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Highlights • HBc-VLP and its derivatives with various foreign epitopes show strong surface hydrophobicity. • Displaying of foreign epitopes influence the surface hydrophobicity of the recombinant VLPs. • Analysis on their surface hydrophobicity provide rational approach for HIC condition selection. • A complete downstream process involving a heat pretreatment, HIC and SEC was developed. • Total recovery of HBc-VLP and three derivatives was from 33.2% to 41.92%, with purity of final products nearly 100%. Abstract Hepatitis B core virus-like particle (HBc-VLP) has become a carrier for expression and presentation of foreign epitopes as vaccine candidates. Efficient purification is necessary for preparation of HBc-VLP and its derivatives with various foreign epitopes. In previous reports, HBc-VLP was mainly purified with ion exchange chromatography. In this study, we developed a platform purification technique based on hydrophobic interaction chromatography (HIC). The underlying principle is the strong hydrophobicity on the surface of HBc-VLP, which was found by mathematical calculation and experimental measurement. Based on HIC, a complete downstream process, from feedstock supernatant to the final pure product, was developed involving a heat pretreatment, an HIC, an ultrafiltration concentration and a size exclusion chromatography. The total recovery of HBc-VLP was 41.92% with nearly 100% purity. HIC was also applicable to three HBc-based vaccine candidates displaying epitope from nuclear protein (NP) and matrix protein 2 (M2e) of the influenza A virus, as well as from ovalbumin (OVA). Optimal HIC condition for these three recombinant VLPs was rationally designed by analysis on their surface hydrophobicity, which was influenced upon insertion of the foreign epitope. By applying the same process developed for HBc-VLP, satisfactory results were achieved for product recovery, purity and host cell DNA removal, thus it is possible to become a platform technique for various HBc-VLP based vaccine candidates. [ABSTRACT FROM AUTHOR]

Removing impurities by purification is important for livestock vaccines to avoid side effects and ensure sufficient immunogenic protection on animals. In this study, three anion-exchange media with similar particle size and ligand density except pore size were applied and compared for purification of inactivated foot-and-mouth disease virus (FMDV) antigen. The dynamic binding capacity for DEAE-POROS (214 nm) and DEAE–650 M (106 nm) were 11.53 and 10.03 mg/mL, while that for DEAE-FF (32 nm) was less than 1/10 of the previous two. The recovery of inactivated FMDV after chromatographic process of these three media was 68.42%, 66.32% and 54.46%, respectively, showing a decrement as the decrease of pore size of media. Possible denaturation of the FMDV on the surfaces of anion exchange media was analyzed by differential scanning calorimetry (DSC). FMDV is known to be prone to dissociate into smaller subunits 12S. In solution, this process is reflected by T m 1 of DSC, which is at about 48.52 °C. When inactivated FMDV were absorbed on DEAE-FF, the T m 1 became 41.73 °C, indicating the increased possibility of dissociation. The T m 1 for DEAE–650 M and DEAE-POROS was 44.04 °C and 45.37 °C, showing less dissociation and improved stability than on DEAE-FF. After DEAE-POROS chromatography, 94% FMDV recovery with 7.7-fold purification was achieved, further polishing by size exclusion chromatography led totally 173-fold increase in purity with average overall recovery of 79%. [ABSTRACT FROM AUTHOR]

Development of acellular pertussis vaccine (aPV) requires purification of several components from Bordetella pertussis. While the components pertussis toxin (PT) and filamentous hemagglutinin (FHA) have been successfully purified, the third component, pertactin, proves to be a difficult target due to its very low concentration. In order to solve its purification problem, we performed the surface potential analysis with GRASP2 program. The results demonstrated that there are two major charge patches, one negative and one positive, which are located separately on this linear protein. For this special feature, we designed a dual ion exchange chromatography strategy including an anionic exchange and a cationic exchange process for separation of pertactin from the heat extract of B. pertussis . The initial anionic exchange chromatography concentrated the product from 1.7% to 14.6%, with recovery of 80%. The second cationic exchange chromatography increased the purity to 33%, with recovery of 83%. The final purification was accomplished by hydrophobic interaction chromatography, yielding a purity of 96%. The total recovery of the three columns was 61%. Characterization of the purified antigen was performed with CD, intrinsic fluorescence, HP-SEC and western-blot, showing that the purified protein kept its natural conformation and immune-reactivity. The rationally designed process proved to be feasible, and it is suitable for large-scale preparation of the third aPV component pertactin. [ABSTRACT FROM AUTHOR]

