Core-shell versus inert polymer grafted adsorbents for the negative chromatography of virus-like particle

Core-shell and polymer grafted adsorbents are new generation media developed for the separation of virus-like particle (VLP) in a negative chromatography. The inert shell and grafted polymer chain are designed to exclude the big biomolecules such as VLP from adsorbing onto the ligands situated on the surface of the adsorbents. Meanwhile, these exclusion layers should be permeable for the smaller impurities which will be adsorbed by the ligands to prevent its presence in the flowthrough fractions. In this study, the performance of these negative chromatography media were compared in the purification of recombinant hepatitis B VLPs (HB-VLPs) from clarified E. coli feedstock. The core-shell adsorbents with different shell thickness (InertShell and InertLayer 1000) and poly[(ethylene glycol) methacrylate] grafted adsorbents (SQ) were studied in a packed bed mode. SQ adsorbed more impurities, thus achieving a higher purity in flowthrough while core-shell adsorbents recovered more HB-VLPs and recorded nearly 100% recovery in InertShell. This suggests the shielding effect of the core shell layer is higher than the inert polymer chain. For core-shell adsorbents, there was a trade-off between the purity and recovery of flow-through HB-VLPs due to the shell thickness. A thicker shell allows more HB-VLP exclusion but less impurities adsorption. Prolonging the residence time of the negative chromatography only resulted in a slight improvement in the impurities adsorption in all adsorbents, but the recovery of HB-VLPs in InertShell was reduced substantially. Atomic force microscopic (AFM) analysis revealed funnel-shaped pore channels on the shell layer which may contribute to the entrapment of HB-VLPs on core-shell adsorbents, thus decreasing the HB-VLP recovery. Overall, SQ performed better than the core-shell adsorbents in handling feedstock with high concentration

Negative chromatography of hepatitis B virus-like particles: comparative study of different adsorbent designs

Purification of virus-like particles (VLPs) in bind-and-elute mode has reached a bottleneck. Negative chromatography has emerged as the alternative solution; however, benchmark of negative chromatography media and their respective optimized conditions are absent. Hence, this study was carried out to compare the performance of different negative chromatography media for the purification of hepatitis B VLPs (HB-VLPs) from clarified Escherichia coli feedstock. The modified anion exchange media, core-shell adsorbents (InertShell and InertLayer 1000) and polymer grafted adsorbents (SQ) were compared. The results of chromatography from packed bed column of core-shell adsorbents showed that there is a trade-off between the purity and recovery of HB-VLPs in the flowthrough fraction due to the shell thickness. Atomic force microscopic analysis revealed funnel-shaped pore channels in the shell layer which may contribute to the entrapment of HB-VLPs. A longer residence time at a lower feed flow rate (0.5ml/min) improved slightly the HB-VLPs purity in all modified adsorbents, but the recovery in InertShell reduced substantially. The preheat-treatment is not recommended for the negative chromatography as the thermal-induced co-aggregation of HCPs and HB-VLPs would flow along with HB-VLPs and thus reduced the HB-VLPs purity in the flowthrough. Further reduction in the feedstock concentration enhanced the purity of HB-VLPs especially in InertLayer 1000 but reduced substantially the recovery of HB-VLPs. In general, the polymer grafted adsorbent, SQ, performed better than the core-shell adsorbents in handling a higher feedstock concentration

Size-selective purification of hepatitis B virus-like particle in flow-through chromatography: types of ion exchange adsorbent and grafted polymer architecture

Hepatitis B virus-like particles expressed in Escherichia coli were purified using anion exchange adsorbents grafted with polymer poly(oligo(ethylene glycol) methacrylate) in flow-through chromatography mode. The virus-like particles were selectively excluded, while the relatively smaller sized host cell proteins were absorbed. The exclusion of virus-like particles was governed by the accessibility of binding sites (the size of adsorbents and the charge of grafted dextran chains) as well as the architecture (branch-chain length) of the grafted polymer. The branch-chain length of grafted polymer was altered by changing the type of monomers used. The larger adsorbent (90 μm) had an approximately twofold increase in the flow-through recovery, as compared to the smaller adsorbent (30 μm). Generally, polymer-grafted adsorbents improved the exclusion of the virus-like particles. Overall, the middle branch-chain length polymer grafted on larger adsorbent showed optimal performance at 92% flow-through recovery with a purification factor of 1.53. A comparative study between the adsorbent with dextran grafts and the polymer-grafted adsorbent showed that a better exclusion of virus-like particles was achieved with the absorbent grafted with inert polymer. The grafted polymer was also shown to reduce strong interaction between binding sites and virus-like particles, which preserved the particles' structure

Development of dual functions negative chromatography adsorbent for the purification of hepatitis B core antigen virus-like particle from Escherichia coli homogenate

