[Preparation of a macroporous anion exchange chromatographic medium by polyamine grafting and evaluation of its protein-adsorption behavior].

The macroporous anion exchange chromatographic medium (FastSep-PAA) was prepared through grafting polyallylamine (PAA) onto polyacrylate macroporous microspheres (FastSep-epoxy). The effects of the synthesis conditions, including the PAA concentration, reaction time, and reaction solution pH, on the ion exchange (IC) of the medium were investigated in detail. When the PAA concentration, reaction time, and reaction solution pH were increased, the IC of the medium increased, and optimal synthesis conditions were then selected in combination with changes of protein binding capacity. A scanning electron microscope was used to examine the surface morphology of the medium. The medium possessed high pore connectivity. Furthermore, the pore structure of the medium was preserved after the grafting of PAA onto the macroporous microspheres. This finding demonstrates that the density of the PAA ligands does not appear to have any discernible impact on the structure of the medium; that is, no difference in the structure of the medium is observed before and after the grafting of PAA onto the microspheres. The pore size and pore-size distribution of the medium before and after grafting were determined by mercury intrusion porosimetry and the nitrogen adsorption method to investigate the relationship between pore size (measured in the range of 300-1000 nm) and protein adsorption. When the pore size of the medium was increased, its protein binding capacity did not exhibit any substantial decrease. An increase in pore size may hasten the mass transfer of proteins within the medium. Among the media prepared, that with a pore size of 400 nm exhibited the highest dynamic-binding capacity (DBC: 70.3 g/L at 126 cm/h). The large specific surface area of the medium and its increased number of protein adsorption sites appeared to positively influence its DBC. When the flow rate was increased, the protein DBC decreased in media with original pore sizes of less than 700 nm. In the case of the medium with an original pore size of 1000 nm, the protein DBC was independent of the flow rate. The protein DBC decreased by 3.5% when the flow rate was increased from 126 to 628 cm/h. In addition, the protein DBC was maintained at 57.7 g/L even when the flow velocity was 628 cm/h. This finding reveals that the diffusion rate of protein molecules at this pore size is less restricted and that the prepared medium has excellent mass-transfer performance. These results confirm that the macroporous polymer anion exchange chromatographic medium developed in this study has great potential for the high-throughput separation of proteins.

Regulation on both pore structure and pressure-resistant property of uniform agarose microspheres for high-resolution chromatography.

How to improve the performance of chromatographic media is very important in chromatography. Uniform agarose microspheres were successfully prepared using membrane emulsification method with a controllable particle size, followed by multi-step crosslinking and dextran-grafting, respectively. To obtain both fine pore structure and good pressure-resistant property, the effects of both dextran-grafting and crosslinking process were studied carefully and also, the preparation conditions were delicately adjusted. Inverse size-exclusion chromatography was used for determining the pore structure of these agarose microspheres. Uniform agarose microspheres with an average particle size of about 8 µm were obtained with regularly spherical, transparent and smooth appearance. By introducing a certain molecular weight of dextran or pentaerythritol glycidyl ether at different crosslinking steps, both the pressure-resistant and the chromatographic properties of microspheres were improved. Both the maximum flow velocity and the corresponding pressure drop increased with the decrease of the molecular weight of dextran, i.e., 99 cm/h and 3.22 MPa, respectively, using dextran T3 (3 kDa). The average pore size of agarose microspheres decreased from 6.04±0.56 nm to 2.50±0.12 nm with the increase of the molecular weight of dextran from dextran T3 (3 kDa) to dextran T100 (100 kDa), with a high resolution obtained for a certain molecular range of model proteins. Also, the pressure-resistant property was highly improved in multi-step crosslinking process, with a maximum flow velocity of 107 cm/h and a corresponding pressure drop of 3.62 MPa obtained after the whole crosslinking steps. The average pore size of agarose microspheres was 3.72±0.32, 3.90±0.21 and 3.60±0.27 nm for the introduction of pentaerythritol glycidyl ether as the crosslinking agent at different steps, respectively. These uniform dextran-grafted agarose microspheres have a finely controllable molecular range with a high resolution compared with traditional ones, which are beneficial for chromatographic selectivity. Therefore, they are very useful for high-resolution chromatography and have wide applications in downstream process.