Driven by the prevalent use of ion exchange chromatography (IEC) for polishing therapeutic proteins, many rules have been formulated to summarize the different dependencies between chromatographic data and various operational parameters of interest based on statically determined interactions. However, the effects of the unfolding of protein structures and conformational stability are not as well understood. This study focuses on how the flexibility of proteins perturbs retention behavior at the molecular scale using microscopic characterization approaches, including hydrogen–deuterium (H/D) exchange detected by NMR and a quartz crystal microbalance (QCM). The results showed that a series of chromatographic retention parameters depended significantly on the adiabatic compressibility and structural flexibility of the protein. That is, softer proteins with higher flexibility tended to have longer retention times and stronger affinities on SP Sepharose adsorbents. Tracing the underlying molecular mechanism using NMR and QCM indicated that an easily unfolded flexible protein with a more compact adsorption layer might contribute to the longer retention time on adsorbents. The use of NMR and QCM provided a previously unreported approach for elucidating the effect of protein structural flexibility on binding in IEC systems. [ABSTRACT FROM AUTHOR]

Protein purification has long been regarded as an art rather than a science. Trial and error is the widely used practice for a protein to be purified from the crude extract. In this study, we carried out a rational approach by analysis of the protein surface properties before setting out to do experiments. The target protein was recombinant human lactoferrin (rHLF), to be purified from milk of transgenic cow. We need to overcome two major problems. One is its possible co-precipitation with casein during initial separation; the other is the difficulty in separating its homologous counterpart, bovine lactoferrin (BLF). By calculation of the surface hydrophobicity, an initial separation step was decided by calcium precipitation, which removed casein but left rHLF in the supernatant. Then the average surface hydrophobicity and electric potential of rHLF were compared with those of BLF. There was a more significant difference in the electric potentials than the average surface hydrophobicity (ASH) between the homologous pairs. Therefore, the purification step should be favored by ion exchange chromatography (IEC) instead of hydrophobic interaction chromatography (HIC). Experiments were performed to verify the prediction. After removing casein, one step cationic ion exchange chromatography realized complete separation of rHLF from BLF with rHLF recovery up to 83%, while the resolution of HIC process was very limited due to the small difference in ASH between them. The laboratory process was then successfully scaled up to pilot-plant scale of 500 l milk of transgenic cow per batch. Average rHLF recovery of 79%and purity of 98.9% were attained for five batches. The purified rHLF displayed bioactivities as good as the natural human-resource lactoferrin standard. [ABSTRACT FROM AUTHOR]

Disassembling of virus-like particles (VLPs) like hepatitis B virus surface antigen (HB-VLPs) during chromatographic process has been identified as a major cause of loss of antigen activity. In this study, dual polarization interferometry (DPI) measurement, together with chromatography experiments, were performed to study the adsorption and conformational change of HB-VLPs on ion exchange surface at three different pHs. Changes in pH values of buffer solution showed only minimal effect on the HB-VLPs assembly and antigen activity, while significantly different degree of HB-VLPs disassembling was observed after ion exchange chromatography (IEC) at different pHs, indicating the conformational change of HB-VLPs caused mainly by its interactions with the adsorbent surface. By creating an ion exchange surface on chip surface, the conformational changes of HB-VLPs during adsorption to the surface were monitored in real time by DPI for the first time. As pH increased from 7.0 to 9.0, strong electrostatic interactions between oppositely charged HB-VLPs and the ion exchange surface make the HB-VLPs spread thinly or even adsorbed in disassembled formation on the surface as revealed by significant decrease in thickness of the adsorbed layer measured by DPI. Such findings were consistent with the results of IEC experiments operated at different pHs, that more disassembled HB-VLPs were detected in the eluted proteins at pH 9.0. At low pH like pH 5.0, however, possible bi-layer adsorption was involved as evidenced by an adsorbed layer thickness higher than average diameter of the HB-VLPs. The “lateral” protein–protein interactions might be unfavorable and would make additional contribution to the disassembling of HB-VLPs besides the primary mechanism related to the protein–surface interactions; therefore, the lowest antigen activity was observed after IEC at pH 5.0. Such real-time information on conformational change of VLPs is helpful for better understanding the real mechanism for the disassembling of VLPs on the solid–liquid interface. [ABSTRACT FROM AUTHOR]

Highlights: [•] Gigaporous microspheres were hydrophilic modified by poly(ethylenimine) (PEI). [•] PEI in the microspheres was in situ branched to resist non-specific adsorption for proteins. [•] The modified microspheres gave more than 90% proteins recovery. [•] The branched PEI in the microspheres could change its construction in different environment of solutions. [•] Dynamic binding capacity of proteins on the modified media didn’t show obviously decrease at the high flow rates. [Copyright &y& Elsevier]

Highlights: [•] γ-Globulin adsorption to PEI-Sepharose was studied and compared with BSA. [•] γ-Globulin capacity increased with ionic capacity (IC) at IC<560mmol/L. [•] The IC where γ-globulin uptake rate started to hop was earlier than that of BSA. [•] γ-Globulin uptake rate at IC=680mmol/L was four times of that at IC<460mmol/L. [•] Both capacity and uptake rate of γ-globulin decreased drastically at IC>680mmol/L. [Copyright &y& Elsevier]

