on-column monitoring of protein purification using spectroscopic techniques.

单克隆抗体（mab）是制药行业的主要兴趣（1,2），因为它们具有高度特异性并且不会穿过血脑屏障。IgG4型抗体已在欧盟和美国被批准用于治疗急性淋巴细胞白血病、A型血友病和特应性皮炎(3)。当宿主细胞蛋白（HCPs）和DNA存在时，可发生免疫原性反应。建议在最终样品中存在<100pg DNA和<100ppm HCPs(4,5)，以防止不良和潜在危险的免疫反应。HCPs和DNA推荐是通过下游处理结合蛋白A亲和层析实现的（5-8）。蛋白A树脂价格昂贵，并且由于污垢、就地清洗方案（CIP）和与单抗纯化相关的蛋白A配体的损失而导致的结合能力损失会产生额外的成本（8-10）。由于连续流方法最适合大规模生产（1,11），因此开发柱上单克隆抗体纯化监测方法变得越来越重要。监测这一过程的方法包括使用共聚焦激光扫描显微镜（12）和衰减全反射傅立叶变换红外（ATR-FTIR）光谱（8,10）等方法的微流柱，尽管这两种方法都尚未达到工业规模。目的：建立ATR-FTIR和共聚焦拉曼显微镜在柱上分析纯化的实验方法，实时观察树脂质量和性能的变化。将这些技术应用于蛋白A树脂和其他用于分离单克隆抗体的色谱技术。在工业环境中使用这些技术。引用:1。杨建军，刘建军。生物医学工程学报，2007;23(5):995-1008.2。李建军，刘建军，李建军，等。中国医药科学，2017;8:344 - 344。Kaplon H, Reichert JM。马伯。2018; 10(2): 183 - 203.4。世卫组织使用动物细胞作为生物制剂生产的体外底物的要求（生物物质要求第50号）。生物制剂,1998,26(3):175 - 93.5。Tarrant RD, Velez-Suberbie ML, Tait AS, Smales CM, Bracewell DG。生物工程学报，2012;28(4):1037-44.6。张建军，刘建军，李建军，等。2008;21(3):1 - 7。张建军，张建军，张建军，等。生物医学工程学报，2018,36(4):1223- 128。Boulet-Audet M, Byrne B, Kazarian SG。生物化学学报，2015;37(3):771 - 779。Close EJ, Salm JR, Iskra T, Sorensen E, Bracewell DG。生物工程学报，2013,39(9):2425-35.10。刘建军，张建军，张建军，等。中国科学进展，2016;6(6):526 - 526。王晓明，王晓明，王晓明，等。中国生物医学工程学报，2015;21(3):644 - 644。王勇，陈强，献明，年荣，徐峰。安博快递。2018;8(1):93。

Monoclonal antibodies (Mabs) are of major interest to the pharmaceutical industry (1, 2), as they are highly specific and do not cross the blood-brain barrier. IgG4 type antibodies have been approved in the EU and US for treating Acute Lymphoblastic Leukaemia, Haemophilia A and Atopic Dermatitis(3). Immunogenic responses can occur when host cell proteins (HCPs) and DNA are present. It is recommended that <100pg DNA and <100ppm HCPs(4, 5) are present in final samples to prevent undesirable and potentially dangerous immune responses.HCPs and DNA recommendations are achieved by downstream processing coupled with Protein A affinity chromatography (5-8). Protein A resins are expensive and additional costs are incurred by loss of binding capacity due to fouling, cleaning in place protocols (CIP) and a loss of protein A ligand associated with Mab purification(8-10).Since continuous flow methodologies are optimal for large scale production (1, 11), it is becoming increasingly important to develop methods to monitor purification of Mabs on-column. Ways to monitor this process include micro flow columns using methods such as confocal laser scanning microscopy(12) and Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy (8, 10) although neither method is industrial scale as yet. Aims: To develop experimental methodologies for analysing purification on-column using ATR-FTIR and Confocal Raman Microscopy to observe changes in resin quality and performance in real time.To apply these techniques to Protein A resin and other chromatography techniques used for isolation of mAbs.To use these techniques in an industrial setting. References:1. Kelley B. Biotechnol Prog. 2007;23(5):995-1008.2. Moorkens E, Meuwissen N, Huys I, Declerck P, Vulto AG, Simoens S. Front Pharmacol. 2017;8:314.3. Kaplon H, Reichert JM. MAbs. 2018;10(2):183-203.4. WHO Requirements for the Use of Animal Cells as in vitro Substrates for the Production of Biologicals (Requirements for Biological Susbstances No. 50). Biologicals. 1998;26(3):175-93.5. Tarrant RD, Velez-Suberbie ML, Tait AS, Smales CM, Bracewell DG. Biotechnol Prog. 2012;28(4):1037-44.6. Shukla AA, Jiang C, Ma J, Rubacha M, Flansburg L, Lee SS. 2008;24(3):615-22.7. Goey CH, Alhuthali S, Kontoravdi C. Biotechnol Adv. 2018;36(4):1223-37.8. Boulet-Audet M, Byrne B, Kazarian SG. Anal Bioanal Chem. 2015;407(23):7111-22.9. Close EJ, Salm JR, Iskra T, Sorensen E, Bracewell DG. Biotechnol Bioeng. 2013;110(9):2425-35.10. Boulet-Audet M, Kazarian SG, Byrne B. Sci Rep. 2016;6:30526.11. Girard V, Hilbold NJ, Ng CK, Pegon L, Chahim W, Rousset F, et al. J Biotechnol. 2015;213:65-73.12. Wang Y, Chen Q, Xian M, Nian R, Xu F. AMB Express. 2018;8(1):93.