Optimization Of Synthesis Of Recombinant Protein A Adsorbent And Evaluation Of Their Properties

BackgroundNatural staphylococcus protein A (Staphylococcal protein, SPA) is a kind of cell protein that found in most of Staphylococcal protein A. The amino terminal of SPA, composed of five highly homologous domains, has the ability to bind the Fcγ fragment of IgG molecule of humans (hIgG) and other mammals without interrupting its antigen-ability, and the carboxyl terminal of it can cross-linked to agarose gel or dextran gel to make SPA affinity packing. This attachment between SPA and hIgG is reversible, when the PH values are within the normal physiological range, the hIgG attached to SPA closely, and when the PH values are reduced to2.3～2.5, the coupling is loose and the hIgG can be washed off. When the PH values revert back to normal physiological range, the SPA can be regenerated for reuse. With this special immunological characteristics, staphylococcus protein A adsorbent has been widely used in immunoadsorption therapy and obtained the good clinical treatment result. As ligand, SPA was immobilized to the solid phase carrier and achieved the purpose of purifying blood by selectively removing pathogenic antibodies in the blood. Recently, industrialization production of rSPA adsorbent has achieved abroad, Immunosorba and Prosorba adsorption column are the most commonly used, that were certificated by commission (FDA), for the treatment of idiopathic thrombocytopenic purpura, systemic lupus erythematosus, rheumatoid arthritis and hemophilia with inhibiting factor[4-6], however, the domestic level of synthesis and purification of rSPA falls behind the developed countries. The application of the staphylococcus protein A adsorbent has restricted because of the high price of current commercial protein A adsorbent. At present, the most widely used protein A immunoadsorption columns were activated by cyanogen bromide and carbonyl imidazole, these methods have some shortcomings. This paper based on the previous recombinant staphylococcus protein A purification in our laboratory, attempts to establish an efficient approach for production of therapeutic-quality recombinant staphylococcus protein A. The rSPA was immobilized on sepharose-6FF activated by means of epichlorohydrin (ECH) and sodium periodate (NaIO4)to make adsorbent. The reaction conditions were optimized, and the influence on immobilizing quantity was observed in the synthesis route, activation density and the capacity of the absorbent. The performance for autoantibody removal and safety of rSPA absorbent were evaluated. The binding capacity of the absorbent for antibodies after10cycles, and non-specific adsorption of plasma components to rSPA absorbent was evaluate. Our purpose is to looking for a synthetic method for low-cost、safe and high-capacity rSPA immunoadsorbent. Our research paved the better foundation for its industrial-scale production and applications in the medicine.Methods1. sepharose-6FF activated by means of epichlorohydrin1.1Effect of concentration of NaOH on the density of epoxy Group A certain amount of sepharose-6FF were loaded into a glass funnel and the gel was washed with lots of reverse osmotic water, then divided the suction-dried gel to7(5g for each), the gel were placed into the conical flask of50ml respectively with the solution of5ml ECH and5ml different concentration of NaOH (1.0,1.5,2,2.5,3,3.5, and4mol/l). the suspension was incubated at40℃for2h with shaking (180rpm).It was then transferred to a glass filter funnel and the gel was washed with lots of reverse osmotic water, and then, suction-dried it. Activated sepharose-6FF samples from each group were collected and measured the density of epoxy group.1.2Effect of the concentration of epichlorohydrin on the density of epoxy GroupsA certain amount of sepharose-6FF were loaded into a glass funnel and the gel was washed with lots of reverse osmotic water, then divided the suction-dried gel to7(5g for each), the gel were placed into the conical flask of50ml respectively with the solution of5ml a certain concentration of NaOH and5ml different concentration of ECH (10%、20%、30%、40%和50%(v/v)). the suspension was incubated at40℃for2h with shaking (180rpm). It was then transferred to a glass filter funnel and the gel was washed with