Yongdong Huang

Chinese Academy of Sciences, Peping, Beijing, China

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Publications (17)26.3 Total impact

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    ABSTRACT: High hydrophilicity of gigaporous microspheres based on a copolymer of poly(glycidyl methacrylate)-co-divinyl benzene (PGMA-DVB) was successfully realized through coating the branched polyethyleneimine (PEI) in PGMA-DVB microspheres. PEI with various molecules weights and different branching agents were identified in terms of protein recovery as evaluation approach. For this evaluation, PEI600 (Mw=600) and poly (ethylene glycol) diglycidyl ether (PEGDE, Mw=400) were used as modification agent and branching agent, respectively. The modified microspheres showed good permeability and revealed a certain mechanical strength. After modification, the protein recovery increased from 40% to >90%. The protein recovery increased with the branched generations and the first and second generations could give the protein recovery of 93% and 96%, respectively. Meanwhile, the ionic capacity also showed a rising trend in the range of 0.11-0.32mmol/mL with the branched generations. But the dynamic binding capacity of protein (bovine serum albumin, BSA as the model protein) increased at first and then decreased. Analysis of the dry microspheres structure by mercury intrusion method as well as observation of the branched PEI on PGMA-DVB membrane in aqueous solution indicated that excess PEI chains with the extended state in the second generation would block the small pores and decrease the accessible surface area. Therefore, the protein capacity on the second generation, on the contrary, was lower than that on the first generation. Meanwhile, it was found that the PEI chains in the modified microspheres changed their construction from the extended to the collapsed state with increase of NaCl concentration. And the corresponding pore size of the modified microspheres increased with salt concentration through low-field nuclear magnetic resonance. Dynamic binding capacity of proteins on the modified supports did not significantly change with increase of the flow rate. The media showed good performance for separation three model proteins at high flow rate of 1084cm/h. This modified gigaporous microspheres had a large potential in application for rapid separation of biomolecules.
    Journal of Chromatography A 04/2014; · 4.61 Impact Factor
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    ABSTRACT: To study the leakage at different solution pH values, IgG Sepharose 6FF®, a commercially available immunoadsorbent, was used as a model. The leaked substance consists of three parts: (1) ligands and its fragments; (2) ligands plus matrix fragments in which ligands are chemically attached to the adsorbent matrix; and (3) matrix fragments. Buffer solution pH values had a great effect on both the kinetics and the amount of ligand leakage. Cross-linking of the adsorbent matrix could reduce both matrix leakage and antibody leakage at pH 3.0, but its effect was limited at pH 11.0 for ligand leakage. © 2013 The Authors. Biomedical Chromatography published by John Wiley & Sons Ltd.
    Biomedical Chromatography 05/2013; · 1.95 Impact Factor
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    ABSTRACT: Novel GSH-AP (phenoxyl agarose coated gigaporous polystyrene, Agap-co-PSt) microspheres were successfully prepared by introducing GSH ligand into hydrophilic AP microspheres pre-activated with 1,4-butanediol diglycidyl ether. The gigaporous structure and chromatographic properties of GSH-AP medium were evaluated and compared with commercial GSH Sepharose FF (GSH-FF) medium. The macropores (100–500 nm) of gigaporous PSt microspheres were well maintained after coating with agarose and functionalized with GSH ligand. Hydrodynamic experiments showed that GSH-AP column had less backpressure and plate height than those of GSH-FF column at high flow velocity, which was beneficial for its use in high-speed chromatography. The presence of flow-through pores in GSH-AP microspheres also accelerated the mass transfer rate of biomolecules induced by convective flow, leading to high protein resolution and high dynamic binding capacity (DBC) of glutathione S-transferase (GST) at high flow velocity. High purity of GST and GST-tagged recombinant human interleukin-1 receptor antagonist (rhIL-1RA) were obtained from crude extract with an acceptable recovery yield within 1.5 min at a velocity up to 1400 cm/h. GSH-AP medium is promising for high-speed affinity chromatography for the purification of GST and GST-tagged proteins.
    Protein Expression and Purification 01/2013; · 1.43 Impact Factor
