Comparison of chromatographic ion-exchange resins IV. Strong and weak cation-exchange resins and heparin resins

A comparative study was performed on heparin resins and strong and weak cation exchangers to investigate the pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and scanning electron microscopy pictures of chromatographic resins. The resins tested include: Heparin Sepharose FF, SP Sepharose FF, CM Sepharose FF, Heparin Toyopearl 650 m, SP Toyopearl 650 m, CM Toyopearl 650 m, Ceramic Heparin HyperD M, Ceramic S HyperD 20, and Ceramic CM HyperD F. Testing was performed with four different proteins: anti-FVII Mab (IgG), aprotinin, lysozyme, and myoglobin. Dependence of pH on retention was generally very low for proteins with high isoelectric point (pI), though some decrease of retention with increasing pH was observed for CM Ceramic HyperD F and S Ceramic HyperD 20. Binding of anti-FVII Mab with pI < 7.5 was observed on several resins at pH 7.5. Efficiency results show the expected trend of increasing dependence of the plate height with increasing flow rate of Ceramic HyperD resins followed by Toyopearl 650 m resins and the highest flow dependence of the Sepharose FF resins corresponding to their pressure resistance. Determination of particle size distribution by two independent methods, coulter counting and SEM, was in good agreement. Binding strength of cation-exchange resins as a function of ionic strength varies depending on the protein. Binding and elution at high salt concentration may be performed with Ceramic HyperD resins, while binding and elution at low salt concentration may be performed with model proteins on heparin resins. Employing proteins with specific affinity for heparin, a much stronger binding is observed, however, some cation exchangers may still be good substitutions for heparin resins. Dynamic capacity at 10% breakthrough compared to static capacity measurements and dynamic capacity displays that approximately 40-80% of the total available capacity is utilized during chromatographic operation depending on flow rate. A general good agreement was obtained between results of this study and data obtained by others. Results of this study may be used in the selection of resins for testing during protein purification process development.     

Comparison of chromatographic ion-exchange resins. III. Strong cation-exchange resins

A comparative study was performed on strong cation-exchangers to investigate the pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and SEM pictures of chromatographic resins. The resins tested included: SP Sepharose XL, Poros 50 HS, Toyopearl SP 550c, SP Sepharose BB, Source 30S, TSKGel SP-5PW-HR20, and Toyopearl SP 650c. Testing was performed with four different proteins: anti-FVII Mab (IgG), aprotinin, lysozyme, and myoglobin. Dependence of pH on retention was generally very low for proteins with high pI. An unexpected binding at pH 7.5 of anti-FVII Mab with pI < 7.5 was observed on several resins. Efficiency results show the expected trend of higher dependence of the plate height with increasing flow rate of soft resins compared to resins for medium and high-pressure operation. Determination of particle size distribution by two independent methods, Coulter counting and SEM, was in very good agreement. The mono-dispersed nature of Source 30S was confirmed. Binding to cation-exchange resins as a function of ionic strength varies depending on the specific protein. Generally, binding and elution at high salt concentration may be performed with Toyopearl SP 550c and Poros 50 HS, while binding and elution at low salt concentration may be performed with Toyopearl SP 650c. A very high binding capacity was obtained with SP Sepharose XL. Comparison of static capacity and dynamic capacity at 10% break-through shows in general approximately 50-80% utilisation of the total available capacity during chromatographic operation. A general good agreement was obtained between this study and data obtained by others. The results of this study may be used for selection of resins for testing in process development. The validity of experiments and results with model proteins were tested using human insulin precursor in pure state and in real feed-stock on Toyopearl SP 550c, SP Sepharose BB, and Toyopearl SP 650c. Results showed good agreement with experiments with model proteins.     

