Proteoform Characterization of HIV-1 Broadly Neutralizing Antibody PGT 121.414.LS Product Through Middle-Up and Bottom-Up Proteomics for Clinical Support.

IF 2.7 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS
Connor E Gould, Qing Ma, Raymond Cha, Valerie A Frerichs, Alan E Friedman, Robin DiFrancesco, Gene D Morse, Troy D Wood
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Abstract

Broadly neutralizing antibody (bNAb) therapies are under development for the prevention and treatment of HIV-1 infection as an alternative to conventional antiretroviral therapy because of their potential for sustained passive immunotherapy. Currently, bNAbs targeting the HIV-1 viral envelope proteins are included in research protocols to evaluate their role in preventing transmission and in combination antiviral regimens to achieve viral suppression. While quantification of bNAbs is of clinical importance, validation of the structural fidelity of therapeutic antibodies is also an important consideration to assess the clinical efficacy. Here, we applied middle-up and bottom-up proteomics workflows for the quality assurance of the primary structure of the HIV-1 bNAb PGT 121.414.LS product. Middle-up and bottom-up proteomics workflows were performed using liquid chromatography coupled to an Orbitrap mass spectrometer. Middle-up analysis of the crystallizable fragment (Fc/2), light chain (Lc), and fragment denaturation (Fd, N-terminal fragment of the heavy chain) regions of PGT 121.414.LS was performed by using IdeS digestion, which revealed proteoform heterogeneity. To complement the middle-up approach, a bottom-up workflow combining trypsin and chymotrypsin digests was performed for detailed glycoform mapping and annotation. The bottom-up results indicated that Lc contained an additional N-terminal Ser residue. For the Fc, two abundant and two lower-abundance glycoforms of the heavy chain were detected that correspond to Asn-312 (Asn-297 in the consensus heavy chain sequence of IgG). For Fd, bottom-up analysis revealed eight sialylated glycoforms and five nonsialylated glycoforms at Asn-124 of the heavy chain. The results emphasize bNAb heterogeneity, which should be considered in affinity binding studies.

HIV-1广泛中和抗体PGT 121.414的蛋白形态特征LS产品通过中向上和自下而上的蛋白质组学进行临床支持。
广泛中和抗体(bNAb)疗法正在开发中,用于预防和治疗HIV-1感染,作为传统抗逆转录病毒疗法的替代方案,因为它们具有持续被动免疫治疗的潜力。目前,针对HIV-1病毒包膜蛋白的bNAbs被纳入研究方案,以评估其在预防传播和联合抗病毒方案中实现病毒抑制的作用。虽然bnab的定量具有临床重要性,但验证治疗抗体的结构保真度也是评估临床疗效的重要考虑因素。在这里,我们应用了中向上和自下而上的蛋白质组学工作流程来保证HIV-1 bNAb PGT 121.414初级结构的质量。LS的产品。中向上和自下而上的蛋白质组学工作流程使用液相色谱耦合到Orbitrap质谱仪。PGT 121.414的可结晶片段(Fc/2)、轻链(Lc)和片段变性区(Fd,重链n端片段)的中游分析。利用IdeS酶切法进行LS分析,发现蛋白质形态具有异质性。为了补充中向上的方法,自下而上的工作流程结合胰蛋白酶和糜凝胰蛋白酶消化进行了详细的糖型映射和注释。自下而上的结果表明Lc含有一个额外的n端Ser残基。对于Fc,检测到两个丰富和两个低丰度的重链糖型,对应于Asn-312 (IgG的一致重链序列为Asn-297)。对于Fd,自下而上的分析显示在重链Asn-124处有8个唾液化糖型和5个非唾液化糖型。结果强调了bNAb的异质性,在亲和结合研究中应考虑到这一点。
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来源期刊
CiteScore
5.50
自引率
9.40%
发文量
257
审稿时长
1 months
期刊介绍: The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role. Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives
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