Development of Comprehensive Online Two-Dimensional Liquid Chromatography/Mass Spectrometry Using Hydrophilic Interaction and Reversed-Phase Separations for Rapid and Deep Profiling of Therapeutic Antibodies

被引:90
作者
Stoll, Dwight R. [1 ]
Harmes, David C. [1 ]
Staples, Gregory O. [2 ]
Potter, Oscar G. [2 ]
Dammann, Carston T. [1 ]
Guillarme, Davy [3 ,4 ]
Beck, Alain [5 ]
机构
[1] Gustavus Adolphus Coll, Dept Chem, St Peter, MN 56082 USA
[2] Agilent Technol, Santa Clara, CA 95051 USA
[3] Univ Geneva, Sch Pharmaceut Sci, Rue Michel Servet 1, CH-1206 Geneva 4, Switzerland
[4] Univ Lausanne, Rue Michel Servet 1, CH-1206 Geneva 4, Switzerland
[5] Inst Rech Pierre Fabre, Ctr Immunol, Pierre Fabre 5,Ave Napoleon III,BP 60497, F-74160 St Julienen En Genevois, France
基金
瑞士国家科学基金会; 美国国家科学基金会;
关键词
MONOCLONAL-ANTIBODIES; MASS-SPECTROMETRY; SYSTEMATIC OPTIMIZATION; DRUG CONJUGATE; OFF-LINE; PROTEIN; PROCYANIDINS; AGGREGATION; ORIGINATOR;
D O I
10.1021/acs.analchem.8b00776
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
Monoclonal antibodies (mAb) and related molecules are being developed at a remarkable pace as new therapeutics for the treatment of diseases ranging from cancer to inflammatory disorders. However, characterization of these molecules at all stages of development and manufacturing presents tremendous challenges to existing analytical technologies because of their large size (ca. 150 kDa) and inherent heterogeneity resulting from complex glycosylation patterns and other post -translational modifications. Multidimensional liquid chromatography is emerging as a powerful platform technology that can be used to both improve analysis speed for these molecules by combining existing one-dimensional separations into a single method (e.g., Protein A affinity separation and size-exclusion chromatography) and increasing the resolving power of separations by moving from one dimension of separation to two. In the current study, we have demonstrated the ability to combine hydrophilic interaction (HILIC) and RP separations in an online comprehensive 2D separation coupled with high resolution MS detection (HILIC X RP-HRMS). We find that active solvent modulation (ASM) is critical for coupling these two separation modes, because it mitigates the otherwise serious negative impact of the acetonitrile-rich HILIC mobile phase on the second dimension RP separation. The chromatograms obtained from these HILIC X RP-HRMS separations of mAbs at the subunit level reveal the extent of glycosylation on the Fc/2 and Fd subunits in analysis times on the order of 2 h. In comparison to previous CEX x RP separations of the same molecules, we find that chromatograms from the HILIC X RP separations are richer and reveal separation of some glycoforms that coelute in the CEX X RP separations.
引用
收藏
页码:5923 / 5929
页数:7
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