Biochar-based molybdenum slow-release fertilizer enhances nitrogen assimilation in Chinese flowering cabbage (Brassica parachinensis).

Low molybdenum (Mo) bioavailability in acidic soil obstructs vegetable nitrogen assimilation and thus increases the health risk of vegetable ingestion due to nitrate accumulation. Constantly providing available Mo in acidic soil is a challenge for decreasing nitrate accumulation in vegetables. In this study, three Mo application methods, including biochar-based Mo slow-release fertilizer (Mo-biochar), seed dressing, and basal application, were investigated to enhance Mo bioavailability in acidic soil and nitrogen assimilation in Chinese flowering cabbage (Brassica parachinensis). The results showed that Mo-biochar constantly and sufficiently supplied Mo nutrients throughout the growing period of Brassica parachinensis, as evidenced by the soil available Mo, plant Mo uptake, and Mo values. The improved Mo supply was attributed to the alleviation of acidic soil (pH from 5.10 to 6.99) and the slow release of Mo adsorbed on biochar. Mo-biochar increased the nitrate reductase (NR) activity by 238.6% and glutamate dehydrogenase activity by 27.5%, indicating an enhancement of the rate-limiting steps of nitrogen assimilation, especially for nitrate reduction and amino acid synthesis. The increase in Mo-containing NR could be directly ascribed to the high level of Mo in Brassica parachinensis. Compared with the control, the nitrate content of Brassica parachinensis decreased by 42.9% due to the nitrate reduction induced by increased NR. Additionally, Mo-biochar was beneficial to vegetable growth and quality. In contrast, the transformation from NO3- to NH4+ was blocked with Mo seed dressing and basal application because of low Mo bioavailability in the soil, resulting in a high nitrate content in Brassica parachinensis. Conclusively, Mo-biochar can slowly release Mo and improve the neutral environment for Mo bioavailability, which is an effective strategy to mitigate the high nitrate accumulation of vegetables planted in acidic soil.

Effects of Microplastics on the Mineral Elements Absorption and Accumulation in Hydroponic Rice Seedlings (Oryza sativa L.).

Environmental pollution caused by microplastics (MPs) have become a non-negligible environmental problem and come into our notice recently. Herein, the nutrient elemental signature (ionome) of rice seedlings exposed to four levels of polyethylene microplastics (PE-MPs), and was analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES) or mass spectroscopy (ICP-MS) to determine the relationship between ionome and MPs. After 21 days of laboratory exposure, no shoots growth difference has shown among any doses of PE-MPs treatments, however, the roots growth was significantly inhibited after the medium and high doses of PE-MPs treatments. Further analysis showed that PE-MPs treatments could decrease the accumulation of one essential mineral element Mn and some non-essential mineral elements accumulation including As and Cd, while increase the accumulation of one essential mineral elements including Na in rice seedlings. This study is the first to document the variation of the rice seedlings ionome after exposing microplastics.

Tailored nanodisc immobilization for one-step purification and reconstitution of cytochrome P450: A tool for membrane proteins' hard cases.

A novel nanodisc-based immobilization method was developed for high-efficient purification and reconstitution of cytochrome P450 in one step. Using membrane scaffold protein containing a histidine tag, charged-nanodiscs were prepared in the form of self-assembly of lipid-protein nanoparticles. Their properties including the particle diameter and its distribution and Zeta potential were controlled well by adjusting molar ratios of phospholipids to membrane scaffold protein. At an optimum lipid-to-membrane scaffold protein molar ratio of 60:1, uniformly regular-shaped and discoidal nanodiscs with an average particle diameter of 10 nm and Zeta potential of -19 mV were obtained. They can be well fractionated by size exclusion chromatography. Charged-nanodiscs were successfully immobilized onto Ni-chelating microspheres via histidine tags with a density of 6.6 mg membrane scaffold protein/mL gel. After being packed in a column, chromatography studies demonstrated that this nanodisc-immobilized chromatographic medium had a specific binding to cytochrome P450 in rat liver microsome. Nanodiscs containing cytochrome P450 can be furthermore eluted from the column with a diameter of about 87.0 nm and height of about 8.0 nm, respectively. The purity of cytochrome P450 after purification increased 25 folds strikingly. This nanodisc-immobilized chromatography method is promising for the one-step purification and reconstitution of membrane protein.

