在抗体药物生产的下游工艺中,一般会在捕获阶段(Protein A亲和层析)之后的精制步骤中,采用两种不同分离模式的层析法进行纯化。疏水层析(HIC)是能有效去除多聚体等杂质的分离模式。
I. R. A. P. Bresolin, N. Lingg, I. T. L. Bresolin and A. Jungbauer
Hydrophobic interaction chromatography as polishing step enables obtaining ultra-pure recombinant antibodies
J. Biotechnology: X6 (2020) 100020, https://doi.org/10.1016/j.btecx.2020.100020
(要点)
·在精制步骤中使用HIC填料TOYOPEARL Phenyl-650M,分离纯化出了超高纯度的抗肿瘤坏死因子抗体(anti-tumor necrosis factor alpha:TNFa、IgG1)。
·对HIC模式下的吸附等温线、穿透曲线、层析分离等实施了评估。
·通过HIC纯化的抗体的纯度是99.9%,回收率是96.2%。并且确认到,抗体组分中混入的多聚体杂质、宿主细胞蛋白(HCP)、DNA都在检验要求范围之内。
·在需要制备超高纯度的抗体时,可在下游纯化工艺中使用该HIC分离法。
● 抗肿瘤坏死因子抗体(anti-tumor necrosis factor alpha:TNFa、IgG1)的纯化
样品:抗肿瘤坏死因子抗体(anti-tumor necrosis factor alpha:TNFa、IgG1)
参照抗体:阿达木单抗 (Adalimumab,商品名:Humira® )
动态吸附载量(TOYOPEARL Phenyl-650M):12.05 g/L
色谱柱尺寸:5 mm I.D. × 2.55 cm、驻留时间:2.5 min(流速:0.2 mL/min)
洗脱溶液:20 mmol/L磷酸钠缓冲溶液(pH 7.4)+ 1 mol/L 硫酸铵
多聚体的分析、定量:TSKgel® G3000SWXL
● 使用疏水填料TOYOPEARL Phenyl-650M对抗体进行超高纯度纯化
(分离出的抗体洗脱峰1和洗脱峰2的纯度测定,请继续阅读下一页)
●
对疏水层析(HIC)纯化后的抗体(Peak 1)进行纯度测定
样品 | 浓度 (g/L)a | 抗体纯度 (%)b | 多聚体 (%)c | HCP (ppm)d | dsDNA (ppb)e |
anti-TNFa IgG1(纯化前) | 2.00 | 98.04 | 1.53 | 342.5 | < 0.41* |
anti-TNFa IgG1 + salt(添加样品) | 2.00 | 98.31 | 1.55 | 173.7 | < 0.41* |
Peak 1(纯化组分) | 0.96 | 99.90 | < 0.06* | < 2.0* | < 0.85* |
Peak 2 | 0.03 | 59.88 | 57.55 | < 63* | < 27* |
Adalimumab (Humira) | 2.00 | 99.56 | 0.33 | n.d. (181.6)** | < 0.41* |
a by UV at 280 nm, b by SEC at 280 nm, c by SEC at 215 nm, d ELISA, e PCR resDNASEQ®
* Below lower limit of detection (LLOD), undiluted sample used
** Data from LC/MS/MS by Oliviero et al., Easy, Fast and Reproducible Analysis of Host Cell Protein (HCP) in Monoclonal Antibody Preparations, Application Note (2019)
Ref.: I. R. A. P. Bresolin et al., J. Biotechnology: X6 (2020) 100020, https://doi.org/10.1016/j.btecx.2020.100020, data modified from original
※通过HIC,获得了比市售抗体药物阿达木单抗更高纯度的抗体组分。
● 抗体纯化时HIC填料选择的要点
·根据抗体的疏水性,选择疏水性适度的HIC填料(官能团、细孔径、粒径等参数的不同),优化抗体的回收率和分离选择性(去除杂质)
·优化分离时的洗脱条件(缓冲液浓度、pH、盐浓度、流速、梯度的倾斜度、温度等)
·设置稳定的线性梯度洗脱条件,使抗体在梯度的中间部分洗脱出来
·HIC填料的疏水性:(低) Ether-650/PPG-600 < Phenyl-650/600 < Butyl-650/600 < Hexyl-650 (高)
● HIC填料和筛选用预装柱产品一览表
