•Aggregation results from interaction between PRN with different refolding state.•Flash-batch dilution refolding is efficient in prevention of aggregate formation.•Aggregates can be removed from monomeric rPRN by Ni-chromatography.•Monomeric rPRN is obtained with purity of 98% and total recovery of 59%.•RPRN shows similar physiochemical properties and immunological response with nPRN.Production of natural pertactin for pharmaceutical use is limited by its low abundance. In this study, recombinant pertactin was highly expressed in the form of inclusion bodies in E. coli. However, up to 75% of the soluble turned out as aggregates when refolding by pulse-fed batch dilution. The conceivable route for aggregate formation was proposed as that the C-terminus of partially folded intermediate with a strong hydrophobic core would intertwine with that region of newly added denatured protein, resulting in aggregation between proteins with different folding states. The key factor for prevention of aggregate formation was to improve the synchronization of refolding. For this purpose, flash-batch dilution was conducted at a scale of 5 L and achieved a monomeric refolding yield of above 70%. Aggregates formed were efficiently removed along with impurities by one-step chromatography of Ni-resin. The purity of monomeric pertactin was >98%. An overall yield was 320 mg per liter fermentation liquor with a total recovery of about 59%. The purified protein was characterized by MALDI-TOF, circular dichroism, fluorescence, HPLC and DSC, and showed similar physiochemical properties compared to its natural counterpart. Animal study showed similar immunological responses and antibodies elicited demonstrated a comparable reactivity.Download high-res image (154KB)Download full-size image

To overcome the deficiency of rapid elimination from blood, the truncated human recombinant ciliary neurotrophic factor was formulated by site-specific attachment of different-sized PEG-maleimide or by cross-linking with human transferrin through a hetero-bi-functional PEG linker (NHS-PEG5k-MAL). The PEGylated CNTF was purified by a two-step chromatography procedure and the transferrin coupling CNTF conjugate was separated through an elegant protocol. The conjugation site on CNTF was identified by peptide mapping analysis and validated that the linkage of the conjugates was specifically happened to Cys17 residue. Although both PEGylated and transferrin coupling CNTF demonstrated decreased cell based residual activity, markedly enhanced pharmacokinetic behaviors in normal male Sprague-Dawley rats were observed, especially for the PEG40k-CNTF with approximately 58-times improvement compared with the unmodified counterpart. The evaluation of the in vivo potency of body weight-losing was performed with normal male C57BL6 mice and the results revealed that both PEGylation and transferrin coupling could achieve improved therapeutic benefits relative to that of CNTF. Besides, PEG20k/40k-CNTF demonstrated more effective than transferrin coupling CNTF (Tf-PEG5k-CNTF) despite that the later showed preferable pharmacokinetic profile and cell based residual activity compared with PEG20k-CNTF. Weekly subcutaneous administration of PEG40k-CNTF with 0.5 mg/kg and 1.0 mg/kg dose resulted in approximately 35% and 50% decrease in food intake during one interval period of injection, indicating that PEG40k-CNTF is the most potential anti-obese agent for therapeutics.Download high-res image (160KB)Download full-size image

•High hydrostatic pressure was first utilized for encapsulation of DOX in HFn.•HHP, combined with additives, can completely inhibited aggregation.•The HFn-DOX NPs showed similar structural features to the hollow cage.•The HFn-DOX NPs demonstrated significant antitumor activity in vitro and in vivo.•This HHP encapsulation strategy could economize on the consumption of DOX.Human ferritin (HFn) nanocaging is becoming an appealing platform for anticancer drugs delivery. However, protein aggregation always occurs during the encapsulation process, resulting in low production efficiency. A new approach using high hydrostatic pressure (HHP) was explored in this study to overcome the problem of loading doxorubicin (DOX) in HFn. At the pressure of 500 MPa and pH 5.5, DOX molecules were found to be encapsulated into HFn. Meanwhile, combining it with an additive of 20 mM arginine completely inhibited precipitation and aggregation, resulting in highly monodispersed nanoparticles with almost 100% protein recovery. Furthermore, stepwise decompression and incubation of the complex in atmospheric pressure at pH 7.4 for another period could further increase the DOX encapsulation ratio. The HFn-DOX nanoparticles (NPs) showed similar morphology and structural features to the hollow cage and no notable drug leakage occurred for HFn-DOX NPs when stored at 4 °C and pH 7.4 for two weeks. HFn-DOX NPs prepared through HHP also showed significant cytotoxicity in vitro and higher antitumor bioactivity in vivo than naked DOX. Moreover, This HHP encapsulation strategy could economize on DOX that was greatly wasted during the conventional preparation process simply through a desalting column. These results indicated that HHP could offer a feasible approach with high efficiency for the production of HFn-DOX NPs.

