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Homogenous Scavenging Resolves Low-Purification Yield/Selectivity Caused by Secondary Binding of Protein-A to Antigen-Binding Antibody Fragments.
Biomacromolecules ( IF 5.5 ) Pub Date : 2019-12-16 , DOI: 10.1021/acs.biomac.9b01516 Palapuravan Anees 1 , Marc A Gauthier 1
Biomacromolecules ( IF 5.5 ) Pub Date : 2019-12-16 , DOI: 10.1021/acs.biomac.9b01516 Palapuravan Anees 1 , Marc A Gauthier 1
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Antigen-binding fragments of antibodies are biotechnologically useful agents for decorating drug delivery systems, for blocking cell-surface receptors in cell culture, for recognizing analytes in biosensors, and potentially as therapeutics. They are typically produced by enzymatic digestion of full antibodies and isolated from the undesirable fragment crystallizable (Fc) by affinity chromatography using Protein-A columns. However, while Protein-A has a strong "classical" interaction with Fc fragments, it can also more weakly bind to an "alternative" site on the heavy chain variable region of antigen-binding fragments. As such, purifying small amounts of antibody fragments by Protein-A chromatography can result in low yield. Moreover, loading larger amounts of antibody fragments onto a Protein-A column can result in poor separation, because of competition of Fc and antigen-binding fragments for immobilized Protein-A. This study demonstrates that Protein-A-based homogeneous scavenging resolves this issue by precisely controlling the stoichiometry of Protein-A to Fc fragments, something that is not possible for conventional flow-type systems, such as affinity chromatography.
中文翻译：
均质清除解决了蛋白A与抗原结合抗体片段的二次结合所引起的低纯化产率/选择性。
抗体的抗原结合片段是生物技术上有用的试剂，用于修饰药物递送系统，阻断细胞培养中的细胞表面受体，识别生物传感器中的分析物，并有可能作为治疗剂。它们通常通过完整抗体的酶消化产生，并使用Protein-A色谱柱通过亲和色谱法从不想要的可结晶片段（Fc）中分离出来。但是，尽管Protein-A与Fc片段具有很强的“经典”相互作用，但它也可以更弱地与抗原结合片段的重链可变区上的“替代”位点结合。这样，通过蛋白A层析纯化少量抗体片段可能导致低产率。而且，由于Fc和抗原结合片段与固定的Protein-A竞争，将大量抗体片段上样到Protein-A色谱柱上会导致分离效果差。这项研究表明，基于蛋白质A的均质清除可通过精确控制蛋白质A与Fc片段的化学计量来解决此问题，这对于常规流式系统（如亲和色谱法）是不可能的。
更新日期：2020-01-04
中文翻译：
均质清除解决了蛋白A与抗原结合抗体片段的二次结合所引起的低纯化产率/选择性。
抗体的抗原结合片段是生物技术上有用的试剂，用于修饰药物递送系统，阻断细胞培养中的细胞表面受体，识别生物传感器中的分析物，并有可能作为治疗剂。它们通常通过完整抗体的酶消化产生，并使用Protein-A色谱柱通过亲和色谱法从不想要的可结晶片段（Fc）中分离出来。但是，尽管Protein-A与Fc片段具有很强的“经典”相互作用，但它也可以更弱地与抗原结合片段的重链可变区上的“替代”位点结合。这样，通过蛋白A层析纯化少量抗体片段可能导致低产率。而且，由于Fc和抗原结合片段与固定的Protein-A竞争，将大量抗体片段上样到Protein-A色谱柱上会导致分离效果差。这项研究表明，基于蛋白质A的均质清除可通过精确控制蛋白质A与Fc片段的化学计量来解决此问题，这对于常规流式系统（如亲和色谱法）是不可能的。
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