Development of Isotopically Labeled Glycoproteins for use as Internal Standards

开发用作内标的同位素标记糖蛋白

• 批准号: 8834650
• 负责人: RON ORLANDO
• 金额: $ 27.03万
• 依托单位: GLYCOSCIENTIFIC, LLC
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8834650
• 关键词: Amino Acid Sequence; Blinded; Cell Line; Cell Nucleus; Complex; Coupled; Development; Diagnostic; Embryo; Erythropoietin; Glycoconjugates; Glycolipids; Glycoproteins; Glycosaminoglycans; Half-Life; Heparin; Human; Immunoglobulin G; Inequality; Institution; Intravenous Immunoglobulins; Investigation; Ions; Label; Laboratories; Laboratory Study; Link; Location; Malignant Neoplasms; Mass Spectrum Analysis; Metabolic; Methodology; Monoclonal Antibodies; Phase; Play; Polysaccharides; Preparation; Procedures; Process; Product Labeling; Production; Prognostic Marker; Protein-Carbohydrate Interaction; Proteome; Reagent; Recombinant Proteins; Reference Standards; Relative (related person); Research Personnel; Resources; Role; Sampling; Scientist; Serum; Site; Structure; System; Therapeutic; Therapeutic Agents; Therapeutic Monoclonal Antibodies; Therapeutic Uses; base; glycosylation; instrument; kidney cell; novel; novel diagnostics; prognostic; public health relevance; response; scale up; stability testing

DESCRIPTION (provided by applicant): The ability to accurately quantitate the glycan chains attached to glycoproteins has wide-ranging implications. Numerous studies over the past 40 years have demonstrated that abnormal glycosylation occurs in virtually all types of human cancers, and demonstrate the potential of using glycan markers in either a diagnostic or a prognostic manner. The glycosylation on recombinant protein therapeutics is also known to have profound effects, with one of the better known examples being the increased serum half-life of erythropoietin (EPO) resulting from glycoengineering. Hence, the quantification of glycoprotein glycans play important roles from the discovery of new diagnostic/prognostic markers to the development of various therapeutic agents. A current impediment for performing quantitative glycomics is the shortage of widely available standard glycoproteins and isotopically labeled reagents to enable accurate quantitation. The issue with glycan quantitation was highlighted by inter-laboratory studies conducted by the Human Proteome Organization (HUPO) and the Association of Biomolecular Resource Facilities (ABRF). Both of these studies demonstrated errors greater than several hundred percent in the analysis of mid-to-low level glycans were compared across participating laboratories. The inability to accurately quantitate low level glycans is particularly worrisome since it is often glycans of low abundance that have the largest impact, as is seen with the therapeutic human intravenous immunoglobulin G (IVIg). The focus of this proposal is to develop a well-characterized standard glycoprotein with isotopically labeled glycans to enable the accurate, robust, and reproducible analysis of N-linked glycans at the relative and potentially absolute level. A monoclonal antibody (mAb) was selected because of the widespread use of these as therapeutic agents coupled with the need for glycan quantification by various regulatory agencies. Here, a known quantity of the isotopically labeled mAb (i-mAb) can be added directly to therapeutic mAb preparation, analyzed by any standard procedure that includes mass spectrometry, and glycan quantitation will be provided by comparing the ratios of the native to isotopically labeled ions. i-mAb is expected to be an excellent internal standard for all therapeutic mAbs because of the similarity in glycan structures and in the amino acid sequence flanking the glycosylation site that is found on most mAb-based therapeutics. Since i-mAb can be added directly to the sample before any processing and is virtually identical to the analyte mAb, this approach is expected to be capable of compensating for a wide range of systematic errors, such as differential losses during sample handling, matrix effects, operator errors/inequalities, instrumental drift/response, etc. It is further anticipated hat i-mAb will enable researchers at different locations to obtain comparable results despite using different instruments, which is not possible with current methodology.

描述(由申请人提供)：准确定量附着在糖蛋白上的糖链的能力具有广泛的意义。过去40年的大量研究表明，糖基化异常发生在几乎所有类型的人类癌症中，并证明了将糖链标志物用于诊断或预测预后的潜力。重组蛋白疗法中的糖基化也具有深远的影响，其中一个较著名的例子是糖工程导致促红细胞生成素(EPO)的血清半衰期增加。因此，糖蛋白多糖的定量从发现新的诊断/预后标记物到开发各种治疗药物都起着重要的作用。目前进行定量糖组分的一个障碍是缺乏广泛可用的标准糖蛋白和同位素标记试剂来实现准确的定量。人类蛋白质组组织(HuPo)和生物分子资源设施协会(ABRF)进行的实验室间研究突显了多糖定量的问题。这两项研究都表明，在对参与实验室的中低水平葡聚糖进行比较时，误差大于几百%。无法准确定量低水平的葡聚糖尤其令人担忧，因为影响最大的往往是低丰度的葡聚糖，就像治疗性人类静脉注射免疫球蛋白G(IVIg)一样。这项建议的重点是开发一种具有良好特性的标准糖蛋白，其具有同位素标记的多糖，以便能够在相对和潜在的绝对水平上对N-连接的多糖进行准确、可靠和可重复性的分析。之所以选择单抗，是因为这些单抗被广泛用作治疗药物，而且需要由不同的监管机构对多糖进行量化。在这里，已知数量的同位素标记的单抗(I-mAb)可以直接添加到治疗性单抗制剂中，通过包括质谱仪在内的任何标准程序进行分析，并通过比较天然离子与同位素标记离子的比例来提供葡聚糖定量。由于I-mAb在糖链结构上的相似性，有望成为所有治疗性mAb的优良内部标准品 以及在大多数基于单抗的治疗药物中发现的糖基化位点两侧的氨基酸序列中。由于I-mAb可以在任何处理前直接加入到样品中，并且实际上与分析物mAb完全相同，这种方法有望能够补偿广泛的系统误差，如样品处理过程中的差值损失、基质效应、操作员误差/不平等、仪器漂移/响应等。进一步预计，I-mAb将使不同地点的研究人员能够获得可比较的结果，尽管使用不同的仪器，这是目前的方法所不可能的。