Rapid and high-capacity loading of IgG monoclonal antibodies by polymer brush and peptides functionalized microspheres.

Fast-throughput and cost reduction of current purification platforms are becoming increasing requests during antibody manufacture. The macroporous-matrix absorbents have presented extensive potentiality in improving operational throughput during purification of macromolecule. And meanwhile the peptide ligand has become a promising alternative to recombinant protein ligands for cost reduction of chromatographic purification. Therefore, here we designed a functionalized microspheres resin with both macroporous matrix of polymerized glycidyl methacrylate and ethylene glycol dimethacrylate (PGMA-EDMA) and peptide ligand of hexapeptide (FYEILH). In order to circumvent the steric effect of peptides and amplify the binding sites on macroporous matrix, the peptide ligand was coupled on a liner PGMA polymer brushes grafted on microspheres. Comparing to the conventional agarose-matrix resin and the general peptide-grafted microspheres, the functionalized microspheres presented excellent permeability and high capacity to rapid loading hIgG by maintaining a stable level of dynamic binding capacity at fast flow rate above 110 column volume per hour (cv/h) and very short residence time below 0.5 min. Such functionalized microspheres provide a facile and broadly applicable strategy to develop the attractive candidate for rapid and cost-reduced purification of antibody.

Manipulation of pore structure during manufacture of agarose microspheres for bioseparation.

Agarose microspheres with a controllable pore structure were manufactured by varying agarose types and crosslinking degrees. Various agarose could tailor the gel formation of microspheres matrix and thus affect the final pore structures. Small pores in microspheres could be fabricated by agarose with a higher molecular weight, which was demonstrated by the packed column with lower distribution coefficient (Kav ) values measured by gel filtration chromatography. Further, higher Kav values also demonstrated that more and larger pores were formed with increasing the crosslinking degree of agarose microspheres. Either using agarose with a high molecular weight or increasing the crosslinking degree would finally lead to the enhancement of the flow rate during flow performance of packed column as necessary for improving separation efficiency. This provides a foundation for high-resolution chromatography with a controllable separation range as beneficial for downstream process.

Nano-FeS incorporated into stable lignin hydrogel: A novel strategy for cadmium removal from soil.

Strategies for reducing cadmium (Cd) content in polluted farmland soils are currently limited. A type of composite with nanoparticles incorporated into a hydrogel have been developed to efficiently remove heavy metals from sewage, but their application in soils faces challenges, such as organic hydrogel degradation due to oxygen exposure and slow Cd2+ release from soil constituents. To overcome these challenges, a composite with superior stability for long-term application in soil is required. In this study, ferrous sulfide (FeS) nanoparticle@lignin hydrogel composites were developed. The lignin-based hydrogels inherited lignin's natural mechanical and environmental stability and the FeS nanoparticles efficiently adsorbed Cd2+ and enhanced Cd2+ desorption from soils by producing H+. The high sorption capacity (833.3 g kg-1) of the composite was attributed to four proposed mechanisms, including cadmium sulfide (CdS) precipitation via chemical reaction (84.06%), lignin complexation (13.19%), hydrogel swelling (0.61%), and nanoparticle sorption (2.15%). In addition, Fe2+ displaced from the composite was gradually oxidized to form solid iron oxide hydroxide, which increased Cd2+ sorption. The composite significantly reduced the total, surfactant-soluble, and fixed Cd in heavily and lightly polluted paddy soils by 22.4-49.6%, 13.5-68.6%, and 40.1-16.6%, respectively, in 7 days.

Fabrication of rigid and macroporous agarose microspheres by pre-cross-linking and surfactant micelles swelling method.