lots of reverse osmotic water, and then, suction-dried it. Activated sepharose-6FF samples from each group were collected and measured the density of epoxy group.1.3Effect of reaction time on the density of epoxy GroupA certain amount of sepharose-6FF were loaded into a glass funnel and the gel was washed with lots of reverse osmotic water, then divided the suction-dried gel to7(5g for each), the gel were placed into the conical flask of50ml respectively with the solution of5ml ECH and5ml a certain concentration of NaOH. The suspension was incubated at40℃for1h、2h、3h and4h with shaking (180rpm).It was then transferred to a glass filter funnel and the gel was washed with lots of reverse osmotic water, and then, suction-dried it. Activated sepharose-6FF samples from each group were collected and measured the density of epoxy group.1.4Effect of temperature on the density of epoxy GroupA certain amount of sepharose-6FF were loaded into a glass funnel and the gel was washed with lots of reverse osmotic water, then divided the suction-dried gel to7(5g for each), the gel were placed into the conical flask of50ml respectively with the solution of5ml ECH and a certain concentration of NaOH. the suspension was incubated at20℃、30℃、40℃and50℃. It was then transferred to a glass filter funnel and the gel was washed with lots of reverse osmotic water, and then, suction-dried it. Activated sepharose-6FF samples from each group were collected and measured the density of epoxy group.2. Sepharose-6FF activated by means of sodium periodate2.1Effect of the concentration of NaIO4on the density of aldehyde groupA certain amount of sepharose-6FF were loaded into a glass funnel and the gel was washed with lots of reverse osmotic water, then divided the suction-dried gel to7(5g for each), the gel were placed into the conical flask of50ml respectively with the solution of5ml varying concentration of NaIO4solution(0.05、0.1、0.2、0.3、0.4、0.5mol/l). The suspension was incubated at37℃/180rpm, for4h. It was then transferred to a glass filter funnel and the gel was washed with lots of reverse osmotic water, till the potassium iodide test paper shows no color,and then, suction-dried it. Activated sepharose-6FF samples from each group were collected and measured the density of epoxy group.2.2Effect of reaction temperature on the density of aldehyde groupA certain amount of sepharose-6FF were loaded into a glass funnel and the gel was washed with lots of reverse osmotic water, then divided the suction-dried gel to 7(5g for each), the gel were placed into the conical flask of50ml respectively with the solution of0.1mol/1sodium periodate. the suspension was incubated at30℃、35℃and40℃for4h. It was then transferred to a glass filter funnel and the gel was washed with lots of reverse osmotic water, till the potassium iodide test paper shows no color,and then, suction-dried it. Activated sepharose-6FF samples from each group were collected and measured the density of epoxy group.2.3Effect of reaction time on the density of aldehyde groupA certain amount of sepharose-6FF were loaded into a glass funnel and the gel was washed with lots of reverse osmotic water, then divided the suction-dried gel to7(5g for each), the gel were placed into the conical flask of50ml respectively with the solution of with0.1mol/1NaIO4solution. the suspension was incubated at40℃for1h、2h、3h and4h with shaking. It was then transferred to a glass filter funnel and the gel was washed with lots of reverse osmotic water, till the potassium iodide test paper shows no color,and then, suction-dried it. Activated sepharose-6FF samples from each group were collected and measured the density of epoxy group.3. Effect of the addition of rSPA on the amount of immobilized rSPA of Activated sepharose-6FF3.1Effect of the addition of rSPA on the amount of immobilized rSPA of sepharose-6FF activated by ECHA certain amount of sepharose-6FF activated by ECH were loaded into a glass funnel and the gel was washed with lots of reverse osmotic water, then divided the suction-dried gel to6(10g for each), the gel were placed into the conical flask of50ml respectively with the solution of glutaraldehyde, and then, immobilize the rSPA with different addition (1、2、3、4、5、6mg/g gel), the suspension was incubated at37℃for24h with shaking, washed the gel, collected