Conventional preparation strategies for antibody–drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab′ fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment–drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab′ fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab′ fragment to form a Fab′-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab′-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

•Combine NMR and QCM approaches on molecular scale to analyze the protein retentions perturbed by structure flexibility in IEC.•Easily unfolding of flexible protein was found by evaluation of protein protection behavior during H/D exchange reaction.•The soft protein with high flexibility tends to longer retention and strong affinity.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.

We explored an intelligent vaccine system via facile approaches using both experimental and theoretical techniques based on the two-dimensional graphene oxide (GO). Without extra addition of bio/chemical stimulators, the microsized GO imparted various immune activation tactics to improve the antigen immunogenicity. A high antigen adsorption was acquired, and the mechanism was revealed to be a combination of electrostatic, hydrophobic, and π–π stacking interactions. The “folding GO” acted as a cytokine self-producer and antigen reservoir and showed a particular autophagy, which efficiently promoted the activation of antigen presenting cells (APCs) and subsequent antigen cross-presentation. Such a “One but All” modality thus induced a high level of anti-tumor responses in a programmable way and resulted in efficient tumor regression in vivo. This work may shed light on the potential use of a new dimensional nano-platform in the development of high-performance cancer vaccines.

•DPI measurements were performed to monitor the adsorption of HB-VLPs on anion exchange surface.•Thickness of the HB-VLPs adsorbed layer varied significantly at different pHs.•HB-VLPs spread thinly or even adsorbed in disassembled formation on the surface at pH 7.0 and 9.0.•Possible bi-layer adsorption was involved at pH 5.0 thus led to severer disassembly of HB-VLPs.•Findings from DPI measurements were consistent with results of IEC experiments operated at different pHs.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.

•ITC study was performed on adsorption and disassembling of HB-VLPs during anion exchange chromatography.•Media with higher ligand density lead more serious disassembling of HB-VLPs.•Thermodynamic model was developed to obtain enthalpies related to adsorption and disassembling of VLPs.•Disassembling of VLPs is spontenous enthalpy-driven process.•As ligand density increase, enthalpy change related to VLPs disassembling become more negative.Chromatographic purification of virus-like particles (VLPs) is important to the development of modern vaccines. However, disassembly of the VLPs on the solid–liquid interface during chromatography process could be a serious problem. In this study, isothermal titration calorimetric (ITC) measurements, together with chromatography experiments, were performed on the adsorption and disassembling of multi-subunits hepatitis B virus surface antigen virus-like particles (HB-VLPs). Two gigaporous ion-exchange chromatography (IEC) media, DEAE-AP-280 nm and DEAE-POROS, were used. The application of gigaporous media with high ligand density led to significantly increased irreversible disassembling of HB-VLPs and consequently low antigen activity recovery during IEC process. To elucidate the thermodynamic mechanism of the effect of ligand density on the adsorption and conformational change of VLPs, a thermodynamic model was proposed. With this model, one can obtain the intrinsic molar enthalpy changes related to the binding of VLPs and the accompanying conformational change on the liquid–solid interface during its adsorption. This model assumes that, when intact HB-VLPs interact with the IEC media, the total adsorbed proteins contain two states, the intact formation and the disassembled formation; accordingly, the apparent adsorption enthalpy, ΔappH, which can be directly measured from ITC experiments, presents the sum of three terms: (1) the intrinsic molar enthalpy change associated to the binding of intact HB-VLPs (ΔbindHintact), (2) the intrinsic molar enthalpy change associated to the binding of HB-VLPs disassembled formation (ΔbindHdis), and (3) the enthalpy change accompanying the disassembling of HB-VLPs (ΔconfHdis). The intrinsic binding of intact HB-VLPs and the disassembled HB-VLPs to both kinds of gigaporous media (each of which has three different ligand densities), were all observed to be entropically driven as indicated by positive values of ΔbindHintact and ΔbindHdis; while the nagative ΔconfHdis values suggested a spontenous enthalpy-driven process for the forming of HB-VLPs disassembled formation at all conditions studied. As ligand density increases, ΔconfHdis became more negative, which was in agreement with the findings from chromatography experiments, that higher ligand density leads to more serious disassembling of HB-VLPs. Results from thermodynamic studies provided us insight understanding on the mechanism of adsorption and conformational change of VLPs, as well as the effect of ligand densities on the structural stability of VLPs during IEC process.