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    ABSTRACT: Conjugation of truncated recombinant staphylokinase (trSak) with polyethylene glycol (PEG) is an effective way to overcome its short plasma half-life and enhance its therapeutic potential. However, conventional amine directed PEGylation chemistry inevitably led to modification at its functionally important N terminus, which resulted in a significantly reduced bioactivity of trSak. In this study, a novel solid phase PEGylation process was developed to shield the N-terminal region of the protein from PEGylation. The process was achieved by oriented adsorption of an N-terminally His-tagged trSak (His-trSak) onto an immobilized metal-ion affinity chromatography (IMAC). His-trSak was efficiently separated and retained on IMAC media before reaction with succinimidyl carbonate mPEG (SC-mPEG, 5, 10 or 20kDa). The IMAC derived mono-PEGylated His-trSak showed structural and stability properties similar to the liquid phase derived conjugate. However, isoelectric focusing electrophoresis analysis revealed that mono-PEGylated His-trSaks via solid phase PEGylation were more homogeneous than those from liquid phase PEGylation. Moreover, tryptic peptide mapping analysis suggested that a complete N-terminal blockage of IMAC bound His-trSak from PEGylation with 10kDa- and 20kDa-SC-mPEG. In contrast, only partial protection of the N-terminal region was obtained for 5kDa-SC-mPEG. Bioactivities of 10kDa- and 20kDa-PEG-His-trSak conjugates without N-terminal PEGylation were significantly higher than those of randomly PEGylated products. This further demonstrated the advantage of our new on-column PEGylation strategy.
    Process Biochemistry - PROCESS BIOCHEM. 01/2012;
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    ABSTRACT: Hydrophobic interaction chromatography (HIC) is often an inevitable step for a satisfying purification in giant vaccine molecules production. But great mass and activity loss associated with poor purity often occur simultaneously. In this paper, high purity and high bioactivity recovery for the HIC process of hepatitis B surface antigen (rHBsAg) purification were achieved through manipulation of surface hydrophobicity of the adsorbent. Spacer arm length and ligand density were regulated, respectively, through which the interaction between the vaccine and the adsorbent was manipulated deliberately. It was found even in a narrow scope, varying spacer arm length and ligand density resulted in purification factor changing from 1 to 96.5, and rHBsAg recovery from 3 to 91%. The optimal purification performance was achieved when the spacer arm was C8 and the ligand density was 9.2 μmol/g suction-dried wet gel with an average distance of ligands of 3.6 nm. This deliberate regulation strategy represents a new approach of improving purification of giant multi-subunit proteins.
    Journal of Separation Science 11/2011; 34(22):3186-93. · 2.59 Impact Factor
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    ABSTRACT: 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.
    Artificial Cells Blood Substitutes and Biotechnology (formerly known as Artificial Cells Blood Substitutes and Immobilization Bi 04/2011; 39(2):79-86. · 0.94 Impact Factor
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    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 0.183 mmol/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 0.020 mmol/ml. Taking the activity recovery of HBsAg, the purification factor and the binding capacity into account, DEAE Sepharose FF with a ligand density of 0.041 mmol/ml was most effective for the purification of HBsAg. Such a strategy may also be beneficial for the purification of macromolecules and multimeric proteins.
    Protein Expression and Purification 12/2010; 74(2):257-63. · 1.43 Impact Factor
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    ABSTRACT: As a virus-like particle, hepatitis B surface antigen (HBsAg) was the primary component of hepatitis B vaccine. HBsAg was maintained by the non-covalent interaction of proteins and lipids. The intact structure of HBsAg particle was vital to its function. However, there was no report about the effects of solvent environment on HBsAg structure. In this paper, we studied the effects of temperature, pH, ionic type and salt concentration on HBsAg structure. The results showed that HBsAg was stable at normal temperature, but began to denature above 60 degrees C. The aggregation of HBsAg at pH 3.0 and 4.0 was nearly irreversible, but partly reversible at pH 5.0. The influence of ionic type on HBsAg was generally in accordance with Hofmeister sequence, except that SO4(2-) caused more aggregation than F-. HBsAg aggregates started to be visible in 0.4 mol/L (NH4)2SO4, and the extent of aggregation increased with the salt concentration. Therefore, caution must be taken when using (NH4)2SO4 in the hydrophobic chromatography purification of HBsAg.
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 12/2010; 26(12):1674-82.
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    ABSTRACT: Endotoxin removal is essential for the safety of biological products. To remove endotoxin efficiently, we used polymyxin B (PMB) affinity adsorbent to remove endotoxin from protein solutions by static adsorption. We studied the effects of spacer length and ligand density of the affinity adsorbent, pH, salt type and concentration, protein type and concentration, endotoxin concentration, and additive on endotoxin removal and protein recovery. Endotoxin content and protein concentration were determined by test and Lowry assay respectively. The results showed that PMB affinity adsorbent had high capacity, high adsorption speed, high removal efficiency and good reusability. In addition, ligand density, pH, salt concentration and the isoelectric point and hydrophobicity of protein all had remarkable influence on the endotoxin removal. Under the optimal conditions, the recoveries of hemoglobin, human serum albumin and lysozyme were 87.2%, 73.4% and 97.3%, respectively, and the corresponding endotoxin removal rates 99.8%, 97.9% and 99.7%, respectively. This study illustrated the effects of solution conditions on the efficiency of endotoxin removal and protein recovery, and would provide useful reference for the efficient removal of endotoxin from biological products.
