用于单克隆抗体纯化的仿生多肽超大孔PGMA微球制备及其性能

以仿生多肽配基FYEILHC为亲和配基、以葡聚糖修饰的聚甲基丙烯酸缩水甘油酯[Dextran-poly(glycidyl methacrylate), Dextran-PGMA]超大孔微球为基质,制备用于单克隆抗体纯化的仿生多肽超大孔PGMA微球,在环氧氯丙烷中滴加2 mol/LNaOH使其表面衍生出环氧基,在表面修饰FYEILHC;用扫描电镜表征微球表面形貌,用AKTA蛋白纯化系统考察了Dextran-PGMA微球和琼脂糖微球对抗体的动态吸附量随线性流速的变化。结果表明,偶联FYEILHC后Dextran-PGMA微球仍能保持其大孔结构,在923 cm/h线性流速下,其对抗体的动态吸附量仅下降约8%,而琼脂糖微球的动态吸附量则迅速下降25%。表明在较高流速下,抗体在Dextran-PGMA微球上的传质性能较好。吸附?用0.1 mol/L NaOH原位清洗重复40次后,Dextran-PGMA微球对抗体的动态吸附量约为(21?1) mg/mL,表明微球具有良好的化学稳定性;血清中回收的抗体纯度为95.0%,表明仿生多肽亲和介质具有从复杂生物样品中纯化抗体的巨大潜力,可满足高流速、高通量抗体分离纯化需求。

Preparation of biomimetic peptide attached supermacroporous poly(glycidyl methacrylate) microspheres for monoclonal antibody purification and its performance

Recently, immunoglobulin G (IgG) has attracted great attentions in clinical medicine, biotechnology, stimulated the development of downstream purification technology of monoclonal antibodies. Now, the protein based affinity ligands during traditional chromatographic purification of IgG exist drawbacks including the high cost and low stability, which leads to the secondary contamination and biological toxicity to purified sample. Also the chromatographic agarose microspheres present shortcomings such as the gel compressibility and the mass transfer limitations. In this work, biomimetic peptide as the protein based ligands alternative, grafting on the supermacroporous dextran-poly(glycidyl methacrylate) microspheres (Dextran-PGMA) to overcome above drawbacks were prepared and to efficiently improve the IgG chromatographic separation. The hydroxy on dextran-grafted polymer chromatographic microspheres were converted to the epoxy groups in epichlorohydrin solution with 2 mol/L NaOH, and the epoxy groups were opened and coupled with biomimetic peptide, FYEILCH, to achieve the purpose of affinity modification. The SEM observed that the pore structure of macroporous was mostly maintained after coupling with the biomimetic peptide. The change of 10% dynamic binding capacity (DBC10%) of FYEILHC based on Dextran-PGMA microspheres and agarose microspheres under different flow rate (92~923 cm/h) was compared. The DBC10% of dextran-PGMA was declined only 8% under 923 cm/h, whereas the DBC10% of agarose was sharply declined about 25%. This phenomenon might be related to better mass transfer for IgG at higher flow rate on the Dextran-PGMA microspheres. The DBC10% of Dextran-PGMA microspheres maintained (21±1) mg/mL after cleaning-in-place (CIP) 40 cycles using 0.1 mol/L NaOH, indicating that the affinity medium had good chemical stability. The purity of recovered antibodies in plasma was 95.0%, demonstrating that this biomimetic peptide based affinity medium had great potential to purify IgG from complex biological samples. This biomimetic peptide grafted supermacroporous poly(glycidyl methacrylate) microspheres could meet the requirement for rapid and high-throughput separation of monoclonal antibody.