The porous structure characteristics of twenty-one porous microspheres with agarose framework were investigated based on the low-field nuclear magnetic resonance (LF NMR) relaxation time (T2) distribution measurements. The feasibility of the technique was confirmed by direct relationship between T2 and the mean pore size of the polymer networks (Rh) of agarose hydrogel which was established using the “Fiber-Cell” model. The expected pore radius distribution curve was obtained and the reliability was validated by comparing them with the results obtained by inverse size-exclusion chromatography. Thereafter, the technique was applied to characterize the pore size distribution (PSD) of soft microspheres and the pore size transformation of microspheres with or without grafted polymers in the process of protein adsorption. All of these results strongly support LF NMR as a promising, rapid and nondestructive technique for the determination of PSDs in many kinds of soft porous microspheres.

Protein structure in nanopores is an important determinant in porous substrate utilization in biotechnology and materials science. To date, accurate residue details of pore curvature induced protein binding and unfolding were still unknown. Here, a multiscale ensemble of chromatography, NMR hydrogen and deuterium (H/D) exchange, confocal scanning and molecular docking simulations was combined to obtain the protein adsorption information induced by pore size and curvature. Lysozyme and polystyrene microspheres within pores in the 14–120 nm range were utilized as models. With pore size increasing, the bound lysozyme presented a tendency of significantly decreased retention, less unfolding and fewer interacted sites. However, such a significant dependence between pore curvature and protein size only existed in a limited micro-pore range comparable to protein sizes. The mechanism behind the above events could be attributed to the diverse protein interaction area determined by pore curvature and size change, by models calculating the binding of lysozyme onto surfaces. Another surface of opposite curvature for nanoparticles was also calculated and compared, the rules were similar but with opposite direction and such a critical size also existed. These studies of proteins on curved interfaces may ultimately help to guide the design of novel porous materials and assist in the discrimination of the target protein from molecular banks.

PEGylation, including nonspecific and site-directed approaches, is a well-established and validated strategy to increase the stability, in vivo plasma retention time, and efficacy of protein pharmaceutics together with a reduction in immunogenicity and hydrophobicity. Site-directed conjugation by PEG-aldehyde is the most widely used method for N-terminal modification; however, the generation of multimodified products is inevitable because of lysine chemistry, which always leads to difficulties in purification and quantification. In this study, we developed a specific PEGylation strategy through the periodation of the N-terminus of interferon beta-1b (IFN-β-1b) followed by the coupling of PEG-hydrazide. The prolonged elimination half-life and significantly diminished immunogenicity of the PEG-hydrazide-modified protein indicated the development of an effective process for improving the pharmacology and immunogenicity of IFN-β-1b. We further conducted comparisons on the selectivity, velocity, yield, and pharmacokinetics of the two methods. The results demonstrated that the hydrazide-based conjugation was a highly specific coupling reaction that only produced homogeneous N-terminal mono-PEGylated conjugate but also generated heterogeneous multimodified products in the aldehyde-based process. In addition, a better PEGylation yield was found for the hydrazide conjugation compared with that of the aldehyde strategy. These investigations supply a practical approach for the site-specific modification of proteins with an N-terminal serine or threonine to achieve improved homogeneity of the conjugates as well as enhanced pharmacological properties.

