Downstream processing of virus-like particles with aqueous two-phase systems: Applications and challenges.

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.

Process development and optimization of continuous capture with three-column periodic counter-current chromatography.

Continuous capture with affinity chromatography is one of the most important units for continuous manufacturing of monoclonal antibody (mAb). Due to the complexity of three-column periodic counter-current chromatography (3C-PCC), three approaches (experimental, model-based, and simplified approaches) were studied for process development and optimization. The effects of residence time for interconnected load (RT C ), breakthrough percentage of the first column for interconnected load (s) and feed protein concentration (c 0 ) on productivity and capacity utilization were focused. The model-based approach was found superior to the experimental approach in process optimization and evaluation. Two phases of productivity were observed and the optimal RT C for the maximum productivity was located at the boundary of the two phases. The comprehensive effects of the operating parameters (RT C , s, and c 0 ) were evaluated by the model-based approach, and the operation space was predicted. The best performance of 34.5 g/L/h productivity and 97.6% capacity utilization were attained for MabSelect SuRe LX resin under 5 g/L concentration at RT C = 2.8 min and s = 87.5%. Moreover, a simplified approach was suggested to obtain the optimal RT C for the maximum productivity. The results demonstrated that model-assisted tools are useful to determine the optimum conditions for 3C-PCC continuous capture with high productivity and capacity utilization.

A novel dextran-grafted tetrapeptide resin for antibody purification.

Short peptide biomimetic affinity chromatography as a novel antibody separation chromatography is a potential alternative to protein A chromatography. However, if directly attaching ligand to matrix, the adsorption capacity and mass transfer rate would be affected by pore blockage and steric effect. Grafting resin is an effective method to solve this problem by using polymer as a bridge between matrix and ligand. In this work, a novel resin was prepared by grafting a tetrapeptide to the dextran-grafted matrix. Then, the adsorption properties for human immunoglobin G and BSA were determined. The results showed the saturation adsorption capacity could reach to 133 mg/g resin at pH 8.9 with a significantly low dissociation constant (0.03 mg/mL). The influence of flow rates to dynamic binding capacity of this resin was less than that of the non-grafted resin. The separation performance of the resin showed monoclonal antibody could be well isolated from the Chinese hamster ovary culture supernatant at pH 9.0 with the purity of 93.0% and yield of 84.7% by one step. Overall, this resin could achieve higher binding capacity by the possible of gaining higher ligand density, indicating its potential significance for separation in larger scale systems.

Rational design of specific ligands for human serum albumin separation and applications.

Human serum albumin is widely used in clinical practice, and the development of new ligands with high affinity is beneficial to improve its separation efficiency. The Site II of human serum albumin is an active binding site of various molecules such as l-tryptophan, which was studied with molecular simulation to obtain insights for the design of new ligands. The results showed that the carboxyl and indolyl groups of l-tryptophan were critical for the binding on Site II. Seven ligands containing carboxyl groups and indolyl groups were designed, and molecular simulation showed that indole-3-pentanoic acid was the best ligand. A new ligand combined indole-3-acetic acid and cysteine was designed for easier resin preparation, and molecular simulation also indicated that the new ligand bound strongly to Site II. Resins with the new ligand designed was prepared and static adsorption experiments indicated that the new resin had high adsorption capacity of human serum albumin and strong salt tolerance. Finally, recombinant human serum albumin was separated from yeast broth with high purity of 90.4% and recovery of 94.2%, which indicated that the new resin had good adsorption selectivity and strong potential for applications.

Poly(glycidyl methacrylate)-grafted hydrophobic charge-induction agarose resins with 5-aminobenzimidazole as a functional ligand.

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.

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

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

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

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

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

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

Protein adsorption behavior and immunoglobulin separation with a mixed-mode resin based on p-aminohippuric acid.

p-Aminohippuric acid is a newly developed ligand for mixed-mode chromatography with a commercial resin name of Nuvia cPrime. In this study, bovine immunoglobulin G and bovine serum albumin were used as two model proteins, and the adsorption isotherms with Nuvia cPrime were investigated under different pH and salt concentrations. The results showed that pH had a strong but different influence on the adsorption of these two proteins. The adsorption capacity for bovine immunoglobulin G and BSA was 170.4 and 28.1 mg/g at pH 6.0, respectively. Different salts also showed varying effects on the protein adsorption. Moreover, the adsorption and elution behaviors of the two proteins in a column were determined under varying pH and salt concentrations. An optimized process showed that feedstock loaded under pH 6.0 with 0.8 M (NH4)2SO4 and eluted under pH 8.0 with 1.0 M NaCl could effectively purify bovine immunoglobulin G from feedstock containing BSA. The purity of bovine immunoglobulin G could reach 99.8% and the recovery was 92.7%. The results demonstrated that the control of pH and salt addition during the loading and elution processes were two key factors in improving separation efficiency with Nuvia cPrime resin.

Evaluation of a PEG/hydroxypropyl starch aqueous two-phase system for the separation of monoclonal antibodies from cell culture supernatant.

