尽管不同的 VH3 结合蛋白 A 树脂对 VH3 Fab 的动态结合能力各不相同,但它们都显示出相似的 VH3 结合介导的产物分离能力。

IF 1 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Protein and Peptide Letters Pub Date : 2024-08-16 DOI:10.2174/0109298665320125240805112024
Lixia Hu, Rongrong Wang, Qinxue Wu, Yan Wan, Yifeng Li
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引用次数: 0

摘要

背景:蛋白 A 树脂已被广泛用于 mAb、双特异性抗体(bsAb)和 Fc 融合蛋白纯化过程中的产物捕获。虽然蛋白 A 配体主要与 Fc 区域结合,但许多配体也能与 VH3 结构域结合。在 mAb/bsAb 纯化过程中,某些截短的副产品可能含有与产品相同的 Fc 区,但 VH3 结构域的数量较少。在这种情况下,VH3 结合蛋白 A 树脂提供了一种根据 VH3 结合价的差异分离副产品的潜在方法。由于不同的 VH3 结合蛋白 A 树脂的配体来自原生蛋白 A 的不同结构域,因此了解它们在分离具有相同 Fc 区域但不同 VH3 结构域数量的物种时是否具有可比能力将非常有意义:本研究旨在探索不同的 VH3 结合蛋白 A 树脂在分离具有相同 Fc 区但不同数量 VH3 结构域的抗体种类方面的潜力:方法:木瓜蛋白酶消化从含 VH3 的 mAb 中释放出 VH3 Fab。消化后,使用 CaptureSelect CH1-XL 和 MabSelect SuRe 亲和色谱法依次纯化释放的 VH3 Fab。纯化的 VH3 Fab 被用作负载材料,以评估五种 VH3 结合蛋白 A 树脂(即 Amshpere A3、Jetted A50、MabCapture C、MabSelect 和 MabSelect PrismA)的动态结合能力(DBC)。使用由产物和截短副产物组成的人工混合物,评估了 VH3 结合蛋白 A 树脂在分离具有相同 Fc 区但不同 VH3 结构域数目的物种方面的潜力。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)用于监测含有相同 Fc 区但不同数量 VH3 结构域的 Fab 的纯化和分离:结果:当载入分离的 VH3 Fab 时,不同的 VH3 结合蛋白 A 树脂显示出不同的 DBC。然而,当这些蛋白 A 树脂用于分离截短的副产品(只含 Fc 区而不含任何 VH3 结构域)和产品(除 Fc 区外还包括一个 VH3 结构域)时,它们在分离这两种物质方面的能力相当:结论:尽管不同的 VH3 结合蛋白 A 树脂对 VH3 Fab 的 DBC 不同,但它们在分离具有相同 Fc 区但不同数量 VH3 结构域的物种时表现出了相当的能力。
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Different VH3-Binding Protein A Resins Show Comparable VH3-Binding Mediated By product Separation Capabilities Despite Having Varied Dynamic Binding Capacities Towards A VH3 Fab.

Background: Protein A resins have been widely used for product capture during mAb, bispecific antibody (bsAb), and Fc-fusion protein purification. While Protein A ligands mainly bind the Fc region, many of them can also bind the VH3 domain. During mAb/bsAb purification, certain truncated byproducts may contain the same Fc region as the product but fewer numbers of the VH3 domain. In such a scenario, VH3-binding Protein A resins provide a potential means for byproduct separation based on the difference in VH3-binding valency. As the ligands of different VH3-binding Protein A resins are derived from distinct domains of the native Protein A, it would be interesting to know whether they possess comparable capabilities for separating species with the same Fc region but different numbers of VH3 domain.

Objective: This study aims to explore the potential of different VH3-binding Protein A resins for separating antibody species with the same Fc region but different numbers of VH3 domain.

Methods: The VH3 Fab was released from a VH3-containing mAb by papain digestion. Post digestion, the released VH3 Fab was purified sequentially using CaptureSelect CH1-XL and MabSelect SuRe affinity chromatography. The purified VH3 Fab was used as the load material to assess the dynamic binding capacity (DBC) of five VH3-binding Protein A resins (i.e., Amshpere A3, Jetted A50, MabCapture C, MabSelect and MabSelect PrismA). The potential of VH3-binding Protein A resins for separating species having the same Fc region but different numbers of VH3 domain was evaluated using an artificial mixture composed of the product and a truncated byproduct, which contained one and zero VH3 domain, respectively (both species contained the same Fc region). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to monitor Fab purification and separation of species containing the same Fc region but different numbers of VH3 domain.

Results: When loaded with an isolated VH3 Fab, different VH3-binding Protein A resins showed varied DBCs. Nevertheless, when these Protein A resins were used to separate a truncated byproduct, which contained the Fc region only without any VH3 domain, from the product, which included one VH3 domain in addition to the Fc region, they showed comparable capabilities for separating these two species.

Conclusion: Although different VH3-binding Protein A resins showed varied DBCs towards a VH3 Fab, they exhibited comparable capabilities for separating species with the same Fc region but different numbers of VH3 domain.

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来源期刊
Protein and Peptide Letters
Protein and Peptide Letters 生物-生化与分子生物学
CiteScore
2.90
自引率
0.00%
发文量
98
审稿时长
2 months
期刊介绍: Protein & Peptide Letters publishes letters, original research papers, mini-reviews and guest edited issues in all important aspects of protein and peptide research, including structural studies, advances in recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, and drug design. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallization and preliminary structure determination of biologically important proteins are considered only if they include significant new approaches or deal with proteins of immediate importance, and preliminary structure determinations of biologically important proteins. Purely theoretical/review papers should provide new insight into the principles of protein/peptide structure and function. Manuscripts describing computational work should include some experimental data to provide confirmation of the results of calculations. Protein & Peptide Letters focuses on: Structure Studies Advances in Recombinant Expression Drug Design Chemical Synthesis Function Pharmacology Enzymology Conformational Analysis Immunology Biotechnology Protein Engineering Protein Folding Sequencing Molecular Recognition Purification and Analysis
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