再生纤维素膜因具备优异的亲水性、良好的生物相容性以及出色的机械性能,成为理想的膜层析载体材料。然而,在以再生纤维素膜为载体进行大分子配基修饰时,传统的直接羟基接枝法存在明显的空间位阻问题。空间位阻导致配基负载量较低且吸附效能受到极大限制,无法充分发挥膜层析技术的优势。本研究提出构建“载体-长链-配基”三级结构的思路,利用聚甲基丙烯酸缩水甘油酯(PGMA)改性再生纤维素膜,制备高载量蛋白A(Protein A)膜层析介质,并应用于蛋白纯化。结果表明,Protein A膜层析介质具有6.5mg/mL的静态吸附载量,且在流速提升10倍(0.5→5mL/min)时动态结合容量仅下降0.03%(3.3→3.1mg/mL)。经0.1mol/L NaOH处理8h后仍保持87%初始载量,展现出优异的耐碱稳定性可复用性,验证了其在生物制药领域大规模应用的潜力。
Regenerated cellulose membrane has become an ideal carrier material for membrane chromatography due to its excellent hydrophilicity,good biocompatibility and excellent mechanical properties.However,when the regenerated cellulose membrane is used as the carrier for macromolecular ligand modification,the traditional direct hydroxyl grafting method suffers obvious steric hindrance.This problem leads to a low ligand load,and the adsorption efficiency is greatly limited,preventing full utilization of the advantages of membrane chromatography technology.In this study,we proposed to construct a “carrier-long chain-ligand” tertiary structure,and used PGMA (poly (glycidyl methacrylate)) to modify regenerated cellulose membrane to prepare a high-load Protein A membrane chromatography medium for protein purification application.The results showed that the medium had a static adsorption capacity of 6.5mg/mL,and the dynamic binding capacity only decreased by 0.03%(3.3→3.1mg/mL) when the flow rate increased by 10 times (0.5→5mL/min).87% of the initial load capacity was still maintained after 8 hours of treatment with 0.1mol/L NaOH,showing excellent alkali stability and reusability,which verified its potential for large-scale application in the field of biopharmaceuticals.
[1] Köhler,Georges,Cesar Milstein.Continuous cultures of fused cells secreting antibody of predefined specificity[J].Nature,1975,256,5517:495-497.
[2] Liu H F,Ma J F,Charles Winter,et al.Recovery and purification process development for monoclonal antibody production[J].Taylor & Francis,2010,2(5):480-499.
[3] Sánchez-Trasviña C,Miguel F,Daniela E O,et al.Purification of modified therapeutic proteins available on the market:an analysis of chromatography-based strategies[J].Frontiers in Bioengineering and Biotechnology,2021,9:717326.
[4] Carter-Franklin J N,Victa C,McDonald P,et al.Fragments of protein A eluted during protein A affinity chromatography[J].Journal of Chromatography A,2007,1163(1/2):105-111.
[5] Ramos-de-la-Peña A M,González-Valdez J,Aguilar O.Protein A chromatography:challenges and progress in the purification of monoclonal antibodies[J].Journal of Separation Science,2019,42(9):1816-1827.
[6] Lącki K M,Riske F J.Affinity chromatography:an enabling technology for large-scale bioprocessing[J].Biotechnology Journal,2020,15(1):1800397.
[7] Saxena A,Tripathi B P,Kumar M,et al.Membrane-based techniques for the separation and purification of proteins:an overview[J].Advances in Colloid and Interface Science,2009,145(1/2):1-22.
[8] Qu Y,Bekard I,Hunt B,et al.The transition from resin chromatography to membrane adsorbers for protein separations at industrial scale[J].Separation & Purification Reviews,2024,53(4):351-371.
[9] Thömmes J,Kula M R.Membrane chromatography-an integrative concept in the downstream processing of proteins[J].Biotechnology Progress,1995,11(4):357-367.
[10] Zeng X,Ruckenstein E.Membrane chromatography:preparation and applications to protein separation[J].Biotechnology Progress,1999,15(6):1003-1019.
[11] Stewart B H,Helen Chan O.Use of immobilized artificial membrane chromatography for drug transport applications[J].Journal of Pharmaceutical Sciences,1998,87(12):1471-1478.
[12] Chen J,Yu B,Cong H,et al.Recent development and application of membrane chromatography[J].Analytical and Bioanalytical Chemistry,2023,415(1):45-65.
[13] Boysen R I,Schwarz L J,Nicolau D V,et al.Molecularly imprinted polymer membranes and thin films for the separation and sensing of biomacromolecules[J].Journal of Separation Science,2017,40(1):314-335.
[14] Zhao Y,Zhu X,Jiang W,et al.Chiral recognition for chromatography and membrane-based separations:recent developments and future prospects[J].Molecules,2021,26(4):1145-1174.
[15] Vogel J H,Nguyen H,Giovannini R,et al.A new large-scale manufacturing platform for complex biopharmaceuticals[J].Biotechnology and Bioengineering,2012,109(12):3049-3058.
[16] Trnovec H,Doles T,Hribar G,et al.Characterization of membrane adsorbers used for impurity removal during the conti-nuous purification of monoclonal antibodies[J].Journal of Chromatography A,2020,1609:460518-460530.
[17] Buonomenna M G,Mousavi S M,Hashemi S A,et al.Water cleaning adsorptive membranes for efficient removal of heavy metals and metalloids[J].Water,2022,14(17):2718-2760.
[18] Kaur J,Sengupta P,Mukhopadhyay S.Critical review of bioadsorption on modified cellulose and removal of divalent hea-vy metals (Cd,Pb,and Cu)[J].Industrial & Engineering Chemistry Research,2022,61(5):1921-1954.
[19] Gupta A,Sharma V,Sharma K,et al.A review of adsorbents for heavy metal decontamination:growing approach to wastewater treatment[J].Materials,2021,14:4702-4746.
[20] Dong H,Tong L,Cheng M,et al.Utilizing electrospun mole-cularly imprinted membranes for food industry:opportunities and challenges[J].Food Chemistry,2024,460:140695-140709.
[21] Donato L,Nasser I I,Majdoub M,et al.Green chemistry and molecularly imprinted membranes[J].Membranes,2022,12(5):472-504.
[22] Alla A J,Stine K J.Recent strategies for using monolithic materials in glycoprotein and glycopeptide analysis[J].Separations,2022,9(2):44-78.
基金资助
国家自然科学基金(22365023);准格尔旗应用技术研究开发项目(2023YY-03)
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