In this study, an aqueous two-phase system (ATPS) with PEG and hydroxypropyl starch (HPS) was used to separate monoclonal antibody (mAb) from Chinese hamster ovary cell culture supernatant. The phase diagram of the PEG/HPS ATPS was determined, and the effects of NaCl addition were investigated. The results showed that NaCl addition could lead to a shift of the binodal curve and that phase separation would occur at higher PEG and HPS concentrations. The effects of NaCl addition, pH, and the load of cell supernatant on the partitioning of mAb in a PEG/HPS ATPS were investigated. It was found that with 6% cell supernatant and 15% NaCl addition at pH 6.0, the yield of mAb in the upper phase was 96.7% with a purity of 96.0%. The back-extraction of mAb with a PEG/phosphate ATPS were also studied, and the results showed that after the two-step extraction with ATPSs the purity of mAb could reach 97.6 ± 0.5% with a yield of 86.8 ± 1.0%, which was comparable to the purification with Protein A chromatography. These results indicate that the two-step extraction with PEG/HPS and PEG/phosphate ATPSs might be a promising alternative for the separation of mAb from cell culture supernatant.

Design of chitosan and its water soluble derivatives-based drug carriers with polyelectrolyte complexes.

Chitosan, the cationic polysaccharide derived from the natural polysaccharide chitin, has been studied as a biomaterial for more than two decades. As a polycationic polymer with favorable properties, it has been widely used to form polyelectrolyte complexes with polyanions for various applications in drug delivery fields. In recent years, a growing number of studies have been focused on the preparation of polyelectrolyte complexes based on chitosan and its water soluble derivatives. They have been considered well-suited as biomaterials for a number of vital drug carriers with targeted/controlled release profiles, e.g., films, capsules, microcapsules. In this work, an overview highlights not only the favorable properties of chitosan and its water soluble derivatives but also the good performance of the polyelectrolyte complexes produced based on chitosan. Their various types of applications as drug carriers are reviewed in detail.

Parameter-by-parameter estimation method for adsorption isotherm in hydrophobic interaction chromatography.

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.

Modeling multi-component separation in hydrophobic interaction chromatography with improved parameter-by-parameter estimation method.

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 (ks,i and keq,i) and nonlinear parameters (ni and qmax,i) of the isotherm can be estimated by the linear regression (LR) and the linear approximation (LA) steps, respectively. The remaining two parameters (kp,i and kkin,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 qmax. Therefore, the inverse method was introduced to further calibrate the parameter qmax, 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 qmax without solving any complex matrix and avoids the problem of matrix unsolvable. In the improved mPbP-HIC method, qmax 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.

Evaluation of dynamic control of continuous capture with periodic counter-current chromatography under 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.

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

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

Poly(hydroxyethyl methacrylate)-based composite cryogel with embedded macroporous cellulose beads for the separation of human serum immunoglobulin and albumin.

A novel super-macroporous monolithic composite cryogel was prepared by embedding macroporous cellulose beads into poly(hydroxyethyl methacrylate) cryogel. The cellulose beads were fabricated by using a microchannel liquid-flow focusing and cryopolymerization method, while the composite cryogel was prepared by cryogenic radical polymerization of the hydroxyethyl methacrylate monomer with poly(ethylene glycol) diacrylate as cross-linker together with the cellulose beads. After graft polymerization with (vinylbenzyl)trimethylammonium chloride, the composite cryogel was applied to separate immunoglobulin-G and albumin from human serum. Immunoglobulin-G with a mean purity of 83.2% and albumin with a purity of 98% were obtained, indicating the composite cryogel as a promising chromatographic medium in bioseparation for the isolation of important bioactive proteins like immunoglobulins and albumins.

Isolation of immunoglobulin G from bovine milk whey by poly(hydroxyethyl methacrylate)-based anion-exchange cryogel.

Bovine milk whey contains several bioactive proteins such as α-lactalbumin, ß-lactoglobulin, and immunoglobulin G (IgG). Chromatographic separation of these proteins has received much attention in the past few years. In this work, we provide a chromatographic method for the efficient isolation of IgG from bovine milk whey using a poly(2-hydroxyethyl methacrylate)-based anion-exchange cryogel. The monolithic cryogel was prepared by grafting 2-(dimethylamino) ethyl methacrylate onto the poly(2-hydroxyethyl methacrylate)-based cryogel matrix and then employed to separate IgG under various buffer pH and salt elution conditions. The results showed that the buffer pH and the salt concentration in the step elution have remarkable influences on the purity of IgG, while the IgG recovery depended mainly on the loading volume of whey for a given cryogel bed. High purity IgG (more than 95%) was obtained using the phosphate buffer with pH of 5.8 as the running buffer and the salt solution in as the elution liquid. With suitable loading volume of whey, the maximum IgG recovery of about 94% was observed. The present separation method is thus a potential choice for the isolation of high-purity IgG from bovine milk whey.

Parameter-by-parameter method for steric mass action model of ion exchange chromatography: Simplified estimation for steric shielding factor.

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(n3) 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.

Model-assisted process development, characterization and design of continuous chromatography for antibody separation.

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.

Improved process design for monoclonal antibody charge variants separation with multicolumn counter-current solvent gradient purification.

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.