•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.Limited binding capacity and low recovery of large size multi-subunits virus-like particles (VLPs) in conventional agarose-gel based chromatographic supports with small pores have long been a bottleneck limiting the large scale purification and application of VLPs. In this study, four anion exchange media including DEAE-Sepharose FF (DEAE-FF), DEAE-Capto, gigaporous DEAE-AP-120 nm and DEAE-AP-280 nm with average pore diameters of 32 nm, 20 nm, 120 nm and 280 nm, respectively, were applied for purification of hepatitis B virus surface antigen (HBsAg) VLPs. Pore size effects of media on the VLPs adsorption equilibrium, adsorption kinetics, dynamic binding capacity (DBC), and recovery were investigated in detail. According to the confocal laser scanning microscopy observation, adsorption of the VLPs in DEAE-FF and DEAE-Capto was mostly confined to a thin shell on the outer surface of the beads, leaving the underlying pore space and the binding sites inaccessibly, while the large pores in gigaporous media enabled the VLPs to access to the interior pore spaces by diffusion transport efficiently. Compared to the most widely used DEAE-FF, gigaporous media DEAE-AP-280 nm gained about 12.9 times increase in static adsorption capacity, 8.0 times increase in DBC, and 11.4 times increase in effective pore diffusivity. Beyond increasing the binding capacity and enhancing the mass transfer, the gigaporous structure also significantly improved the stability of the VLPs during intensive adsorption-desorption process by lowing the multi-point interaction between the VLPs and binding sites in the pores. At 2.0 mg/mL-media loading quantity, about 85.5% VLPs were correctly self-assembled after the chromatography with DEAE-AP-280 nm media; oppositely about 85.2% VLPs lost their normal assembly with DEAE-FF due to irreversible disassembly. Comparative investigation was made to study the purifying performance of these four chromatographic media for actual VLPs purification from recombinant Hansenula polymorpha. DEAE-AP-280 nm media were demonstrated the best results showing the highest recovery of 68.33% and purification fold of 3.47, at 2.98 mg protein/mL-media loading quantity and a flow rate of 240 cm/h.

•HHP could completely solubilize and refold rhCNTF IBs at enhanced concentration.•HHP refolding of rhCNTF IBs shows higher productivity than the dilution method.•HHP refolding yields of rhCNTF IBs are independent of protein concentrations.•HHP method favors the refolding of monomeric proteins without disulfide bond.•Purified rhCNTF shows similar structure and in vitro bioactivity to native species.Protein refolding from inclusion bodies (IBs) often encounters a problem of low recovery at high protein concentration. In this study, we demonstrated that high hydrostatic pressure (HHP) could simultaneously achieve high refolding concentration and high refolding yield for IBs of recombinant human ciliary neurotrophic factor (rhCNTF), a potential therapeutic for neurodegenerative diseases. The use of dilution refolding obtained 18% recovery at 3 mg/mL, even in the presence of 4 M urea. In contrast, HHP refolding could efficiently increase the recovery up to almost 100% even at 4 mg/mL. It was found that in the dilution, hydrophobic aggregates were the off-path products and their amount increased with the protein concentration. However, HHP could effectively minimize the formation of hydrophobic aggregates, leading to almost complete conversion of the rhCNTF IBs to the correct configuration. The stable operation range of concentration is 0.5–4.0 mg/mL, in which the refolding yield was almost 100%. Compared with the literatures where HHP failed to increase the refolding yield beyond 90%, the reason could be attributed to the structural difference that rhCNTF has no disulfide bond and is a monomeric protein. After purification by one-step of anionic chromatography, the purity of rhCNTF reached 95% with total process recovery of 54.1%. The purified rhCNTF showed similar structure and in vitro bioactivity to the native species. The whole process featured integration of solubilization/refolding, a high refolding yield of 100%, a high concentration of 4 mg/mL, and a simple chromatography to ensure a high productivity.