Comparison of chromatographic ion-exchange resins. II. More strong anion-exchange resins

A comparative study was performed on strong anion exchangers to investigate the pH dependence, titration curves, efficiency, binding strength, particle size distribution, and static and dynamic capacity of the chromatographic resins. The resins tested included Q Sepharose XL, UNO Q-1, Poros 50 HQ, Toyopearl QAE 550c, Separon HemaBio 1000Q, Q-Cellthru Bigbeads Plus, Q Sepharose HP and Toyopearl SuperQ 650s. Testing was performed with five different proteins: anti-Factor VII monoclonal antibody (immunoglobulin G), aprotinin, bovine serum albumin, lipolase and myoglobin. The dependence of pH on retention varies from generally low to very high for proteins with a low isoelectric point (pl). An unexpected binding at pH 7-8 of aprotinin with pI >11 was observed on Separon HemaBio 1000Q. No link between pH dependence on retention and titration curves of the different resins was observed. Efficiency results show the expected trend of higher dependence of the plate height with increasing flow-rate of soft resins compared to resins for medium- and high-pressure operation. No or a very small difference in particle size distribution was obtained between new and used resins. Binding to anion-exchange resins as a function of ionic strength varies to some extent depending on the specific protein. Generally, binding and elution at high salt concentration may be performed with Q Sepharose XL, Toyopearl QAE 550c, Q Sepharose HP and Poros 50 HQ, while binding and elution at low salt concentration may be performed with Q-Cellthru Bigbeads Plus. A very high binding capacity was obtained with Q Sepharose XL. Comparison of static capacity and dynamic capacity at 10% breakthrough shows approx. 50-80% utilization of the total available capacity during chromatographic operation. A general good agreement was obtained between this study and data obtained by the suppliers. The results of this study may be used for selection of resins for testing in process development.     

Comparison of chromatographic ion-exchange resins. I. Strong anion-exchange resins

A comparative study has been undertaken on various strong anion-exchangers to investigate the pH dependence, titration curves, efficiency, binding strength, and dynamic capacity of the chromatographic resins. The resins tested included: Macro-Prep 25Q, TSK-Gel Q-5PW-HR, Poros QE/M, Q Sepharose FF, Q HyperD 20, Q Zirconia, Source 30Q, Fractogel EMD TMAE 650s, and Express-Ion Q. Testing was performed with five different proteins: Anti-FVII Mab (IgG), aprotinin, BSA, lipolase, and myoglobin. The dependence of pH on retention varies from generally low to very high for proteins with low pI. No direct link between pH dependence on retention and titration curves of the different resins was observed. Efficiency results show the expected trend of lower dependence of the plate height with increasing flow-rate of resins for medium and high pressure operation compared to the soft resins. Binding to the anion-exchange resins as a function of ionic strength may vary depending on the specific protein. Generally, binding and elution at a high salt concentration may be performed with Poros QE/M or Macro-Prep 25Q, while binding and elution at low salt concentration may be done with TSK-Gel Q-5PW. Dynamic capacities are strongly dependent on the specific protein employed and for some resins dependent on the flow-rate. A general good agreement was obtained between this study and data obtained by suppliers for the dynamic capacity. The results of this study may be used for selection of resins for testing in process development, however, the data does not tell anything about specific selectivity differences or resolution between a target protein and a given impurity. None of the resins studied here should be regarded as good or bad, but more or less suitable for a specific purpose, and only testing for the specific application will determine which one is the optimal resin.     

Comparison of chromatographic ion-exchange resins V. Strong and weak cation-exchange resins

Strong and weak cation-exchangers were compared for a number of chromatographic parameters, i.e. pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and scanning electron microscopy (SEM) pictures. Chromatographic resins investigated were Fractogel EMD SO3- (M), Fractogel EMD SE Hicap (M), Fractogel EMD COO- (M), MacroPrep 25S, MacroPrep High S, MacroPrep CM, CM HyperZ, and Matrex Cellufine C-500. Testing was done with three proteins: Anti-FVII Mab (IgG), aprotinin, and lysozyme. For lysozyme and aprotinin with pI above experimental pH, dependence of pH on retention was generally low, though some pronounced decrease of retention with increasing pH was observed for CM HyperZ. For Anti-FVII Mab with pI<7.5, binding was observed on several resins at pH 7.5. Efficiency results present the expected trend of increasing dependence of plate height as a function of increasing flow rate, and the highest flow dependence was observed for Fractogel EMD COO-. Particle size distribution was determined by two independent methods, coulter counting and SEM pictures, with fair agreement. Binding strength data of cation-exchange resins as a function of ionic strength depends on the protein, but binding and elution at high salt concentration may in general be performed with MacroPrep resins. Comparison of dynamic capacity data at 10% break-through and static capacity measurements shows that a very diverse utilization of approximately 25-90% of the total available capacity is employed during chromatographic operation. The effect of competitive binding from yeast fermentation components on dynamic binding capacity of aprotinin was studied showing a significant decrease in binding capacity. Sepharose FF, Toyopearl 650 M, and Ceramic HyperD F strong and weak cation-exchange resins were included in this study. Resins with good pure aprotinin capacity also performed well for aprotinin in fermentation broth, but the highest relative capacity was obtained with MacroPrep High S having a fairly low pure component dynamic capacity. Results of this paper may be used in the selection of resins for further testing in biopharmaceutical protein purification process development.     