货号产品名称粒径(mm)规格*(mL)备注0016173TOYOPEARL Ether-650M40 - 90100散装填料(瓶)0021302TOYOPEARL PPG-600M40 - 90100散装填料(瓶)0014478TOYOPEARL Phenyl-650M40 - 90100散装填料(瓶)0021888TOYOPEARL Phenyl-600M40 - 90100散装填料(瓶)0007477TOYOPEARL Butyl-650M40 - 90100散装填料(瓶)0021449TOYOPEARL Butyl-600M40 - 90100散装填料(瓶)0019956TOYOPEARL SuperButyl-550C50 - 150100散装填料(瓶)0019026TOYOPEARL Hexyl-650C50 - 150100散装填料(瓶)0043276TSKgel Ether-5PW (20)15 - 2525散装填料(瓶)0043176TSKgel Ether-5PW (30)20 - 4025散装填料(瓶)0043277TSKgel Phenyl-5PW (20)15 - 2525散装填料(瓶)0043177TSKgel Phenyl-5PW (30)20 - 4025散装填料(瓶) | 货号产品名称粒径(mm)规格(mL)备注0045220SkillPak TOYOPEARL Ether-650M40 - 901 mL×5支预装柱0045218SkillPak TOYOPEARL PPG-600M40 - 901 mL×5支预装柱0045217SkillPak TOYOPEARL Phenyl-650M40 - 901 mL×5支预装柱0045216SkillPak TOYOPEARL Phenyl-600M40 - 901 mL×5支预装柱0045215SkillPak TOYOPEARL Butyl-650M40 - 901 mL×5支预装柱0045214SkillPak TOYOPEARL Butyl-600M40 - 901 mL×5支预装柱0045219SkillPak TOYOPEARL Hexyl-650C50 - 1501 mL×5支预装柱0045233SkillPak HIC(TOYOPEARL Butyl-650M, Phenyl-650M,PPG-600M, Hexyl-650C, Ether-650M)-1 mL×5种×1支预装柱0045234SkillPak HIC(TOYOPEARL Butyl-600M, Phenyl-600M,PPG-600M, Hexyl-650C, Ether-650M)-1 mL×5种×1支预装柱0045235SkillPak HIC(TOYOPEARL Butyl-650M, Phenyl-650M, PPG-600M)-1 mL×3种×2支预装柱0045236SkillPak HIC(TOYOPEARL Butyl-600M, Phenyl-600M, PPG-600M)-1 mL×3种×2支预装柱0045237SkillPak HIC(TOYOPEARL Phenyl-650M, PPG-600M, Hexyl-650C)-1 mL×3种×2支预装柱0045238SkillPak HIC(TOYOPEARL Phenyl-600M, PPG-600M, Hexyl-650C)-1 mL×3种×2支色谱柱0045256SkillPak TOYOPEARL Ether-650M40 - 905 mL×1支预装柱0045254SkillPak TOYOPEARL PPG-600M40 - 905 mL×1支预装柱0045253SkillPak TOYOPEARL Phenyl-650M40 - 905 mL×1支预装柱0045252SkillPak TOYOPEARL Phenyl-600M40 - 905 mL×1支预装柱0045251SkillPak TOYOPEARL Butyl-650M40 - 905 mL×1支预装柱0045250SkillPak TOYOPEARL Butyl-600M40 - 905 mL×1支预装柱0045255SkillPak TOYOPEARL Hexyl-650C50 - 1505 mL×1支预装柱 | |
*不同级别的填料中,有不同粒径和不同包装规格的产品。 SkillPak分为1 mL色谱柱(7 mm I.D.×2.5 cm)和5 mL色谱柱(8 mm I.D.×10 cm)。 1 mL型号:进口:使用“货号0017955 拉紧螺栓型(带堵头)”,不可使用“货号0016566 便携式接头·1/16"(PEEK)”。 出口:可直接连接内螺纹接头,连接外螺纹接头时需使用管套(“货号0017898 拉紧螺栓型管套”)等配件。 5 mL型号:进口和出口:使用“货号0016566 便携式接头·1/16"”或“货号0017955 拉紧螺栓型(带包头)”。 |
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