•Two off-path components were identified for proinsulin aspart refolding.•Arginine and urea have different efficacy and possible mechanisms on the refolding.•Only oxidant was necessary for the proinsulin aspart's disulfide bonds formation.•Selenocystamine could significantly increase the refolding yield.Successfully recovering proinsulin's native conformation from inclusion body is the crucial step to guarantee high efficiency for insulin's manufacture. Here, two by-products of disulfide-linked oligomers and disulfide-isomerized monomers were clearly identified during proinsulin aspart's refolding through multiple analytic methods. Arginine and urea are both used to assist in proinsulin refolding, however the efficacy and possible mechanism was found to be different. The oligomers formed with urea were of larger size than with arginine. With the urea concentrations increasing from 2 M to 4 M, the content of oligomers decreased greatly, but simultaneously the refolding yield at the protein concentration of 0.5 mg/mL decreased from 40% to 30% due to the increase of disulfide-isomerized monomers. In contrast, with arginine concentrations increasing up to 1 M, the refolding yield gradually increased to 50% although the content for oligomers also decreased. Moreover, it was demonstrated that not redox pairs but only oxidant was necessary to facilitate the native disulfide bonds formation for the reduced denatured proinsulin. An oxidative agent of selenocystamine could increase the yield up to 80% in the presence of 0.5 M arginine. Further study demonstrated that refolding with 2 M urea instead of 0.5 M arginine could achieve similar yield as protein concentration is slightly reduced to 0.3 mg/mL. In this case, refolded proinsulin was directly purified through one-step of anionic exchange chromatography, with a recovery of 32% and purity up to 95%. All the results could be easily adopted in insulin's industrial manufacture for improving the production efficiency.

•HBsAg particles expressed by CHO and Hansenula polymorpha were compared by AF4-MALLS.•Stabilities of CHO-HBsAg and Hans-HBsAg under different solution environments were described.•CHO-HBsAg was more homogeneous and more stable under different solution environments than Hans-HBsAg.The stabilities of two commercially available virus like particles, CHO-HBsAg expressed by Chinese hamster ovary (CHO) cells and Hans-HBsAg expressed by Hansenula polymorpha (Hans), were compared using AF4-MALLS under different treatment processes. The initial molecular weight and hydrodynamic diameter of CHO-HBsAg measured with AF4-MALLS were 4727 kDa and 29.4 nm, while those of Hans-HBsAg were 3039 kDa and 22.8 nm respectively. In salt solution of 2 M ammonium sulfate, the molecular weight and size of CHO-HBsAg had little change, and its antigenicity remained 95%, while those of Hans-HBsAg changed greatly, resulting in aggregation and 75% antigenicity loss. In freeze–thaw operations, Hans-HBsAg aggregated heavily. Most of the aggregates precipitated and the rest soluble aggregates reached 105–106 kDa in molecular weight. The antigenicity of Hans-HBsAg decreased to 26.9% after five freeze–thaw cycles. For CHO-HBsAg, there was no obvious aggregation in freeze–thaw, and the antigenicity retained above 98%. In heating process, Hans-HBsAg gradually aggregated to large particles with temperature and the antigenicity decreased to 10% when the temperature reached 80 °C. In contrast, CHO-HBsAg would not aggregate with temperature, remained 92% antigenicity at 80 °C. The study demonstrated that CHO-HBsAg appeared to be a superior vaccine antigen in term of particle stability and constant antigenicity, which are important in production, transportation and storage.

