Analytical Sciences, Amgen, Inc., 4000 Nelson Rd., Longmont, CO 80503, USA.
Glycobiology. 2010 May;20(5):629-47. doi: 10.1093/glycob/cwq012. Epub 2010 Jan 27.
Regulatory agencies' expectations for biotherapeutic approval are becoming more stringent with regard to product characterization, where minor species as low as 0.1% of a given profile are typically identified. The mission of this manuscript is to demonstrate a multi-method approach toward de novo glycan characterization and quantitation, including minor species at or approaching the 0.1% benchmark. Recently, unexpected isomers of the Man(5)GlcNAc(2) (M(5)) were reported (Prien JM, Ashline DJ, Lapadula AJ, Zhang H, Reinhold VN. 2009. The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap mass spectrometry (MS). J Am Soc Mass Spectrom. 20:539-556). In the current study, quantitative analysis of these isomers found in commercial M(5) standard demonstrated that they are in low abundance (<1% of the total) and therefore an exemplary "litmus test" for minor species characterization. A simple workflow devised around three core well-established analytical procedures: (1) fluorescence derivatization; (2) online rapid resolution reversed-phase separation coupled with negative-mode sequential mass spectrometry (RRRP-(-)-MS(n)); and (3) permethylation derivatization with nanospray sequential mass spectrometry (NSI-MS(n)) provides comprehensive glycan structural determination. All methods have limitations; however, a multi-method workflow is an at-line stopgap/solution which mitigates each method's individual shortcoming(s) providing greater opportunity for more comprehensive characterization. This manuscript is the first to demonstrate quantitative chromatographic separation of the M(5) isomers and the use of a commercially available stable isotope variant of 2-aminobenzoic acid to detect and chromatographically resolve multiple M(5) isomers in bovine ribonuclease B. With this multi-method approach, we have the capabilities to comprehensively characterize a biotherapeutic's glycan array in a de novo manner, including structural isomers at >/=0.1% of the total chromatographic peak area.
监管机构对生物治疗药物批准的期望越来越严格，需要对产品特征进行描述，通常可以鉴定出低至给定图谱的 0.1%的微量物质。本文的任务是展示一种从头开始的聚糖特征描述和定量分析的多方法方法，包括接近或达到 0.1%基准的微量物质。最近，报道了（Prien JM、Ashline DJ、Lapadula AJ、Zhang H、Reinhold VN。2009. 牛核糖核酸酶 B 的高甘露糖聚糖异构体通过离子阱质谱（MS）的特征化。J Am Soc Mass Spectrom. 20:539-556）出人意料的 Man(5)GlcNAc(2)（M(5)）异构体。在本研究中，对商业 M(5)标准中发现的这些异构体的定量分析表明，它们丰度较低（<总含量的 1%），因此是微量物质特征描述的典型“试金石”。围绕三个核心成熟分析程序设计了一个简单的工作流程：（1）荧光衍生化；（2）在线快速分辨反相分离与负模式顺序质谱（RRRP-(-)-MS(n)）；（3）纳米喷雾顺序质谱（NSI-MS(n)）的全甲基化衍生化，提供全面的聚糖结构确定。所有方法都有局限性；然而，多方法工作流程是一种在线临时解决方案/解决方案，可以减轻每种方法的个别缺点，为更全面的特征描述提供更大的机会。本文首次证明了 M(5)异构体的定量色谱分离，并使用市售的 2-氨基苯甲酸的稳定同位素变体来检测和色谱分离牛核糖核酸酶 B 中的多个 M(5)异构体。通过这种多方法方法，我们有能力以从头开始的方式全面描述生物治疗药物的聚糖图谱，包括总色谱峰面积的>=/0.1%的结构异构体。
