In-process glycoprofiling during recombinant therapeutic glycoprotein production

描述(由申请人提供)：生物制品市场是制药行业增长最快的部门，美国食品和药物管理局(FDA)以及欧洲药品管理局(EMEA)要求所有此类治疗性糖蛋白和抗体的糖谱必须广泛表征，并符合许可时定义的规格。目前，细胞培养材料的糖谱分析包括大量的样品纯化和质谱仪(MS)分析，通常需要一周或几周才能完成。如此长的交货期使细胞培养过程中的纠正行动变得不可能，而导致规格不合格的唯一途径是批量拒收，成本在500万至5000万美元之间。强大、快速和简单的糖谱分析方法的可获得性将从根本上改变生物制药研发和商业制造。我们将开发一种基于多重悬浮阵列(MSA)技术和糖特异性试剂相结合的能够在过程中进行糖谱分析的创新方法。我们的GlycoProf MSATM方法将为监测蛋白质末端糖基化模式提供一种快速、经济、易操作的方法。它将作为一个关键的早期预警系统，以防止拍品拒绝。此外，它还将协助优化工艺开发条件。在GlycoProf MSATM方法中，流式细胞术被用来检测葡聚糖与连接到光谱独特的微球上的葡聚糖特定试剂之间的结合。通过将各个试剂组合成一个多路阵列，可以在基础细胞仪上在不到一分钟的时间内获得整个分析结果。糖谱由每个特定的糖-MSA相互作用的相对荧光强度提供。GlycoProf MSATM方法不会取代对最终产品的全面表征，但将解决生产过程中对快速糖谱分析的未得到满足的需求。我们将使用显示真核末端序列的多糖为GlycoProf MSATM方法建立最佳性能指标。此外，我们将建立GlycoProf MSATM方法重现糖蛋白糖谱的能力，其中糖链已被设计为显示统一的糖基化模式。最后，我们将确定一种治疗性糖蛋白的糖谱，并与独立分析的结果进行比较。 与公共卫生相关：抗体等治疗性糖蛋白(生物制品)解决了目前小分子药物无法满足的关键保健需求，它们构成了制药业增长最快的部门。生物制品的蛋白质和多糖含量都必须根据FDA的指南进行广泛的表征。为了克服目前用于糖谱分析的费时费力的方法，我们正在开发一种快速、可靠和成本效益高的方法，其优点是适合于生物生产中的应用。

DESCRIPTION (provided by applicant): The biologics market is the most rapidly growing sector of the pharmaceutical industry, and the US Food and Drug Administration (FDA), as well as the European Medicines Agency (EMEA), requires that the glycoprofiles of all such therapeutic glycoproteins and antibodies be extensively characterized and fall within the specifications defined at the time of licensure. Currently, glycoprofiling of cell culture material involves extensive sample purification followed by mass spectrometric (MS) analysis, often requiring one or more weeks for completion. These long lead times make corrective action during cell cultivation impossible and the only recourse to specification failures is lot rejection, costing between $5-$50M. The availability of robust, rapid, and simple analytical methods for glycoprofiling would fundamentally alter biopharmaceutical R&D and commercial manufacturing. We will develop an innovative approach capable of in-process glycoprofiling based on combining multiplexed suspension array (MSA) technology with glycan-specific reagents. Our GlycoProf MSATM approach will provide a rapid, cost-effective, and easy to operate method for monitoring protein terminal glycosylation patterns. It will serve as a critical early warning system to protect against lot rejection. In addition, it will assist in the optimization of process development conditions. In the GlycoProf MSATM approach, flow cytometry is used to detect binding between glycans and glycan- specific reagents that are conjugated to spectrally-unique microspheres. By combining the individual reagents into a multiplexed array, the entire analysis can be obtained in less than a minute on a basic cytometer. The glycoprofile is provided by the relative fluorescence intensities for each specific glycan-MSA interaction. The GlycoProf MSATM approach will not supplant full characterization of a final product, but will address the unmet need for rapid glycoprofiling during production. We will establish optimal performance metrics for the GlycoProf MSATM method using glycans that display eukaryotic terminal sequences. Additionally, we will establish the ability of the GlycoProf MSATM method to reproduce the glycoprofiles for glycoproteins in which the glycan chains have been engineered to exhibit uniform glycosylation patterns. Finally, we will determine the glycoprofile of a therapeutic glycoproteins and compare to the results from an independent analysis. PUBLIC HEALTH RELEVANCE: Therapeutic glycoproteins (biologics) such as antibodies address critical health care needs that are currently unmet by small molecule drugs, and they constitute the most rapidly growing sector of the pharmaceutical industry. Both the protein and glycan content of biologics must be extensively characterized according to FDA guidelines. To overcome the laborious and time-consuming methods currently employed for glycoprofiling, we are developing a rapid, robust, and cost-effective method that has the advantage of being suitable for application during biologic production.