Measurement of in Vivo Drug Load Distribution of Cysteine-Linked Antibody鈥揇rug Conjugates Using Microscale Liquid Chromatography Mass Spectrometry

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• 作者：Shawna Mae Hengel ; Russell Sanderson ; John Valliere-Douglass ; Nicole Nicholas ; Chris Leiske ; Stephen C. Alley
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：April 1, 2014
• 年：2014
• 卷：86
• 期：7
• 页码：3420-3425
• 全文大小：311K
• 年卷期：v.86,no.7(April 1, 2014)
• ISSN：1520-6882

文摘

Analysis of samples containing intact antibody鈥揹rug conjugates (ADC) using mass spectrometry provides a direct measurement of the drug-load distribution. Once dosed, the drug load distribution changes due to a combination of biological and chemical factors. Liquid chromatography鈥搈ass spectrometry (LC鈥揗S) methods to measure the in vivo drug load distribution have been established for ADCs containing native disulfide bonds (lysine-linked or cysteine-linked). However, because of an IgG reduction step in conjugation processes, using LC鈥揗S to analyze intact cysteine-linked ADCs requires native conditions, thus limiting sensitivity. While this limitation has been overcome at the analytical scale, to date, these methods have not been translated to a smaller scale that is required for animal or clinical doses/sampling. In this manuscript, we describe the development of ADC specific affinity capture reagents for processing in vivo samples and optimization of native LC鈥揗S methods at a microscale. These methods are then used to detect the changing drug load distribution over time from a set of in vivo samples, representing to our knowledge the first native mass spectra of cysteine-linked ADCs from an in vivo source.