A novel combined method of pre-cross-linking and surfactant micelles swelling was proposed in this study to fabricate highly cross-linked and macroporous agarose (HMA) microspheres. Agarose was chemically modified by allylglycidyl ether (AGE) as heterobifunctional cross-linker via its active glycidyl moieties before gel formation and pre-cross-linking was achieved. By this means, the effective concentration of cross-linker presented in agarose gel increased significantly, and thus cross-linking with a high-efficiency was achieved. Further to enhance the intraparticle mass transfer of agarose microspheres, the surfactant micelles swelling method was utilized to create interconnected macropores. Under the optimal condition, HMA microspheres with homogeneous reticular structure and pore size of hundreds nanometers were successfully prepared. They exhibited a low backpressure with a flow velocity as high as 1987 cm/h, which was much higher than that of commercial Sepharose 4 F F. HMA microspheres were then derivatized with carboxymethyl (CM) groups and applied in ion-exchange chromatography. As expected, CM-HMA column separated model proteins effectively even at a flow velocity three times higher than that of commercial CM-4 F F. Visualization of dynamic protein adsorption by confocal laser scanning microscope (CLSM) revealed that the intraparticle mass transfer of CM-HMA microspheres was intensified due to its macroporous structure. All of the results indicated the newly developed agarose microspheres were a promising medium for high-speed chromatography.

A novel rProtein A chromatographic media for enhancing cleaning-in-place performance.

Protein A chromatography has been a popular method for purification of therapeutic monoclonal antibodies (mAb). Protein A chromatographic media using alkali-resistant rProtein A ligands from site-directed coupling method have been pursued for both high dynamic capacities and excellent stabilities. However, the mechanism of rProtein A leaking under cleaning-in-place (CIP) conditions is not very clear and difficulties have been commonly encountered when improving the media's chromatographic performance. We investigated the chromatographic performance of site-directed coupled rProtein A chromatographic media during CIP procedure. Trace amount of ligands leaked during the chromatographic media's incubation in 0.5 M NaOH was detected, explaining for the decline of chromatographic media's CIP performance. Decrease of rProtein A's concentration in 0.5 M NaOH was consistent with chromatographic media's binding capacity. A novel rProtein A chromatographic media were prepared by site-directed coupling a newly-constructed alkali-resistant rProtein A to highly cross-linked agarose-based matrix. The media had a dynamic binding capacity of 63.2 mg hIgG/mL higher than 48.1 mg hIgG/mL of the commercial one, and the CIP performance was improved greatly with the remained dynamic binding capacity increased from 86% to 95% of the initial value after 40 CIP cycles.

Mechanisms of rice straw biochar effects on phosphorus sorption characteristics of acid upland red soils.

An important pathway for biochar to alter the availability of soil phosphorus (P) is to change P sorption characteristics of the soil. The aim of this study was to understand the mechanisms of biochar effects on P sorption in acid upland red soils in the presence of different concentrations of exogenous P. Rice straw biochar (RSB) was prepared and applied at rates of 0, 1%, 3%, and 5% (w/w) to three red soils (MZ1, MZ2, and QY1) differing in initial pH (pH = 4.31, 4.82, and 5.68, respectively). The P sorption characteristics of these red soils were described using the Langmuir and Temkin equations and their relationships with soil basic physicochemical properties were analyzed. Furthermore, a representative red soil (MZ2) was selected to analyze the zeta potential of soil colloids and the chemical properties of sorption equilibrium solution, in order to understand their relationships with P sorption characteristics. Results showed that within a certain range of P concentration in the equilibrium solution, the amount of P sorbed by the three red soils decreased and the corresponding amount of P desorbed increased with increasing amendment rate of RSB. RSB showed the greatest effect on P desorption characteristics of MZ2 soil in the presence of higher exogenous P concentration. With increasing RSB amendment rate, the maximum P sorption of MZ1 soil decreased, while those of MZ2 and QY1 soils increased after an initial decrease. Phosphate sorption equilibrium constant and maximum P buffer capacity of each soil first increased and then decreased. However, a single physicochemical property could not interpret complex changes in multi-factors that jointly determine the P sorption characteristics of red soils. In the case of MZ2 soil, RSB amendment shifted the zeta potential of soil colloids to the negative direction; this decreased the positive charge and increased the negative charge on the soil surface, thus reducing P sorption in the MZ2 soil. In the presence of the same concentration of exogenous P, RSB amendment altered the pH, dissolved organic C (DOC), humification index (HIX), and maximum fluorescence intensity (Fmax) in the sorption equilibrium solution. In most cases, the amount of P sorbed by the MZ2 soil was negatively correlated with the pH value, DOC concentration, HIX value, and Fmax value of humic-like dissolved organic matter (DOM), and positively correlated with the Fmax value of protein-like DOM (P < 0.05 or P < 0.01). The relative fractional distribution of the contents for humic-like and protein-like DOM might determine the difference in the P sorption characteristics of MZ2 soil. In conclusion, different amendment rates of RSB affected the release of phosphate from soil surfaces into the solution by altering basic physicochemical and electrochemical properties of red soils and chemical properties of sorption equilibrium solution.