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 11/2010; 26(11):1584-95.
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    ABSTRACT: Novel ion exchange adsorbents were synthesized by immobilizing sulfopropyl derivative onto homemade highly cross-linked agarose beads. The effects of different ligand densities (from 0.05 to 0.24 mol/L) on static and dynamic adsorption of the adsorbents were investigated using lysozyme as a model protein. Based on these results, rHLF was purified from the transgenic milk by our SP media. 1 mL high density (0.24 mol/L) adsorbent could handle 50 mL rHLF-containing milk. The mass recovery of rHLF was 86.5% and the purity was 98.5%. CD spectra demonstrated that the native structure of rHLF was not affected in the purification process. The biological functions of the purified rHLF, including iron binding, releasing and antimicrobial activities were then investigated. The results showed that rHLF had comparable iron binding and releasing activity to that of native HLF. 5 g/L concentration of rHLF significantly inhibited the growth of Escherichia coli. These studies lay a solid foundation for the wide application of our self-prepared ion exchange adsorbents in protein purification.
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 11/2010; 26(11):1576-83.
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    ABSTRACT: The low recovery of pertussis toxin (PT) and the low resolving efficiency of chromatography, due to the instability of PT in low salt condition, are the main challenges for its purification. We aplied 2 mol/L urea to prevent the aggregation and disassociation of PT during the purification by ion-exchange chromatography (IEC) and gel filtration chromatography (GFC). The effect of urea on the purification of PT was studied by ELISA assay and non-reduced SDS-PAGE. The activity recoveries of PT and filamentous hemagglutinin (FHA) in IEC and GFC, the resolution efficiency in GFC and the purities of PT and FHA were improved obviously by adding 2 mol/L urea in the mobile phase. The results highlight the potential application of urea in the acellular pertussis vaccine (APV) manufacture procedure.
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 08/2008; 24(7):1279-84.
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    ABSTRACT: The high expression level of recombinant hepatitis B surface antigen obtained from Hansenula polymorpha yeast cell (Hans-HBsAg) made it possible to produce HBsAg vaccine in a large scale and by cost-effective process. However, the present available purification process was somewhat tedious, time-consuming and difficult to scale up. To improve the purification efficiency and simplify the purification process, an integrated chromatographic process was developed and optimized. The downstream process included ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC). A series of chromatographic adsorbents were evaluated for their performances on the purification of Hans-HBsAg, and then the suitable adsorbents for IEC and HIC were screened out, respectively. After clarification by centrifugation, the supernatant of cell disruption (SCD) was purified by standard chromatographic steps, IEC on DEAE Sepharose FF, HIC on Butyl-S-QZT and GFC on Sepharose 4FF. Furthermore, HBsAg recovery, purification factor (PF) and purity during the downstream process were evaluated with enzyme-linked immunosorption assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size-exclusion chromatography (HPSEC). The results demonstrated that in the scale of 550ml SCD, the total HBsAg recovery and PF of the whole procedure were about 21.0+/-0.9% and 80.7+/-8.4 (n=3) respectively, with the purity of above 99%. This new downstream process was efficient, reproducible and relatively easy to be scaled up.
    Protein Expression and Purification 01/2008; 56(2):301-10. · 1.43 Impact Factor
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    ABSTRACT: To improve the purification efficiency of recombinant hepatitis B surface antigen derived from Hansenula polymorpha (Hans-HBsAg), a serial of absorbents for hydrophobic interaction chromatography with the controllable ligand density and spacer arm were synthesized, then developed and further applied to purify Hans-HBsAg. The absorbent, Butyl-S QZT with the ligand density of 25 μmol/(g wet gel) and spacer arm of 3C, was screened out and its physical and chemical properties were evaluated. High rigidity and low backpressure (<0.06 MPa) were obtained at the flow rate up to 20 ml/min. Moreover, it has the stable chemical characteristics of subjecting to high concentrations of acid, alkali and detergents. This HIC absorbent was further applied to purify Hans-HBsAg with the recovery 94% and purification-fold 9 under the optimized operation condition at pH 6.5 and concentration of ammonium sulfate 7.5%. Finally, the HIC adsorbent of Butyl-S QZT was applied in the integrated three-step chromatographic purification process to purify Hans-HBsAg. About 140 mg of purified Hans-HBsAg was obtained from 1 l cell disruption supernatant at the total recovery of 27% and the purification-fold of 151.8. Based on the assay of SDS-PAGE and SEC-HPLC, the purity of the purified HBsAg was over 99% to meet the requirement for the further inoculation use.
