In-process glycoprofiling during recombinant therapeutic glycoprotein production

描述（由申请人提供）：生物制剂市场是制药行业增长最快的领域，美国食品药品监督管理局（FDA）以及欧洲药品管理局（EMEA）要求对所有此类治疗性糖蛋白和抗体的糖谱进行广泛表征，并在许可时定义的质量标准范围内。目前，细胞培养材料的糖谱分析涉及广泛的样品纯化，然后进行质谱（MS）分析，通常需要一周或多周才能完成。这些长的交付周期使得在细胞培养期间不可能采取纠正措施，并且对规格失败的唯一求助是批次拒收，成本在500万至5000万美元之间。强大的，快速的，简单的糖分析方法的可用性将从根本上改变生物制药的研发和商业生产。我们将开发一种创新的方法，能够在过程中糖分析的基础上结合多重悬浮阵列（MSA）技术与聚糖特异性试剂。我们的GlycoProf MATM方法将提供一种快速、经济、易于操作的方法来监测蛋白质末端糖基化模式。它将作为一个重要的早期预警系统，以防止批次拒收。此外，它将有助于优化工艺开发条件。在GlycoProf MATM方法中，流式细胞术用于检测聚糖与缀合至光谱独特微球的聚糖特异性试剂之间的结合。通过将单个试剂组合成多重阵列，可以在基本细胞仪上在不到一分钟的时间内获得整个分析。通过每种特定聚糖-MSA相互作用的相对荧光强度提供糖谱。GlycoProf MATM方法不会取代最终产品的完整表征，但将解决生产过程中快速糖谱分析的未满足需求。我们将使用显示真核生物末端序列的聚糖建立GlycoProf MATM方法的最佳性能指标。此外，我们将建立GlycoProf MATM方法重现糖蛋白糖谱的能力，其中聚糖链已被工程化以表现出均匀的糖基化模式。最后，我们将确定治疗性糖蛋白的糖谱，并与独立分析的结果进行比较。 公共卫生相关性：治疗性糖蛋白（生物制剂），如抗体，解决了目前小分子药物无法满足的关键医疗保健需求，它们构成了制药行业增长最快的部门。生物制剂的蛋白质和聚糖含量必须根据FDA指南进行广泛表征。为了克服目前用于糖谱分析的费力和耗时的方法，我们正在开发一种快速，稳健和具有成本效益的方法，该方法具有适合在生物生产过程中应用的优点。

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.