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（由申请方提供）：准确定量与糖蛋白连接的聚糖链的能力具有广泛的意义。过去40年的大量研究表明，异常糖基化发生在几乎所有类型的人类癌症中，并证明了使用聚糖标记物进行诊断或预后的潜力。还已知重组蛋白治疗剂的糖基化具有深远的影响，其中一个更好的已知实例是由糖工程产生的促红细胞生成素（EPO）的血清半衰期增加。因此，从发现新的诊断/预后标志物到开发各种治疗剂，糖蛋白聚糖的定量起着重要作用。目前进行定量糖组学的障碍是缺乏广泛可用的标准糖蛋白和同位素标记的试剂，以实现准确的定量。由人类蛋白质组组织（HUPO）和生物分子资源设施协会（ABRF）进行的实验室间研究强调了聚糖定量的问题。这两项研究均表明，在参与实验室之间比较了中低水平聚糖分析的误差大于几百%。无法准确定量低水平聚糖尤其令人担忧，因为通常低丰度的聚糖具有最大的影响，如治疗性人静脉注射免疫球蛋白G（IVIg）所示。本提案的重点是开发一种具有同位素标记聚糖的良好表征的标准糖蛋白，以便在相对和潜在绝对水平上对N-连接聚糖进行准确、稳健和重现性分析。选择单克隆抗体（mAb）是因为其作为治疗剂的广泛使用以及各种监管机构对聚糖定量的需求。在此，可以将已知量的同位素标记的mAb（i-mAb）直接添加到治疗性mAb制剂中，通过包括质谱法的任何标准程序进行分析，并且将通过比较天然离子与同位素标记的离子的比率来提供聚糖定量。由于聚糖结构的相似性，预期i-mAb是所有治疗性mAb的优良内标 和位于糖基化位点侧翼的氨基酸序列中，所述糖基化位点存在于大多数基于mAb的治疗剂上。由于i-mAb可在任何处理前直接加入样品中，且与分析物mAb几乎相同，因此预期该方法能够补偿广泛的系统误差，例如样品处理期间的差异损失、基质效应、操作员误差/不等性、仪器漂移/响应，进一步预期i-mAb将使不同地点的研究人员能够在使用不同仪器的情况下获得可比较的结果，这在当前方法学下是不可能的。