Comparison between mass transfer properties of weak-anion-exchange resins with graft-functionalized polymer layers and traditional ungrafted resins

Glycidylmethacrylate was grafted to Toyopearl HW-65M and subsequently modified with diethylamine to obtain a weak anion exchanger. The degree of grafting was varied from 11 to 50%. The binding capacity for bovine serum albumin was 11 mg/ml for the lowest degree of grafting and 97 mg/ml for the highest degree of grafting. The maximum binding capacity was observed at 27% degree of grafting. The mass transfer properties of the grafted resins and an ungrafted resin(Toyopearl DEAE 650M) were investigated assuming rectangular isotherms. Simple models for reaction kinetics, pore- and surface diffusion and film diffusion were used to describe the concentration-time data in batch mode. The data were best fitted by a pore diffusion model. The estimated pore diffusion coefficients (D(P)) for bovine serum albumin were fitted by a polynome to the degree of grafting with an maximum value at 27% of D(P) = 1.95-10(-11) m2/s. Compared to published data of other ungrafted resins and to the molecular diffusion coefficient of bovine serum albumin in free solution of D(P) = 5.6 10(-11) m2/s, the diffusion in grafted layers seems to be accelerated. The breakthrough curves for columns packed with various resins showed a decrease in sharpness with increasing degree of grafting which could not be described by a simple pore diffusion model using the calculated transport coefficients from batch mode. The shape of the breakthrough curves could be well described by a combined film and pore diffusion model. For the ungrafted Toyopearl DEAE 650M resin the breakthrough curve is more favorable and the influence of film diffusion to the mass transfer is reduced. It can be concluded that grafting will increase the capacity and the pore diffusion in batch mode but in column operation the grafting layer has a film resistance which plays an important role in the overall mass transfer.     

Impact of ionic strength on adsorption capacity of chromatographic particles employed in separation of monoclonal antibodies

The influence of ionic strength on the adsorption capacity of seven commercial adsorbents used in downstream processing of monoclonal antibodies was examined. Affinity (MabSelect, Poros 50A High Capacity, ProSep-vA High Capacity), hydrophobic charge-induction (MEP HyperCel), and cation exchange adsorbents (FractoGel EMD SE Hicap (M), SP Sepharose Fast Flow, Ceramic HyperD F) were used to study the adsorption of polyclonal human immunoglobulin G at optimal pH values. The ionic strength, adjusted by sodium chloride concentrations in the range of 0–225 mM, strongly decreased the adsorption capacity of the cation exchangers. Equilibrium data were described in the form of the dependence of the ratio of protein concentrations in the solid and liquid phases on the total concentration of cation counter ions. They were successfully fitted and interpreted through a stoichiometric ion-exchange model.     

Preparation and characterization of prototypes for multi-modal separation media aimed for capture of negatively charged biomolecules at high salt conditions