the cleaning liquid and measure the volume of it. Wash again, then use5%sodium borohydride as the blocking agent, at last, use5%sodium borohydride as reductive agent. The amount of rSPA in cleaning liquid was measured by BCA Protein Quantitation Kit, then, work out the amount of immobilized rSPA.3.2Effect of the addition of SPA on the amount of immobilized SPA of sepharose-6FF activated by NaIO4.A certain amount of sepharose-6FF activated by NaIO4were loaded into a glass funnel and the gel was washed with lots of reverse osmotic water, then divided the suction-dried gel to6(10g for each), the gel were placed into the conical flask of50ml respectively with the different addition of SPA (1、2、3、4、5、6mg/g gel), the suspension was incubated at37℃for24h with shaking, washed the gel, collected the cleaning liquid and measure the volume of it. Wash again, then used5%sodium borohydride as reductive agent. The amount of rSPA in cleaning liquid was measured by BCA Protein Quantitation Kit. Work out the amount of immobilized rSPA.3.3Take the unactivated sepharose-6FF as the control group, treated it as shown in3.2.4. Comparison of the adsorption of IgG to different adsorbent.2.8g adsorbent activated by means of epichlorohydrin、2.8g adsorbent activated by means of NaIO4and2.8g unactivated sepharose-6FF were preformed plasmic adsorption experiment respectively. The method was shown below:(1) Loaded2.8g different adsorbents into the chromatographic column (d=1.0cm, V=10ml), connected constant flow pump and ultraviolet detector, and the wave length of ultraviolet detector was280nm.(2) Wash the column with10ml equilibrium liquid,20ml eluant, and30ml equilibrium liquid in sequence (flow rate:5ml/min). (3)Loaded20ml plasma sample into the chromatographic column (flow rate:2.5ml/min), collected the solution of elution peak, adjust the PH value to7, the adsorption selectivity of the adsorbent was assessed by analyzing the abundance of eluted human immunoglobulin after adsorption of plasma.(4)Wash the column with equilibrium liquid and reverse osmosis water in sequence, and store it at4℃.5. Comparison of the adsorption of plasma protein of different absorbents(1) Took the absorbents activated by means of ECH and NaIO4(under the conditions of6mg/g gel addition of rSPA) as the experimental group, and the unactivated sepharose-6FF as contrast group,2.8g for each, treated as4.(2) Analysis the adsorption selectivity of the absorbent was assessed by analyzing the abundance of eluted plasma protein after adsorption of plasma.6. Effect of the number of cycles of reuse on the adsorption of IgG(1) Took adsorbents (2.8g for each) from ECH group and NaIO4group respectively and treated as shown in4.(2) After performed the experiment of adsorption of plasma for one time,and then wash the column with10ml equilibrium liquid,20ml eluant, and30ml balance liquid in sequence (flow rate:5ml/min), then looping10times in all and collected the eluate and measured the abundance of eluted IgG.7. Comparison of the weight loss of SPA of different adsorbents(1) Took adsorbents from ECH group and NaIO4group respectively filled the chromatographic column (75ml), and then, connect constant flow pump and pipes.(2)Used3L saline to wash the immune adsorption columns and pipes (flow rate:50ml/min), and then wash it with1L saline (20ml/min), collect the1L fluids, and measure the weight loss of SPA of different adsorbents by Protein A ELISA kit.8. Statistics All values are mean±SEM.The significance of differences among mean values was determined by one-way ANOVA, pairwise comparisons using LSD method for multiple sets of data, the repeated measurement was determined by repeated measure ANOVA.Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS17.0.The accepted level of significance was P<0.05.Result1. sepharose-6FF activated by epichlorohydrin1.1Effect of concentration of NaOH on the density of epoxy GroupThe epoxy Group of6samples of suction-dried sepharose-6FF with concentration of NaOH (1、1.5、2、2.5、3、3.5、4mol/l) were measured.