Uniform polysaccharide composite microspheres with controllable network by microporous membrane emulsification technique.

High resolution has been constantly pursued in both preparative and analytical chromatography. Chromatographic media are a key factor during the entire separation process. Tailor-made chromatographic media have gained more attention because of their adjustable structure appropriate for application. Uniform polysaccharide composite microspheres were prepared with a mixture of agarose and dextran solution by membrane emulsification technique for the first time. Their pore structure was deliberately regulated by adjusting both the polysaccharide composition and the molecular weight of dextran. Compared with pure agarose microspheres, polysaccharide composite microspheres had a higher separation resolution and their separation range was controllable. By increasing agarose concentration and decreasing dextran concentration at the same time during the preparation of composite microspheres, the mean pore size increased first and then decreased later, and also the pore size distribution became narrower. By increasing the molecular weight of dextran, the pores became smaller with a narrower pore size distribution. Microspheres with a composition of 10% agarose/2% dextran T40 or 8% agarose/4% dextran T150 showed a higher separation resolution for proteins within range of low molecular weight. Furthermore, the mechanical strength of this composite microsphere was improved by adjusting its composition. Atomic force microscope (AFM) results showed that pores were distributed evenly on both the surface and the inner part of microspheres, beneficial for the passage of biomolecules. These novel uniform polysaccharide composite microspheres have great potential for high-resolution bioseparation. Graphical abstract ᅟ.

Enhanced binding by dextran-grafting to Protein A affinity chromatographic media.

Dextran-grafted Protein A affinity chromatographic medium was prepared by grafting dextran to agarose-based matrix, followed by epoxy-activation and Protein A coupling site-directed to sulfhydryl groups of cysteine molecules. An enhancement of both the binding performance and the stability was achieved for this dextran-grafted Protein A chromatographic medium. Its dynamic binding capacity was 61 mg immunoglobulin G/mL suction-dried gel, increased by 24% compared with that of the non-grafted medium. The binding capacity of dextran-grafted medium decreased about 7% after 40 cleaning-in-place cycles, much lower than that of the non-grafted medium as decreased about 15%. Confocal laser scanning microscopy results showed that immunoglobulin G was bound to both the outside and the inside of dextran-grafted medium faster than that of non-grafted one. Atomic force microscopy showed that this dextran-grafted Protein A medium had much rougher surface with a vertical coordinate range of ±80 nm, while that of non-grafted one was ±10 nm. Grafted dextran provided a more stereo surface morphology and immunoglobulin G molecules were more easily to be bound. This high-performance dextran-grafted Protein A affinity chromatographic medium has promising applications in large-scale antibody purification.

Functional hydrophilic polystyrene beads with uniformly size and high cross-linking degree facilitated rapid separation of exenatide.

A high cross-linking polystyrene(PSt)-based anion-exchange material with uniformly size, high ion exchange capacity, and high hydrophilicity was synthesized by a novel surface functionalization approach in this study. Uniformly sized PSt microspheres were prepared by the membrane emulsion polymerization strategy, and then modified by (1) conversing resid ual surface vinyl groups to epoxy groups followed by quaternization, and (2) decorating aromatic ring matrix including nitration, reduction and attachment of glycidyltrimethylammonium chloride. The 3-D morphology and porous features of microspheres were observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The surface of the modified PSt became roughness but the particle size remained same. Meanwhile, FT-IR spectra and laser scanning confocal microscope (LCSM) indicated that the modification groups had been successfully covalently coated onto the PSt microspheres. Modified PSt microspheres showed greatly improved hydrophilicity and biocompatibility with 0.387mmol/mL ion exchange capacity (IEC). In the application evaluation procedure, exenatide can be purified from 42.9% (peptide crudes) to 88.6% by modified PSt column with 97.1% recovery yield. This modified PSt microspheres had a large potential in application for efficient separation of peptides.

Microscopic insight into role of protein flexibility during ion exchange chromatography by nuclear magnetic resonance and quartz crystal microbalance approaches.

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.

Efficient fabrication of high-capacity immobilized metal ion affinity chromatographic media: The role of the dextran-grafting process and its manipulation.