Several prototypes of multi-modal ligands suitable for the capture of negatively charged proteins from high conductivity (28 mS/cm) mobile phases were coupled to Sepharose 6 Fast Flow. These new prototypes of multi-modal anion-exchangers were found by screening a diverse library of multi-modal ligands and selecting anion-exchangers resulting in elution of test proteins at high ionic strength. Candidates were then tested with respect to breakthrough capacity of BSA in a buffer adjusted to a high conductivity (20 mM Piperazine and 0.25 M NaCl, pH 6.0). The recovery of BSA was also tested with a salt step (from 0.25 to 2.0 M NaCl using 20 mM Piperazine as buffer, pH 6.0) or with a pH-step to pH 4.0. We have found that non-aromatic multi-modal anion-exchange ligands based on primary or secondary amines (or both) are optimal for the capture of proteins at high salt conditions. Furthermore, these new multi-modal anion-exchange ligands have been designed to take advantage not only of electrostatic but also hydrogen bond interactions. This has been accomplished through modification of the ligands by the introduction of hydroxyl groups in the proximity of the ionic group. Experimental evidence on the importance of the relative position of the hydroxyl groups on the ligand in order to improve the breakthrough capacity of BSA has been found. Compared to strong anion-exchangers such as Q Sepharose Fast Flow the new multi-modal weak anion-exchangers have breakthrough capacities of BSA at mobile phases of 28 mS/cm and pH 6.0 that are 20-30 times higher. The new multi-modal anion-exchangers can also be used at normal anion-exchange conditions and with either a salt step or a pH-step to acidic pH can accomplish the elution of proteins. In addition, the functional performance of the new anion-exchangers was found to be intact after treatment in 1.0 M sodium hydroxide solution for 1 week. A number of multi-modal anion-exchange ligands based on aromatic amines exhibiting high breakthrough capacity of BSA have been found. With these ligands recovery was often found to be low due to strong non-electrostatic interactions. However, for phenol derived anion-exchange media the recovery can be improved by desorption at high pH.     

P450 monooxygenase in biotechnology. I. Single-step, large-scale purification method for cytochrome P450 BM-3 by anion-exchange chromatography.

An efficient single-step purification protocol for recombinant cytochrome P450 BM-3 from Bacillus megaterium, expressed in E. coli, was developed. Functional crude protein was obtained by disintegrating induced E. coli DH5 alpha and removing cell debris by centrifugation. After investigating different anion-exchange matrices, elution salts and the elution procedures involving an AKTAexplorer system, adsorption of the crude extract from lysed E. coli to Toyopearl DEAE 650M anion exchanger, followed by a two-step elution using NaCl, proved sufficient to isolate almost pure protein without inactivation (up to 93% P450 BM-3 content) in yields that ranged between 79-86%. The purification method could be scaled up 1500-fold and higher without further optimization to a 6-1 production-scale column containing Toyopearl DEAE 650M anion exchanger.     

Matrix-assisted refolding of autoprotease fusion proteins on an ion exchange column

Refolding of proteins must be performed under very dilute conditions to overcome the competing aggregation reaction, which has a high reaction order. Refolding on a chromatography column partially prevents formation of the intermediate form prone to aggregation. A chromatographic refolding procedure was developed using an autoprotease fusion protein with the mutant EDDIE from the N^pro autoprotease of pestivirus. Upon refolding, self-cleavage generates a target peptide with an authentic N-terminus. The refolding process was developed using the basic 1.8-kDa peptide sSNEVi-C fused to the autoprotease EDDIE or the acidic peptide pep6His, applying cation and anion exchange chromatography, respectively. Dissolved inclusion bodies were loaded on cation exchange chromatographic resins (Capto S, POROS HS, Fractogel EMD SO₃⁻, UNOsphere S, SP Sepharose FF, CM Sepharose FF, S Ceramic HyperD F, Toyopearl SP-650, and Toyopearl MegaCap II SP-550EC). A conditioning step was introduced in order to reduce the urea concentration prior to the refolding step. Refolding was initiated by applying an elution buffer containing a high concentration of Tris–HCl plus common refolding additives. The actual refolding process occurred concurrently with the elution step and was completed in the collected fraction. With Capto S, POROS HS, and Fractogel SO₃⁻, refolding could be performed at column loadings of 50 mg fusion protein/ml gel, resulting in a final eluate concentration of around 10–15 mg/ml, with refolding and cleavage step yields of around 75%. The overall yield of recovered peptide reached 50%. Similar yields were obtained using the anion exchange system and the pep6His fusion peptide. This chromatographic refolding process allows processing of fusion peptides at a concentration range 10- to 100-fold higher than that observed for common refolding systems.     

