Dillon Thomas M, Bondarenko Pavel V, Rehder Douglas S, Pipes Gary D, Kleemann Gerd R, Ricci Margaret Speed
Pharmaceutics Department, Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA 91320, USA.
J Chromatogr A. 2006 Jul 7;1120(1-2):112-20. doi: 10.1016/j.chroma.2006.01.016. Epub 2006 Jan 30.
An enhanced analytical RP-HPLC/MS method was developed for monitoring the stability and production of intact and fragmented monoclonal antibodies (MAbs). The use of high column temperatures (70-80 degrees C), organic solvents with high eluotropic strength coefficients (isopropyl and n-propyl alcohols), and Zorbax StableBond columns, were critical for good recovery and resolution of immunoglobulin G1 (IgG1) and IgG2 monoclonal antibodies. Using this method, cleavage products of a degraded IgG1 antibody were clearly separated and identified by in-line electrospray ionization time-of-flight (ESI-TOF) mass spectrometry generating exact masses and unique terminal ladder sequences. The glycosylation profile, including mapping of the terminal galactose and fucose heterogeneity of the N-linked sugars, was determined by mass spectrometry of intact MAbs. In addition, we discovered that several IgG2 MAbs exhibited greater structural heterogeneity compared to IgG1s. Mass spectral characterization data and reduction data suggested that the heterogeneity is disulfide related. This reversed-phase LC/MS method represents a key advancement in monitoring intact MAb production and stability.
开发了一种增强型分析反相高效液相色谱/质谱方法，用于监测完整和片段化单克隆抗体（MAb）的稳定性和生产情况。使用高柱温（70 - 80摄氏度）、具有高洗脱强度系数的有机溶剂（异丙醇和正丙醇）以及Zorbax StableBond色谱柱，对于免疫球蛋白G1（IgG1）和IgG2单克隆抗体的良好回收率和分离度至关重要。使用该方法，通过在线电喷雾电离飞行时间（ESI - TOF）质谱法清晰地分离并鉴定了降解的IgG1抗体的裂解产物，生成了精确质量和独特的末端梯状序列。通过完整MAb的质谱法确定了糖基化谱，包括N - 连接糖的末端半乳糖和岩藻糖异质性的图谱。此外，我们发现与IgG1相比，几种IgG2 MAb表现出更大的结构异质性。质谱表征数据和还原数据表明这种异质性与二硫键有关。这种反相液相色谱/质谱方法代表了监测完整MAb生产和稳定性方面的一项关键进展。