Advancement in genetic engineering has allowed the expression of virus-like particle (VLP) in various recombinant hosts that can be used as vaccine to replace the attenuated or inactivated virus. However, the large particle size of VLP has rendered its adsorption onto the outer surface of adsorbent, hence reduce the recovery of VLP using bind and elute mode chromatography. Furthermore, the harsh condition during binding and elution processes could also damage the structure of VLP. Whereas in negative chromatography, small size impurities are retained by the adsorbent and the large size VLPs are allowed to flow through the column. As a result, the limitations in VLP purification using bind and elute mode chromatography can be avoided in negative chromatography. This work demonstrates the development of poly[oligo(ethylene glycol) methacrylate](POEGMA) grafted anion exchange adsorbent for the negative chromatography of hepatitis B VLP(HB-VLP). Firstly, the size selective adsorption of the adsorbent was performed on a feedstock consisted of 3 different sizes of protein. The large size HB-VLP was mostly (88%) excluded from the adsorbent along with 79% of medium size BSA, while the small size insulin was retained in the column. Further increased in temperature led to the collapse of the POEGMA chain, thus enhanced the adsorption of medium size BSA, while retaining the HB-VLP exclusion. POEGMA grafted anion exchange adsorbent was used for negative chromatography of HB-VLP from E. coli clarified lysate. The anion exchange adsorbents were grafted with POEGMA of different chain lengths; namely SQ (shorter chain length) and LQ (longer chain length). A shorter chain length of POEGMA (SQ) allowed a better penetration of host cell proteins (HCPs) through the POEGMA layer. Furthermore, the exclusion of HB-VLP from the surface of SQ allowed more HCPs to be adsorbed which resulted in 86% removal of impurities. The single step negative chromatography of HB-VLP has shown better performance compared to previous studies of anion exchange chromatography of HB-VLP. The grafted POEGMA conformation changes with temperature. The increase in temperature increased the purity and recovery of HB-VLP in flowthrough. The collapsed of POEGMA chains at higher temperature enhanced the HCPs adsorption while retaining its HB-VLP exclusion. This work demonstrates for the first time the role of thermo-responsive polymer in the preparative negative chromatography of HB-VLP. SQ was further benchmarked against inert layer coated anion exchange adsorbents (InertShell and InertLayer 1000), two negative chromatography adsorbent prototypes. The HB-VLP purity obtained under similar operation condition for SQ (62%) was much higher than the inert layer coated adsorbents. Preheat-treatment was found to deteriorate the performance of negative chromatography. Using a lower feed concentration has no significant effect over SQ. Therefore, SQ is more capable in handling higher feed concentration compared to InertLayer 1000. In general, the POEGMA grafted adsorbent is capable to remove substantial amount of impurities (HCPs) and suitable for primary step. Extra purification step is required to remove the remained HCPs and the residual DNA which may co-flowthrough with HB-VLP. Further work is recommended to enhance the shielding effect of POEGMA on HB-VLP while allowing more HCPs for adsorption

Dual Responsive Pickering Emulsion Stabilized by Poly[2-(dimethylamino)ethyl methacrylate] Grafted Cellulose Nanocrystals

A weak polyelectrolyte, poly­[2-(dimethylamino)­ethyl methacrylate] (PDMAEMA), was grafted onto the surface of cellulose nanocrystals via free radical polymerization. The resultant suspension of PDMAEMA-grafted-cellulose nanocrystals (PDMAEMA-<i>g</i>-CNC) possessed pH-responsive properties. The grafting was confirmed by FTIR, potentiometric titration, elementary analysis, and thermogravimetric analysis (TGA); the surface and interfacial properties of the modified particles were characterized by surface tensiometer. Compared to pristine cellulose nanocrystals, modified CNC significantly reduced the surface and interfacial tensions. Stable heptane-in-water and toluene-in-water emulsions were prepared with PDMAEMA-<i>g</i>-CNC. Various factors, such as polarity of solvents, concentration of particles, electrolytes, and pH, on the properties of the emulsions were investigated. Using Nile Red as a florescence probe, the stability of the emulsions as a function of pH and temperature was elucidated. It was deduced that PDMAEMA chains promoted the stability of emulsion droplets and their chain conformation varied with pH and temperature to trigger the emulsification and demulsification of oil droplets. Interestingly, for heptane system, the macroscopic colors varied depending on the pH condition, while the color of the toluene system remained the same. Reversible emulsion systems that responded to pH were observed and a thermoresponsive Pickering emulsion system was demonstrated

Size-selective purification of hepatitis B virus-like particle in flow-through chromatography: Types of ion exchange adsorbent and grafted polymer architecture

Hepatitis B virus‐like particles expressed in Escherichia coli were purified using anion exchange adsorbents grafted with polymer poly(oligo(ethylene glycol) methacrylate) in flow‐through chromatography mode. The virus‐like particles were selectively excluded, while the relatively smaller sized host cell proteins were absorbed. The exclusion of virus‐like particles was governed by the accessibility of binding sites (the size of adsorbents and the charge of grafted dextran chains) as well as the architecture (branch‐chain length) of the grafted polymer. The branch‐chain length of grafted polymer was altered by changing the type of monomers used. The larger adsorbent (90 μm) had an approximately twofold increase in the flow‐through recovery, as compared to the smaller adsorbent (30 μm). Generally, polymer‐grafted adsorbents improved the exclusion of the virus‐like particles. Overall, the middle branch‐chain length polymer grafted on larger adsorbent showed optimal performance at 92% flow‐through recovery with a purification factor of 1.53. A comparative study between the adsorbent with dextran grafts and the polymer‐grafted adsorbent showed that a better exclusion of virus‐like particles was achieved with the absorbent grafted with inert polymer. The grafted polymer was also shown to reduce strong interaction between binding sites and virus‐like particles, which preserved the particles’ structure

Advances in fabricating spherical alginate hydrogels with controlled particle designs by ionotropic gelation as encapsulation systems

International audienceAlginate is a biopolymer that has exceptional gelling properties, which allow easy gel formation under safe and mild conditions. Consequently, it is often used to encapsulate a variety of cargos, such as cells, enzymes, and lipids, and is typically employed as a model to study hydrogel-based encapsulation systems. Since the first use of alginate in the encapsulation field in the 1970s, many methods have been developed to produce alginate hydrogel particles of different sizes, structures, and morphologies. This review provides an overview of the current progress in the fabrication of alginate hydrogels with various particle designs, including a discussion of dispersion techniques to pre-template alginate particles, gelation mechanisms, considerations in selecting suitable fabrication methods, and future directions