with specially-designed ligands is a promising way to improve adsorption selectivity and process efficiency of IgM purification from complex feedstock. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cellulase is a complex enzyme consisted of exoglucanase, endoglucanase and β-glucosidase. An enzyme composition affects the synergistic activity. To enhance the activity of halostable cellulase from a marine Aspergillus niger , activities of three enzyme compositions were analyzed respectively. It was found that endoglucanase and exoglucanase in the cellulase had the relative low activities corresponding to β-glucosidase. Thus, an expression system of cellulase in the marine A . niger was constructed. Piromyces rhizinflata cellulase was efficiently expressed by a constructed vector with promoter glaA . Exoglucanase and endoglucanase was increased from 0.21 U/ml and 4.51 U/ml of the original strain to 0.81 U/ml and 19.10 U/ml of the transformant, respectively. FPA increased nearly 7.5 folds from 0.63 U/ml to 4.69 U/ml. Glucose released by hydrolyzing wheat straw with cellulase from the transformant was 1.68 folds higher than that with cellulase from the original strain under high salinity condition. The results illustrated that P . rhizinflata cellulase could be well expressed in marine A. niger . The cellulase from the transformant not only showed higher activity but also remained the halostable ability, indicating that an appreciate proportion of enzyme-composition in cellulase was very important to cellulase activity. [ABSTRACT FROM AUTHOR]

Adsorption selectivity is critical important for mixed-mode chromatography with specially-designed ligands. Human immunoglobulin G (hIgG), Fc and Fab fragments were used in the present work to evaluate adsorption behavior and binding selectivity of four mixed-mode resins with the ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. The resins showed an obvious pH-dependent adsorption behavior. High adsorption capacities were found at neutral pH for hIgG, Fc and Fab, and almost no adsorption happened under acidic conditions. An adsorption selectivity index was proposed to evaluate separation efficiency. High specificity of hIgG/Fc was found at pH 8.9 for MEP resin, and for W-ABI resin at pH 8.0 and 8.9. In addition, isothermal titration calorimetry was used to evaluate ligand-protein interactions. Finally, the separation of hIgG and Fc (1:1) was optimized with mixed-mode resins, and the best separation performance was obtained with W-ABI-based resin. Loading at pH 8.0 resulted in the flow through of Fc with purity of 90.4% and recovery of 98.8%, while elution at pH 3.6 provided hIgG with purity of 99.7% and recovery of 86.5%. [ABSTRACT FROM AUTHOR]

• New platform was developed to optimize continuous process from whole process level. • Effects of capture mode and sub-lot number on continuous process were studied. • Pilot-scale continuous process designed was verified with high process efficiency. • Cost comparison showed some obvious benefits of continuous process. Continuous process is a promising alternative for tradition batch process in biomanufacturing, which has higher productivity and lower material consumption. However, despite the maturation of necessary technologies for continuous process, there are few discussion about optimization of full continuous process. One possible reason is that the continuous process is such a complex and interacted process that the traditional experiment-based optimization approach is not sufficient anymore. To address that problem, the process simulation tool SuperPro Designer and continuous capture chromatography model were integrated into a model-assisted design platform for better development of continuous process. The influences of different continuous capture operation modes and sub-lot number under various upstream conditions were analyzed for pilot-scale production. The best combination of operation mode and sub-lot number were determined for the highest equipment utilization without any conflict. After the process optimization, the equipment utilization of continuous process was significantly improved to 27.3%. Then, a pilot-scale case study was carried out to validate the proposed approach. The results showed that the scaling up and process design of continuous process were successful. No time conflict and process failure happened and the final product met the release specification. Finally, the cost of goods was evaluated with SuperPro Designer, and the results showed a 17.4% cost reduction for optimized continuous downstream process compared to batch process, which is promoting for the future industrial applications. [ABSTRACT FROM AUTHOR]