    PROCESS BIOCHEMISTRY 05/2007; 42(5):751-756. · 2.41 Impact Factor
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    ABSTRACT: This investigation focused on the structure change of Hepatitis B virus surface antigen (HBsAg) in the process of ultrafiltration (UF). Based on the assay of high performance size exclusion chromatography combining with on-line multi-angle laser light scattering (HPSEC-MALLS) and enzyme-linked immunosorbent assay (ELISA), the HBsAg assemblies were found to aggregate into large-size HBsAg aggregation with only about 20% HBsAg activity of the normal HBsAg assembly. The secondary structure of large size HBsAg aggregation was monitored by circular dichroism spectroscopy (CD) and demonstrated that the content of α-helix in HBsAg decreased from 48.2% to 34.4% and the content of γ-turn increased from 29.6% to 38.7% due to aggregation. The lipid structure of large size HBsAg aggregation was also changed markedly by the assay of infrared spectroscopy (IR) at the wavenumber 1750 cm−1 which is corresponding to ester acyl.
    PROCESS BIOCHEMISTRY 03/2007; · 2.41 Impact Factor
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    ABSTRACT: The molecular weight and size of recombinant Hepatitis B surface antigen (HBsAg) derived from Chinese hamster ovary (CHO) and the Hansenula polymorph have been characterized by high-performance size exclusion chromatography with multi-angle laser light scattering (HPSEC-MALLS). The average molecular weight of CHO-derived HBsAg particle (CHO-rHBsAg) (4921 kDa) was higher than that of H. polymorpha yeast strain (Hans-rHBsAg) (3010 kDa). The size of CHO-rHBsAg (22.1 nm) is nearly the same as that of native HBsAg compared to 18.1 nm for Hans-rHBsAg. The average monomer numbers were found to be 155 for CHO-rHBsAg and 86 for Hans-rHBsAg, respectively. The data obtained support the assumption that the higher immunogenicity of CHO-derived HBsAg is related to its more favorable macromolecular assembly structure.
    Journal of Chromatography B 08/2006; 838(2):71-7. · 2.49 Impact Factor
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    ABSTRACT: Ion-exchange chromatography (IEC) with the lowest step recovery (<50%) is still a bottleneck of the essentially four-step chromatographic procedure for the purification of recombinant hepatitis B surface antigen (r-HBsAg) from transformed Chinese hamster ovary (CHO) cell line. The low recovery is mainly resulted from the change of the assemble structure of HBsAg particles such as disassociation or aggregation on the surface of the absorbent during binding and eluting procedures. To improve the chromatographic efficiency of IEC, the influence of ligand density from 0.041 to 0.130 mmol diethylaminoethyl (DEAE)/ml absorbent on IEC of r-HBsAg was investigated. The retention time of HBsAg particles depended highly on ligand density. Higher HBsAg recovery and purification factor (PF) were obtained from the absorbents with lower ligand density. Under the optimum chromatographic conditions, the HBsAg recovery was increased from about 44% to 66% and the PF was increased from 9 to 13 by decreasing the ligand density from 0.130 to 0.041 mmol DEAE/ml absorbent.
    Process Biochemistry. 01/2006; 41(11):2320-2326.
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    ABSTRACT: About 10% of the Chinese population are chronic carriers of hepatitis B virus (HBV). Thus, the development of a highly efficient process for the preparation of a vaccine based on a recombinant hepatitis B surface antigen (HBsAg) is very important to the Chinese national immunization program. To this end, the ion exchange chromatography recovery of CHO-HBsAg from a recombinant Chinese hamster ovary cell line was shown to increase from about 55 to 80% by the addition of 1% poly(ethylene glycol) (PEG 10,000) to the mobile phase. Furthermore, based on analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the intact glycoprotein form of CHO-HBsAg was completely preserved by the addition of PEG. In the absence of PEG the glycoprotein form of CHO-HBsAg was also spread out into the high salt elution fraction. High-performance size-exclusion chromatography with on-line multiangle-laser-light scattering (HPSEC-MALLS) analysis was performed to monitor the status of the CHO-HBsAg aggregate structure assembly, particle size and molecular weight distribution after each purification step, and the results showed further that the presence of PEG facilitated the separation and recovery of intact glycoprotein form of CHO-HBsAg and promoted their assembly to proper virus-like particles, which are both important features and prerequisites of their immunogenicity.
    Journal of Chromatography A 12/2005; 1095(1-2):119-25. · 4.61 Impact Factor

Publication Stats

41 Citations
26.30 Total Impact Points

Institutions

  • 2005–2014
    • Chinese Academy of Sciences
      • Institute of Process Engineering
      Peping, Beijing, China
  • 2005–2013
    • Northeast Institute of Geography and Agroecology
      • Institute of Process Engineering
      Beijing, Beijing Shi, China
  • 2010
    • Beijing University of Chemical Technology
      Peping, Beijing, China
  • 2008
    • University of Science and Technology, Beijing
      • Department of Environmental Engineering
      Peping, Beijing, China