蛋白A亲和色谱材料因其与免疫球蛋白G(IgG)的特异性相互作用而广泛应用于临床医学和生物医学中。既是矩阵的特征,比如它的结构和形态,表面改性方法有助于填充材料的亲和性能。具体的,有序,蛋白质A的定向固定可以减少其与基质的空间位阻并保留其生物活性位点。在这项研究中,使用琼脂糖和聚甲基丙烯酸缩水甘油酯(PGMA)球体作为底物获得了四种类型的亲和色谱材料,多官能环氧和马来酰亚胺基团用于固定蛋白A。乙二胺浓度的影响,反应pH值,缓冲液浓度,等条件对蛋白A的偶联效率和IgG的吸附性能进行了评价。通过使用0.2和1.6mol/L乙二胺将琼脂糖和PGMA基质的部分环氧基转化为氨基来制备多官能环氧材料,分别。使用5mmol/L硼酸盐缓冲液(pH8)作为反应溶液将蛋白A偶联到多官能环氧材料上。当蛋白A通过马来酰亚胺基团固定在底物上时,琼脂糖和PGMA底物用25%(v/v)乙二胺活化16小时以将所有环氧基团转化为氨基。然后通过添加溶解在二甲基亚砜(DMSO)中的3mg/mL的3-马来酰亚胺苯甲酰基-N-羟基琥珀酰亚胺酯(MBS),然后悬浮在5mmol/L的硼酸盐缓冲液(pH8)中,将马来酰亚胺材料转化为氨基改性的材料。马来酰亚胺基团与重组蛋白A的巯基的C-末端特异性反应以实现在琼脂糖和PGMA底物两者上的高度选择性固定。通过优化蛋白A的键合条件,提高了亲和材料对IgG的吸附性能,例如矩阵类型,基质粒径,和蛋白质A含量,并测定各亲和材料对IgG的吸附性能。随后在不同流速下评估通过马来酰亚胺方法使用琼脂糖或PGMA作为基质制备的蛋白A亲和材料的柱压。与用琼脂糖制备的材料相比,用PGMA作为基质制备的亲和材料表现出优异的机械强度。此外,对于这种亲和材料,观察到流速与80mL/min的柱压之间具有良好的线性关系。随后,研究了PGMA基质的粒径对IgG结合能力的影响。在相同的蛋白A含量下,当粒径为44-88μm时,亲和材料在PGMA基质上的动态结合能力高于使用其他粒径时。通过合成具有1、2、4、6、8和10mg/mL的不同蛋白A偶联量的亲和材料,比较了使用多官能环氧和马来酰亚胺改性材料制备的亲和材料的性能。然后测定每种材料对牛IgG的动态和静态结合能力。将制备的亲和材料装入色谱柱中,从牛初乳中纯化IgG。尽管所有材料都显示出对IgG的特异性吸附选择性,通过用马来酰亚胺将蛋白A固定在PGMA基质上制备的亲和材料显示出明显更好的性能,并且在较低的蛋白接枝量下实现了更高的动态结合能力。当蛋白接枝量为15.71mg/mL时,牛IgG的动态结合能力为32.23mg/mL,人IgG的动态结合能力达到54.41mg/mL。碱处理160次循环后,材料的动态结合能力达到初始值的94.6%,表明其良好的稳定性。所开发的方法适用于蛋白质A亲和色谱材料的生产,在蛋白质固定化和免疫吸附材料合成领域显示出巨大的潜力。
Protein A affinity chromatographic materials are widely used in clinical medicine and biomedicine because of their specific interactions with immunoglobulin G (IgG). Both the characteristics of the matrix, such as its structure and morphology, and the surface modification method contribute to the affinity properties of the packing materials. The specific, orderly, and oriented immobilization of protein A can reduce its steric hindrance with the matrix and preserve its bioactive sites. In this study, four types of affinity chromatographic materials were obtained using agarose and polyglycidyl methacrylate (PGMA) spheres as substrates, and multifunctional epoxy and maleimide groups were used to fix protein A. The effects of the ethylenediamine concentration, reaction pH, buffer concentration, and other conditions on the coupling efficiency of protein A and adsorption performance of IgG were evaluated. Multifunctional epoxy materials were prepared by converting part of the epoxy groups of the agarose and PGMA matrices into amino groups using 0.2 and 1.6 mol/L ethylenediamine, respectively. Protein A was coupled to the multifunctional epoxy materials using 5 mmol/L borate buffer (pH 8) as the reaction solution. When protein A was immobilized on the substrates by maleimide groups, the agarose and PGMA substrates were activated with 25% (v/v) ethylenediamine for 16 h to convert all epoxy groups into amino groups. The maleimide materials were then converted into amino-modified materials by adding 3 mg/mL 3-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) dissolved in dimethyl sulfoxide (DMSO) and then suspended in 5 mmol/L borate buffer (pH 8). The maleimide groups reacted specifically with the C-terminal of the sulfhydryl group of recombinant protein A to achieve highly selective fixation on both the agarose and PGMA substrates. The adsorption performance of the affinity materials for IgG was improved by optimizing the bonding conditions of protein A, such as the matrix type, matrix particle size, and protein A content, and the adsorption properties of each affinity material for IgG were determined. The column pressure of the protein A affinity materials prepared using agarose or PGMA as the matrix via the maleimide method was subsequently evaluated at different flow rates. The affinity materials prepared with PGMA as the matrix exhibited superior mechanical strength compared with the materials prepared with agarose. Moreover, an excellent linear relationship between the flow rate and column pressure of 80 mL/min was observed for this affinity material. Subsequently, the effect of the particle size of the PGMA matrix on the binding capacity of IgG was investigated. Under the same protein A content, the dynamic binding capacity of the affinity materials on the PGMA matrix was higher when the particle size was 44-88 μm than when other particle sizes were used. The properties of the affinity materials prepared using the multifunctional epoxy and maleimide-modified materials were compared by synthesizing affinity materials with different protein A coupling amounts of 1, 2, 4, 6, 8, and 10 mg/mL. The dynamic and static binding capacities of each material for bovine IgG were then determined. The prepared affinity material was packed into a chromatographic column to purify IgG from bovine colostrum. Although all materials showed specific adsorption selectivity for IgG, the affinity material prepared by immobilizing protein A on the PGMA matrix with maleimide showed significantly better performance and achieved a higher dynamic binding capacity at a lower protein grafting amount. When the protein grafting amount was 15.71 mg/mL, the dynamic binding capacity of bovine IgG was 32.23 mg/mL, and the dynamic binding capacity of human IgG reached 54.41 mg/mL. After 160 cycles of alkali treatment, the dynamic binding capacity of the material reached 94.6% of the initial value, indicating its good stability. The developed method is appropriate for the production of protein A affinity chromatographic materials and shows great potential in the fields of protein immobilization and immunoadsorption material synthesis.