Temperature influence on the dynamic binding capacity of a monolithic ion-exchange column

This work investigates the influence of temperature on the binding capacity of bovine serum albumin (BSA), soybean trypsin inhibitor and L-glutamic acid to a CIM (DEAE) weak anion-exchange disk monolithic column. The binding capacity was determined experimentally under dynamic conditions using frontal analysis. The effect on the dynamic binding capacity of dimers present in the BSA solution has been evaluated and a closed-loop frontal analysis was used to determine the equilibrium binding capacities. The binding capacity for both BSA and soybean trypsin inhibitor increased with increasing temperature. In the case of L-glutamic acid, an increase in the binding capacity was observed with temperature up to 20 degrees C. A further increase in temperature caused a decrease of the dynamic binding capacity.     

Separation of aromatic compounds on pellicular anion-exchange resins

Aromatic compounds including phenols, carboxylic acids and aldehydes, can be rapidly separated on pellicular anion-exchange resins in the acetate form by elution with a sodium acetate solution. Like the aromatic carboxylic acids, hydroxybenzaldehydes are held as counter ions in the resin. The affinity of the resin for corresponding anions as well as that for sorbed non-electrolytes is largely influenced by the aromatic ring and non-polar aliphatic substituents. Formation of hydrogen bonds between the resin and phenolic groups is less important.     

Stationary phase effects on the dynamic affinity of low-molecular-mass displacers

In this paper, the selectivity of a variety of cation-exchange stationary phases was investigated using a homologous series of displacer molecules based on pentaerythritol. These displacers were derived from pentaerythritol and contained either four trimethyl ammonium groups [pentaerythrityl-(trimethylammonium chloride)4, PE(TMA)4], benzene rings [pentaerythrityl-(benzyl dimethylammonium chloride)4, PE(DMABzCl)4], heptyl groups [pentaerythrityl-(heptyl dimethylammonium iodide)4, PE(DMAHepI)4] or cyclohexyl groups [pentaerythrityl-(cyclohexyl dimethylammonium iodide)4, PE(DMACyI)4]. This series enabled us to probe the secondary interactions that can play a role in the affinity of low-molecular-mass displacers for different stationary phases. The relative affinities of these displacers were examined using a displacer ranking plot based on the steric mass action (SMA) isotherm model. While hydrophobicity and aromaticity played important roles in generating the affinity to the hydrophilized polystyrene-divinylbenzene (Source 15S) and polymethacrylate-based (Toyopearl 650M) resins, these secondary interactions had a minimal impact on the selectivity in agarose resins coated with dextran (SP Sepharose XL), "gel in a shell" (S Ceramic HyperD F), and monolithic (Bio-Rad Uno S6) cation-exchange materials. Further, the results with a tentacular stationary phase (Fractogel EMD) suggest that the alkyl chains on PE(DMAHepI)4 play an important role in increasing the affinity, possibly because of strong interactions between the alkyl moiety and the polymer matrix as well as between the charged groups and the polyelectrolyte tentacles. The results of this study provide insight into the design of high affinity, low-molecular-mass displacers for different cation-exchange stationary phase materials.     

Analytical and preparative separation of PEGylated lysozyme for the characterization of chromatography media

The effect of PEGylation on cation exchange chromatography was studied with poly(ethylene glycol) of different chain lengths (5 kDa, 10 kDa and 30 kDa) using lysozyme as a model system. A stable binding via reduction of a Schiff base was formed during random PEGylation on lysine residues with methoxy-PEG-aldehyde. A purification method for PEGylated proteins using cation exchange chromatography was developed, and different isoforms of mono-PEGylated lysozyme were isolated. TSKgel SP-5PW and Toyopearl GigaCap S-650M showed the best performance of all tested cation exchange resins, and the separation of PEGylated lysozyme could be also scaled up to semi-preparative level. Size-exclusion chromatography, SDS-PAGE and MALDI-TOF mass spectrometry were used for analysis. Separated mono-PEGylated lysozyme of different sizes was used to determine dynamic binding capacities (DBC) and selectivity of cation exchange chromatography resins. An optimization of binding conditions resulted in a more than 20-fold increase of DBC for Toyopearl GigaCap S-650M with 30 kDa mono-PEGylated lysozyme.     

