Wetterhall Magnus, Ander Mats, Björkman Tomas, Musunuri Sravani, Palmgren Ronnie, Rodrigo Gustav
Cytiva, BioProcess, Björkgatan 30, SE-75184 Uppsala, Sweden.
Cytiva, BioProcess, Björkgatan 30, SE-75184 Uppsala, Sweden.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jan 1;1162:122473. doi: 10.1016/j.jchromb.2020.122473. Epub 2020 Dec 8.
In this study, the enhanced alkaline stability of Protein A ligands and resins designed by protein engineering approaches is demonstrated. High throughput PreDictor™ plates were used to evaluate and compare the human Immunoglobulin G (IgG) static binding capacities (SBC) of MabSelect SuRe™ and MabSelect™ PrismA affinity chromatography (AC) resins after continuous incubation in 0.1-2.0 M NaOH for 1-72 h. The alkaline effect on the Protein A affinity ligand was studied by high resolution mass spectrometry (MS). The IgG binding capacity of the investigated AC resins show expected declining trends with increasing NaOH concentrations and incubation times. The decrease is larger for MabSelect SuRe than for MabSelect PrismA and occur at lower NaOH concentrations. MabSelect SuRe display high remaining binding capacity even after 72 h continuous incubation in 0.1 M NaOH, while higher concentrations induce an accentuated decline with incubation time. The MabSelect PrismA resin shows almost no effect on the binding capacity even after 72 h incubation in 0.5 M NaOH. Decline in capacity is first observed after 48 h incubation in 1.0 M NaOH, thus displaying the extreme alkaline stability of the PrismA affinity ligand. The MS analysis of the ligands, including a Protein A single B-domain, SuRe-domain and PrismA-domain clearly illustrate the increasing alkaline stability (B-domain < SuRe < PrismA) as the ligand has been refined using a protein engineering approach. Deamidation and ligand degradation could be monitored in relation to NaOH incubation conditions. Enzymatic digestion of MabSelect SuRe and MabSelect PrismA resins after alkaline incubation and LC-MS/MS peptide mapping facilitates identification and quantification of specific deamidation sites on the affinity ligand.
在本研究中，展示了通过蛋白质工程方法设计的蛋白A配体和树脂增强的碱稳定性。使用高通量PreDictor™板评估和比较了MabSelect SuRe™和MabSelect™ PrismA亲和色谱（AC）树脂在0.1 - 2.0 M NaOH中连续孵育1 - 72小时后的人免疫球蛋白G（IgG）静态结合容量（SBC）。通过高分辨率质谱（MS）研究了碱对蛋白A亲和配体的影响。所研究的AC树脂的IgG结合容量随NaOH浓度和孵育时间的增加呈现出预期的下降趋势。MabSelect SuRe的下降幅度大于MabSelect PrismA，且在较低的NaOH浓度下就会出现下降。即使在0.1 M NaOH中连续孵育72小时后，MabSelect SuRe仍显示出较高的剩余结合容量，而较高浓度的NaOH会导致结合容量随孵育时间加剧下降。即使在0.5 M NaOH中孵育72小时后，MabSelect PrismA树脂对结合容量几乎没有影响。在1.0 M NaOH中孵育48小时后首次观察到容量下降，从而显示出PrismA亲和配体的极端碱稳定性。对包括蛋白A单个B结构域、SuRe结构域和PrismA结构域在内的配体进行MS分析，清楚地表明随着使用蛋白质工程方法对配体进行优化，其碱稳定性不断提高（B结构域 < SuRe < PrismA）。可以根据NaOH孵育条件监测脱酰胺作用和配体降解。对碱孵育后的MabSelect SuRe和MabSelect PrismA树脂进行酶解以及LC-MS/MS肽图谱分析，有助于鉴定和定量亲和配体上的特定脱酰胺位点。