Novel high-capacity Ni(2+) immobilized metal ion affinity chromatographic media were prepared through the dextran-grafting process. Dextran was grafted to an allyl-activated agarose-based matrix followed by functionalization for the immobilized metal ion affinity chromatographic media. With elaborate regulation of the allylation degree, dextran was completely or partly grafted to agarose microspheres, namely, completely dextran-grafted agarose microspheres and partly dextran-grafted ones, respectively. Confocal laser scanning microscope results demonstrated that a good adjustment of dextran-grafting degree was achieved, and dextran was distributed uniformly in whole completely dextran-grafted microspheres, while just distributed around the outside of the partly dextran-grafted ones. Flow hydrodynamic properties were improved greatly after the dextran-grafting process, and the flow velocity increased by about 30% compared with that of a commercial chromatographic medium (Ni Sepharose FF). A significant improvement of protein binding performance was also achieved by the dextran-grafting process, and partly dextran-grafted Ni(2+) chelating medium had a maximum binding capacity for His-tagged lactate dehydrogenase about 2.5 times higher than that of Ni Sepharose FF. The results indicated that this novel chromatographic medium is promising for applications in high-efficiency and large-scale protein purification.

Quantification of bindings of organometallic ruthenium complexes to GSTπ by mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has been widely used to identify binding sites of metal complexes to proteins. However, the MS quantification of the metal-protein coordination remains a challenge. We have recently demonstrated by ESI-MS analysis that organometallic ruthenium complexes [(η(6)-arene)Ru(en)Cl](+) (arene=p-cymene (1), biphenyl (2) or 9,10-dihydrophenanthrene (3); en=ethylenediamine) bound to human glutathione-S-transferase π (GSTπ) at Cys15 and Cys48 within the G-site, and Cys102 and Met92 on the interface of the GSTπ dimer, showing inhibitory potency against the enzyme (J. Inorg. Biochem., 128 (2013) 77-84). Herein, we developed a mass spectrometric method to quantify the binding stoichiometry of the three complexes to GSTπ. The differences in signal intensities of the heavy-labelled peptides produced by tryptic digestion of the ruthenated GSTπ complexes and the respective light-labelled peptides in the tryptic digest of equimolar GSTπ were used to calculate the binding stoichiometry at specific residues. The results indicated that the pre-complexation of GSTπ with its substrate GSH significantly reduced the bindings of the ruthenium complexes at Met92 and Cys102, but had little impact on the bindings at Cys15 and Cys48. As the inhibitory activities of the ruthenium complexes against GSTπ are similar to those against GSTπ in complexation with GSH, these results suggest that the inhibition of the ruthenium complexes on GSTπ is attributed to the ruthenation at Cys15 and Cys48. The present work provides not only insights into the understanding on the inhibitory mechanism of ruthenium complexes GSTπ, but also a general method for quantitative characterization of metal-protein interactions.

Hydrophilic modification gigaporous resins with poly(ethylenimine) for high-throughput proteins ion-exchange chromatography.

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.

A novel gigaporous GSH affinity medium for high-speed affinity chromatography of GST-tagged proteins.

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 (100-500nm) 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 1.5min at a velocity up to 1400cm/h. GSH-AP medium is promising for high-speed affinity chromatography for the purification of GST and GST-tagged proteins.

[Purification of recombinant human antithrombin III expressed in a goat mammary bioreactor].

Antithrombin III (AT III) is the most important anti-clotting substance. Recombinant human antithrombin III (rhAT III) expressed in transgenic goat milk attracts more and more attention. Develop an effective purification route for rhAT III is vital to its industrial production. An efficient purification method was developed for the rapid purification of rhAT III by isoelectric precipitation and heparin affinity chromatography. First, casein was effectively removed by isoelectric precipitation. rhAT III was further purified by heparin affinity chromatography. In the process of heparin affinity chromatography, the effects of pH and temperature on the stability of rhAT III were studied, and the effects of operating conditions, elution gradient, flow rate and sample loaded, on the purification efficiency were also studied. Under the optimized conditions, the protein recovery of rhAT III was about 90% with purity over 99%, while its activity recovery was about 50%. Such a purification process is very simple and effective, and it would provide a valuable reference for the further scaling-up of industrial production.