•Dual-IEC strategy for purification of pertactin was designed based on surface charge analysis.•Pertactin with the purity of 96% and the total recovery of 61% was obtained with the dual-IEC strategy.•Charged patches on protein surface were demonstrated critical for binding onto IEC resins.•Rational development approach was applied in development of protein purification scheme.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.

•Assembly of three important antigen particles were characterized by SE-HPLC.•SE-HPLC was used to analyze 146S content in FMDV whole virus particle vaccine preparations.•Expression level of the HBcAg-VLPs during cell culture was quantitatively monitored by SE-HPLC.•Product consistency and storage stability of HBsAg vaccine preparations were monitored by SE-HPLC.•SE-HPLC has potentials as a versatile alternative technology for product control of vaccines.The assembly of antigen structure is often crucial to the potency of vaccines. Currently adopted methods like animal testing and ultracentrifugation take long time and are difficult to automate for multiple samples. Here we develop a size-exclusion high-performance liquid chromatography (SE-HPLC) method to characterize the assembly of antigen structure during both manufacturing process and storage. Three important vaccine antigens including inactivated foot and mouth disease virus (FMDV), which is a virus vaccine; and two virus-like particles (VLPs) vaccines involving hepatitis B core antigen (HBcAg) VLPs, and hepatitis B surface antigen (HBsAg) VLPs, were successfully analyzed using commercially available TSK gel columns with pore size above 45 nm. Combined with other analytical methods including SDS–PAGE, dynamic light scattering, wavelength scan, and multi-angle laser light scattering, the SE-HPLC method was proven to be a simple, rapid, and reliable tool for antigen particles assembly analysis. Specifically, for FMDV whole virus particle, SE-HPLC was used to analyze 146S content in vaccine preparations and the thermal dissociation of the 146S. For HBcAg-VLPs that are expressed in recombinant Escherichia coli, its expression level during cell culture process was quantitatively monitored by SE-HPLC. The SE-HPLC also showed applicability for quality check of HBsAg vaccine preparations by monitoring the product consistency of different lot number and the product stability during storage. Results shown in this work clearly demonstrated that SE-HPLC method has potential as a versatile alternative technology for control of the final product by both manufacturers and the regulatory agencies.

•Effects of solution pH and temperature on the dissociation of 146S was investigated.•HPSEC was used for rapid and quantitative determination of 146S.•The most stable pH for FMDV was between 7.5 and 8.0.•Half life of FMDV at 45 °C was prolonged from less than 30 min to more than 3 days be adding 20% sucrose.•Stabilization mechanism were analyzed by DSC.The inactivated foot and mouth disease virus (FMDV), which has a sedimentation coefficient of 146S, is crucial to the efficacy of vaccine preparations, but extremely unstable in vitro. It is prone to dissociate into smaller particles referred to as 12S with a concomitant decrease in immunogenicity; therefore, it is of great importance to find the best condition for stabilizing the FMDV. In the present work, the effects of solution pH and temperature on the dissociation of 146S was investigated and potential stabilizers were screened, with aid of high-performance size-exclusion chromatography (HPSEC) for rapid and quantitative determination of 146S, together with differential scanning calorimetry (DSC) technology for thermal stability analysis. The most stable pH was found between 7.5 and 8.0. Among excipients tested, sucrose and glycerol provided the best protection, such that the half-life of 146S in solution at 45 °C could be prolonged from less than 30 min to more than 3 days by adding 20% sucrose. The stabilization mechanism was confirmed using DSC analysis, which showed that the transition temperature related to 146S dissociation was increased by 5.4 °C in the presence of 20% sucrose. The physical stabilization effects afforded by these stabilizers would allow for the retaining of effective 146S antigens during transportation and storage under relative harsh condition.