Highlights: [•] Pore size effects of media on adsorption capacity, kinetics and activity of VLPs were investigated. [•] Gigaporous media show higher capacity and faster kinetics for VLPs than agarose media. [•] Gigaporous structure also improved the structural stability and activity of VLPs. [•] Mechanism of pore size effects on the aggregation and/or disassociation of VLPs was discussed. [•] Gigaporous media exhibit the best performance in many aspects compared to agarose media. [ABSTRACT FROM AUTHOR]

Human plasma fraction IV is an intermediate precipitate during the production of human serum albumin using cold ethanol method. Haptoglobin locates in this fraction can be purified for various applications. A new process integration of polyethylene glycol (PEG) precipitation and ion-exchange chromatography (IEC) was developed for purification of haptoglobin, which could effectively purify the haptoglobin from 16.6% to 95%. The recovery of the new process was 58.2% in comparison to 30.3% of the conventional affinity chromatography. Furthermore, 175 mg haptoglobin production in a scaled-up process showed the method to be simple, fast, and low-cost. [ABSTRACT FROM AUTHOR]

Abstract: Ion-exchange chromatography (IEC) using commercial ionic absorbents is a widely used technique for protein purification. Protein adsorption onto ion-exchange adsorbents often involves a multipoint adsorption. In IEC of multimeric proteins or “soft” proteins, the intense multipoint binding would make the further desorption difficult, even lead to the destruction of protein structure and the loss of its biological activity. In this paper, DEAE Sepharose FF adsorbents with controllable ligand densities from 0.020 to 0.183mmol/ml were synthesized, and then the effect of ligand density on the static ion-exchange adsorption of bovine serum albumin (BSA) onto DEAE Sepharose FF was studied by batch adsorption technique. Steric mass-action (SMA) model was employed to analyze the static adsorption behavior. The results showed that the SMA model parameters, equilibrium constant (K a), characteristic number of binding sites (υ) and steric factor (σ), increased gradually with ligand density. Thus, it was feasible to regulate BSA multipoint adsorption by modulating the ligand density of ion-exchange adsorbent. Furthermore, IEC of hepatitis B surface antigen (HBsAg) using DEAE Sepharose FF adsorbents with different ligand densities was carried out, and the activity recovery of HBsAg was improved from 42% to 67% when the ligand density was decreased from 0.183 to 0.020mmol/ml. Taking the activity recovery of HBsAg, the purification factor and the binding capacity into account, DEAE Sepharose FF with a ligand density of 0.041mmol/ml was most effective for the purification of HBsAg. Such a strategy may also be beneficial for the purification of macromolecules and multimeric proteins. [ABSTRACT FROM AUTHOR]

• Mechanism of bio-macromolecule denaturation in IEC was different at different pHs. • Sucrose increased stability of bio-macromolecule on column and improved recovery. • Denaturation was closely related to on column thermo-stability of bio-macromolecule. • Higher on column thermo-stability, less denaturation and higher recovery. • DSC helps to understand denaturation of bio-macromolecule during chromatography. Destruction of assembly structures has been identified as a major cause for activity loss of virus and virus-like particles during their chromatographic process. A deep insight into the denaturation process at the solid-liquid interfaces is important for rational design of purification. In this study, in-situ differential scanning calorimetry (DSC) was employed to study the dissociation process of inactivated foot-and-mouth disease virus (FMDV) during ion exchange chromatography (IEC) at different levels of pH. The intact FMDV known as 146S and the dissociation products were quantified by high performance size exclusion chromatography (HPSEC) and the thermo-stability of 146S on-column was monitored in-situ by DSC. Serious dissociation was found at pH 7.0 and pH 8.0, leading to low 146S recoveries of 12.3% and 43.7%, respectively. The elution profiles from IEC and HPSEC combined with the thermal transition temperatures of 146S dissociation (T m1) from DSC suggested two denaturation mechanisms that the 146S dissociation occurred on-column after adsorption at pH 7.0 and during elution step at pH 8.0. By appending different excipients including sucrose, the improvement of 146S recovery and reduced dissociation was found highly correlated to increment of 146S stability on-column detected by DSC. The highest recovery of 99.9% and the highest T m1 of 54.49 °C were obtained at pH 9.0 with 20% (w/v) sucrose. According to chromatographic behaviors and T m1 , three different dissociation processes in IEC were discussed. The study provides a perspective to understand the denaturation process of assemblies during chromatography, and also supplies a strategy to improve assembly recovery. [ABSTRACT FROM AUTHOR]