Chelate-forming resins prepared by modification of anion-exchange resins

The properties of some chelate-forming resins prepared from common anion-exchange resins by treatment with reagents bearing chelate-forming and ion-exchange groups have been studied. A resin prepared from the sulphonic acid derivative of dithizone (DzS) was found to be superior to other chelate-forming resins. Resins loaded with DzS, tetraphenylporphinetrisulphonic acid or zincon were stable in 1M sodium chloride. Resins prepared from sulphonazo III, arsenazo III, thiosalicylic acid or p-mercaptobenzenesulphonic acid were found to be unstable when exposed to sodium chloride solution.     

Preparation of polymeric monoliths by copolymerization of acrylate monomers with amine functionalities for anion-exchange capillary liquid chromatography of proteins

Two novel polymeric monoliths for anion-exchange capillary liquid chromatography of proteins were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion-exchanger and quaternary amine as a strong anion-exchanger, respectively. An alternative weak anion-exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. Important physical and chromatographic properties of the synthesized monoliths were characterized. The dynamic binding capacities of the three monoliths (24 mg/mL, 56 mg/mL and 32 mg/mL of column volume, respectively) were comparable or superior to values that have been reported for various other monoliths. Chromatographic performance was also similar to that provided by a modified poly(GMA-ethylene glycol dimethacrylate) monolith. Separation of standard proteins was achieved under gradient elution conditions using these monolithic columns. Peak capacities of 34, 58 and 36 proteins were obtained with analysis times of 20–30 min. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, additional surface modifications were avoided and the PEGDA crosslinker helped to improve the biocompatibility of the monolithic backbone.     

Affinity resins for the purification of opioid-binding materials

Two new affinity resins for the purification of opioid-binding materials were prepared. One was AH-Sepharose coupled with [D-Ala2, D-Leu5]enkephalin and the other was AF-Amino Toyopearl with [D-Ala2, Leu5]enkephalin. Solubilized-opioid receptors from rat brain were treated with these affinity resins and the materials with opioid-binding activities were purified. On sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the purified materials showed one major band with a molecular weight of 62000-64000. The results suggested that the prepared resins are useful tools for the purification of opioid receptors.     

Isolating the whole complex of target proteins of FK506 using affinity resins from novel solid phases

The development of novel solid phases enabled us to create affinity resins that could be used to isolate the whole complex of target proteins responsible for the immunosuppressive effects of FK506 from rat brain lysate, whereas the affinity resins from commercially available matrices could not achieve this isolation. The results illustrate the enhanced effectiveness of the affinity resin made from this novel material at identifying the target protein of the bioactive compound compared to resins made from the well-known materials Affigel or Toyopearl. This effectiveness arises because the novel material is hydrophilic enough to reduce nonspecific binding proteins and because it has a higher density of ligands that capture the nonubiquitous target protein. Electronic Supplementary Material Supplementary material is available for this article at     

Mixed-matrix membrane adsorbers for protein separation

The separation of two similarly sized proteins, bovine serum albumin (BSA) and bovine hemoglobin (Hb) was carried out using a new type of ion-exchange mixed-matrix adsorber membranes. The adsorber membranes were prepared by incorporation of various types of Lewatit ion-exchange resins into an ethylene-vinyl alcohol copolymer porous structure. The obtained heterogeneous matrices, composed of solid particles surrounded by the polymeric film, display high static and dynamic protein adsorption capacities. The effect of operational parameters such as filtration flow-rate, pH, and ionic strength on the protein separation performances was investigated for cation- as well as anion-exchange adsorber membranes. An average separation factor was calculated by numerical integration of the protein concentration in the permeate curve during the filtration run. High average separation factor values were obtained for BSA-Hb separation at physiological ionic strength with a filtration flow-rate up to 20 1/h per m2, until the protein breakthrough point at 10% of the feed concentration.     

Sinopak-s-DEAE高效弱阴离子交换色谱填料的研究

以多孔硅胶为基质,用改进的合成方法制备了Sinopak-s-DEAE高效弱阴离子交换色谱填。考察了反应条件对填料合成的影响,并以标准蛋白为样品进行了色谱行为的研究,结果表明：所制备的填料对蛋白质的分离性能良好,且对蛋白质的非特异性吸附小。