•We characterize the size of HBsAg VLP and its aggregates by AF4 for the first time.•Mechanism of the aggregation process in NaCl and (NH4)2SO4 salt solution was described.•The antigenicity of HBsAg in different salt solution environment was researched.The phenomenon of aggregation of virus-like particles (VLPs) in salt solution and the corresponding effect upon antigenicity was reported. Asymmetrical flow field-flow fractionation (AF4) combined with multi-angle laser light scattering (MALLS) was used to characterize the size and the aggregation behavior of hepatitis B surface antigen (HBsAg). The average diameter of HBsAg VLP was 22.8 ± 0.4 nm and it tended to aggregate in salt solution to form large particles and the antigenicity changed accordingly. In 0–4 M NaCl solution, part of HBsAg molecules aggregated rapidly into oligomeric particles (OP), whose diameter distributed from 25 to 40 nm, and the antigenicity slightly decreased about 10%. The aggregation reaction is reversible. After removing NaCl, both size and antigenicity could recover to normal level (92–96%). By contrast, the aggregation process is more complicated in (NH4)2SO4 solution. Most of HBsAg particles aggregated into OP and further aggregated into polymeric particles (PP). The diameter of the PP could reach 40 to 140 nm. The concentration of (NH4)2SO4 had remarkable influence upon the rate of aggregation. When concentration of (NH4)2SO4 was below 1 M, most of HBsAg aggregated only into OP in 1 h. While with concentration of (NH4)2SO4 above 1 M, most of particles formed PP within 1 h. The aggregation process to PP was irreversible. After removing (NH4)2SO4, the large aggregates could not recover to normal particles and the remaining antigenicity was below 30%.

•A KGM-based microcarrier was prepared for anchorage-dependent animal cell culture.•Five cell lines adhered well and grew to high cell density on KGM microcarriers.•Vero cells achieved more than 2 × 106 cells/ml on KGM microcarriers in spinner culture.•Cell density and rabies virus titer were comparable in KGM and Cytodex 1 culture.•The KGM microcarrier could be a promising candidate for various cell cultures.A new type of microcarrier was prepared for anchorage-dependent animal cell culture. Similar to the commercial product Cytodex 1, the newcomer is also spherical in micro-size range with ionic group DEAE, but the matrix is konjac glucomannan (KGM) from a natural plant. The optimal anion exchange capacity (AEC) was found to be 2.1 to 2.4 mmol/g dry microspheres. Five cell lines were cultured in static mode with the KGM microcarriers, including Vero, CHO-K1, MDCK, Wish and L929 cells. These cells adhered well and grew to high cell density comparable with or even better than those did on Cytodex 1. A further comparison was made with Vero cell culture in spinner mode. It was found that Vero cells achieved more than 2 × 106 cells/ml, and showed faster cell adhesion and higher specific growth rate on the KGM microcarriers than on Cytodex 1. In 5 L bioreactor perfusion culture, Vero cells grew above 7.0 × 106 cells/ml and the rabies virus titer was about 7.7 lgLD50/ml, compared to Cytodex 1 in the same culture condition. The results suggest that the new type of microcarrier could be a promising candidate for various cell cultures.

Protein structure in nanopores is an important determinant in porous substrate utilization in biotechnology and materials science. To date, accurate residue details of pore curvature induced protein binding and unfolding were still unknown. Here, a multiscale ensemble of chromatography, NMR hydrogen and deuterium (H/D) exchange, confocal scanning and molecular docking simulations was combined to obtain the protein adsorption information induced by pore size and curvature. Lysozyme and polystyrene microspheres within pores in the 14–120 nm range were utilized as models. With pore size increasing, the bound lysozyme presented a tendency of significantly decreased retention, less unfolding and fewer interacted sites. However, such a significant dependence between pore curvature and protein size only existed in a limited micro-pore range comparable to protein sizes. The mechanism behind the above events could be attributed to the diverse protein interaction area determined by pore curvature and size change, by models calculating the binding of lysozyme onto surfaces. Another surface of opposite curvature for nanoparticles was also calculated and compared, the rules were similar but with opposite direction and such a critical size also existed. These studies of proteins on curved interfaces may ultimately help to guide the design of novel porous materials and assist in the discrimination of the target protein from molecular banks.