Tugcu Nihal, Roush David J, Göklen Kent E
BioPurification Development, Merck Research Laboratories, PO Box 2000, Rahway, New Jersey 07065, USA.
Biotechnol Bioeng. 2008 Feb 15;99(3):599-613. doi: 10.1002/bit.21604.
The large scale production of monoclonal antibodies presents a challenge to design efficient and cost effective downstream purification processes. We explored a two stage resin screening approach to identify the best candidates to be utilized for the platform purification of monoclonal antibodies. The study focused on commercially available affinity resins including Protein A, mimetic and mixed-mode interaction resins as well as ion exchangers used in polishing steps. An initial screening using pure proteins was followed by a final screening where selected resins were utilized for the purification of MAbs in complex mixtures. Initial screenings aimed to measure the theoretical upper limit for dynamic binding capacity (DBC) at 1% breakthrough and productivity. We confirmed that DBC of affinity, mimetic and mixed-mode resins was a strong function of the linear velocity used for loading. Productivities >27 g/(L-h), were obtained for rProtein A FF, Mabselect and Prosep rA Ultra at 2 min residence time. For the cation exchangers, we identified UNOsphere S and Fractogel SO(3) as the best candidates for our purification based on DBC. For anion exchangers operated in flowthrough mode, Q Sepharose XL and UNOsphere Q were selected from the initial screening based on DBC and resolution of IgG from BSA. Finally, a three step purification scheme was implemented using the selected affinity and ion exchangers for the purification of IgG from complex feedstocks. We found that Mabselect followed by UNOsphere Q and UNOsphere S provided the best purification scheme for our applications based on productivity.
单克隆抗体的大规模生产对设计高效且具有成本效益的下游纯化工艺提出了挑战。我们探索了一种两阶段树脂筛选方法，以确定用于单克隆抗体平台纯化的最佳候选树脂。该研究聚焦于市售的亲和树脂，包括蛋白A、模拟亲和和混合模式相互作用树脂，以及用于精制步骤的离子交换剂。首先使用纯蛋白进行初步筛选，随后进行最终筛选，在最终筛选中，将选定的树脂用于复杂混合物中单克隆抗体的纯化。初步筛选旨在测定1%穿透率和生产率时动态结合容量（DBC）的理论上限。我们证实，亲和、模拟亲和和混合模式树脂的DBC是加载所用线性流速的强函数。在2分钟停留时间下，rProtein A FF、Mabselect和Prosep rA Ultra的生产率>27 g/(L·h)。对于阳离子交换剂，基于DBC，我们确定UNosphere S和Fractogel SO(3)是我们纯化的最佳候选者。对于以流通模式运行的阴离子交换剂，基于DBC以及从牛血清白蛋白中分离IgG的分辨率，从初步筛选中选择了Q Sepharose XL和UNosphere Q。最后，实施了三步纯化方案，使用选定的亲和树脂和离子交换剂从复杂原料中纯化IgG。我们发现，基于生产率，先使用Mabselect，然后使用UNosphere Q和UNosphere S为我们的应用提供了最佳纯化方案。
