Nano-scale liquid chromatography/mass spectrometry and on-the-fly orthogonal array optimization for quantification of therapeutic monoclonal antibodies and the application in preclinical analysis.
Nano-scale liquid chromatography/mass spectrometry and on-the-fly orthogonal array optimization for quantification of therapeutic monoclonal antibodies and the application in preclinical analysis.
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纳米级液相色谱/质谱和直接正交阵列优化，用于定量治疗性单克隆抗体以及在临床前分析中的应用。
DOI：
10.1016/j.chroma.2012.06.007
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发表时间：
2012-08-17
期刊：
影响因子：
--
通讯作者：
Qu J
中科院分区：
文献类型：
--
作者：
Duan X;Dai L;Chen SC;Balthasar JP;Qu J
Therapeutic monoclonal antibodies (mAb) constitute a group of highly effective agents for treating various refractory diseases. Nonetheless it is challenging to achieve selective and accurate quantification of mAb in pharmaceutical matrices, which is required by PK studies. Liquid chromatography/mass spectrometry under selected reaction monitoring mode (LC/SRM-MS) is emerging as an attractive alternative to immunoassays because of the high specificity and multiplexing capacity it provides, but may fall short in terms of sensitivity, reliability and quantitative accuracy. Moreover, the strategy for optimization of the MS conditions for many candidates of signature peptides (SP) and the selection of the optimal SP for quantification remains elusive. In this study, we employed a suite of technical advances to overcome these difficulties, which include i) a nano-LC/SRM-MS approach to achieve high analytical sensitivity, ii) a high-resolution nano-LC/LTQ/Orbitrap for confident identification of candidate peptides, iii) an on-the-fly orthogonal array optimization (OAO) method for the high-throughput, accurate and reproducible optimization for numerous candidate peptides in a single LC/MS run without using synthesized peptides, iv) a comprehensive evaluation of stability of candidates in matrix using the optimized SRM parameters, v) the use of two unique SP for quantification of one mAb to gauge possible degradation/modification in biological system and thus enhancing data reliability (e.g. rejection of data if the deviation between the two SP is greater than 25%) and vi) the utilization of purified target protein as the calibrator to eliminate the risk of severe negative biases that could occur when a synthesized peptide is used as calibrator. To show a proof of concept, this strategy is applied in the quantification of cT84.66, a chimeric, anti-CEA antibody, in preclinical mouse models. A low detection limit of the mAb down to 3.2 ng/mL was achieved, which is substantially more sensitive than established immunoassay methods for anti-CEA antibodies. The quantitative method showed good linearity (within the range of 12.9 ng/mL to 32.3 µg/mL in plasma), accuracy and precision. Additionally, the ultra-low sample consumption (2 µL plasma per preparation) permits the acquisition of an entire set of time course data from the same mouse, which represents a prominent advantage for PK study using small-animal models. The developed method enabled an accurate PK investigation of cT84.66 in mice following intravenous and subcutaneous administrations at relatively low doses over an extended period of time. The strategy employed in this study can be easily adapted to the sensitive and accurate analysis of other mAb and therapeutic proteins.
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影响因子：
7.4
作者：
Hagman, Charlotte;Ricke, Darrell;Bitsch, Francis
通讯作者：
Bitsch, Francis
影响因子：
4.4
作者：
Anderson, NL;Anderson, NG;Pearson, TW
通讯作者：
Pearson, TW
影响因子：
7.4
作者：
Cao, Jin;Covarrubias, Vanessa M.;Straubinger, Robert M.;Wang, Hao;Duan, Xiaotao;Yu, Haoying;Qu, Jun;Blanco, Javier G.
通讯作者：
Blanco, Javier G.
影响因子：
4.4
作者：
Duan X;Young R;Straubinger RM;Page B;Cao J;Wang H;Yu H;Canty JM;Qu J
通讯作者：
Qu J
影响因子：
64.8
作者：
Mellman, Ira;Coukos, George;Dranoff, Glenn
通讯作者：
Dranoff, Glenn