(one-way ANOVA, F=278.466, P=0.00). The result showed that the density of epoxy Group increased with increasing concentration (from1mol/1to3.5mol/l), the density of epoxy Group, in order,33.53±0.10、37.32±0.87、39.43±0.56、44.34±0.62、45.91±078、49.51±0.75. In3.5mol/l group, the density of epoxy Group was highest, and In4mol/l group, the density of epoxy Group has was no significant difference compared to3.5mol/l group (49.11±0.45, P=0.45>0.05)1.2Effect of concentration of ECH on the density of epoxy GroupThe epoxy Group of5samples of suction-dried sepharose6FF with concentration of ECH (10%、20%、30%、40%和50%(v/v) were measured,(one-way ANOVA, F=400.13, P=0.00). The result showed that the density of epoxy Group increased with increasing concentration (from10%to30%), the density of epoxy Group followed by(10%ECH,4.37±0.45;20%ECH,28.87±0.47;30%ECH,42.82±1.80),In30%test group, the density of epoxy Group was highest. Compared with30%, the40%group and50%group have no significant difference in the density of epoxy Group (40%ECH, P=0.36>0.05;50%ECH, P=0.39>0.05); so was the density of epoxy Group between40%ECH group and50%ECH group (P=0.953)。1.3Effect of reaction time on the density of epoxy GroupThe epoxy Group of6samples of suction-dried sepharose-6FF with different reaction time (1h,2h,3h and4h)were measured.(one-way ANOVA, F=113.123, P=0.00). The result showed that, in2h test group, the density of epoxy Group is highest (41.78±0.23), following by1h和3h test group (1h,40.02±0.25;3h,40.29±0.08P=0.207>0.05), and then, lowest in4h test group (38.17±0.34)1.4Effect of reaction temperature on the density of epoxy GroupThe epoxy Group of4samples of suction-dried sepharose-6FF with different reaction temperature (20℃、30℃、40℃、50℃)were measured.(one-way ANOVA, F=337.817, P=0.00). The result showed that:in40℃test group, the density of epoxy Group is highest (45.21±0.79), following by30℃(39.96±0.17),, and then,20℃test group (33.99±0.40),lowest in50℃test group (27.66±1.11)2. Sepharose-6FF activated by NaIO42.1. Effect of the concentration of NaIO4on the density of aldehyde groupThe aldehyde group of5samples of suction-dried sepharose-6FF with concentration of NaIO4(0.05、0.1、0.2、0.3、0.4、0.5mol/l) were measured.(one-way ANOVA, F=2100.896, P=0.00). The result showed that the density of epoxy Group increased with increasing concentration (from0.05to O.lmol/1), the density of epoxy Group, in order,0.05mol/I,57.32±0.60;0.lmol/l,109.42±0.85. In0.1mol/l group, The aldehyde group was highest, and0.2to0.5mol/l test group, The aldehyde group decreased with increasing concentration, in order,0.2mol/l group,99.92±0.90;0.3mol/l group,95.25±0.24;0.4mol/l group,93.55±0.28;0.5mol/1group,91.88±0.82.2.2. Effect of reaction temperature on the density of aldehyde group The aldehyde group of4samples of suction-dried sepharose-6FF with different reaction temperature (30℃、35℃and40℃)were measured.(one-way ANOVA, F=134.14, P=0.00). The result showed that:in35℃test group, the density of epoxy Group is highest (106.24±1.11), following by30℃(92.03±1.50), lowest in40℃(85.41±2.02)2.3. Effect of reaction time on the density of aldehyde group The aldehyde group of5samples of suction-dried sepharose-6FF with different reaction time (lh,2h,3h,4h and5h)were measured.(one-way ANOVA, F=2206.16, P=0.00). The result showed that, from1h to4h, the density of epoxy Group increased with increasing concentration, in order,(1h,38.32±0.63;2h,77.82±0.22;3h,96.09±0.19;4h,107.59±1.37), in5h group, the density of epoxy Group is no significant different from4h group(5h,101.70±1.84,P=0.902).3. The effect of addition of rSPA on the amount of immobilized rSPA of different activated Sepharose-6FF and the adsorption of IgG of different absorbents.3.1The effect of addition of rSPA on the amount of immobilized rSPA of different activated Sepharose-6FFThe amount of immobilized rSPA with different addition of rSPA (1、2、3、4、5和6mg/g gel)were measured(one-way ANOVA). The result showed that the rSPA can not immobilized to unactivated Sepharose-6FF; the amount of immobilized SPA in ECH group and NaIO4group were compared. when rSPA was lmg/g gel, ECH group (0.87±0.01) has no significant difference with NaIO4group (0.86±0.12)(F=1.471, P=0.292>0.05); when rSPA was2mg/g gel, ECH group (1.77±0.02) was higher than NaIO4group (1.27±0.05)(F=288.80, p=0.00); when rSPA was3mg/g gel, ECH group (2.82±0.05) was higher than NaIO4group (1.80±0.02)(F=1224.01, p=0.00); when rSPA was4mg/g gel, ECH group (3.93±0.04) was higher than NaIO4group (2.25±0.03)(F=3577.69, p=0.00); when rSPA was5mg/g gel, ECH groups (4.91±0.35) was higher than NaIO4group (2.77±0.51)(F=3572.88,p=0.00); when rSPA was6mg/g gel, ECH group (5.90±0.46) was higher than NaIO4group (3.31±0.17)(F=9384.50,p=0.00)3.2Effect of the addition of rSPA on the adsorption of IgG of different absorbents.The adsorption of IgG of different absorbents with different addition of rSPA (1、2、4、5和16mg/g gel)were measured (one-way ANOVA). The result showed that:when SPA was1mg/g gel, ECH group (8.52±0.03) was lower than NaI04group (10.54±0.11)(F=881.46, P=0.00); when SPA was2mg/g gel, ECH group (10.80±0.03) was lower than NaIO4group (12.67±0.04)(F=3869.41, p=0.00); when SPA was3mg/g gel, ECH group (15.37±0.05) was lower than NaIO4group (18.87±0.09)(F=3744.41, p=0.00);when SPA was4mg/g gel, ECH group (19.00±0.10) was lower than NaIO4group(20.29±0.76)(F=336.04,, p=0.00); when SPA was5mg/g gel, ECH group(22.19±0.28) was lower than NaIO4group (23.01±0.21)(F=17.07,p=0.01); when SPA was6mg/g gel, ECH group (23.73±0.10) was lower than NaIO4group (24.49±0.03)(F=173.86,p=0.00)4. Comparison of the adsorption of plasma protein to different absorbentThe adsorption of IgG showed a significant difference in different groups (one-way ANOVA, F=15313.06,P=0.00). The result showed that, the adsorption of IgG in NaIO4group is highest (21.49±0.10), following by ECH group (20.04±0.08), and then, lowest in control group (3.54±0.03).The adsorption of IgM in NaIO4group (0.38±0.02) is higher than ECH group (0.32±0.02)(one-way ANOVA, F=13.50, P=0.02<0.05); the adsorption of IgA in NaIO4group (1.60±0.04) is higher than ECH group (1.43±0.02)(one-way ANOVA, F=229976.23, P=0.00);There was a significant difference in the adsorption of HSA among three groups (one-way ANOVA, F=229976.23, P=0.00).NaIO4group (18.53±0.02, P=0.00) and ECH group (18.49±0.02, P=0.00) had made significant difference with control group (2.13±0.03).But, there was no significant difference between NaIO4group and ECH group. The adsorption function of all groups toward FIB、HAS、triglyceride、cholestero、lHDL、LDL is slighter.6. Effect of the number of cycles of reuse on the adsorption of IgGThe results showed that the adsorbent activated by means of epichlorohydrin and the adsorbent activated by means of NaIO4both could be used repeatedly (repeated measures-ANOVA F=0.933, P=0.506>0.05). The capacity to binding IgG of two kinds of absorbent was significantly different from each other(F=182.734, P=0.00),which were still20mg/ml gel after reused for10times. The interaction between absorbents and frequency of use on the adsorption of IgG showed no significant difference(F=1.041, P=0.427).7. The weight loss of SPA of different protein A adsorbents.The weight loss of SPA of ECH group(3.42±0.12)was significantly lower than NaIO4group (0.657±0.07)(one-way ANOVA, F=1121.408, P=0.00)Conclusion1. The optimal conditions for the activation of Sepharose by ECH:30%ECH、3.5mol/l NaOH,40℃,2h. Under the optimal conditions for activation, the maximum amount of epoxy group was about65umol/ml.2.The optimal conditions for the activation of Sepharose by NaIO4:0.1mol/l NaIO4,35℃,4h. Under the optimal conditions for activation, the maximum amount of aldehyde group was about120umol/ml.3. At the same addition of rSPA, the adsorbent activated by means of ECH has a greater ability to binding rSPA, but the adsorbent activated by means of NaIO4has a greater ability to binding IgG. The maximum adsorption of the adsorbent activated by means of ECH can reach about24mg/ml gel, while the adsorbent activated by means of NaIO4can reach about26mg/ml gel.4. The rSPA adsorbent activated by means of ECH and the rSPA adsorbent activated by means of NaI04both have a greater ability to binding IgG、IgM and IgA, which has a significant difference in the adsorption between the two kinds of adsorbents, but there was no significant difference in the adsorption of HSA between them and the adsorption function of two kinds of adsorbent toward FIB、HAS、 triglyceride、cholesterol、HDL、LDL were slighter.5. The capacity to binding IgG of the rSPA adsorbent activated by means of ECH and the rSPA adsorbent activated by means of NaIO4both showed no significant difference In the experiment of10times, which were still20mg/ml gel after reused for10times.6. The weight loss of SPA of the adsorbent activated by means of ECH was markedly lower than the adsorbent activated by means of NaIO4.