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Real-Time Analysis on Drug-Antibody Ratio of Antibody-Drug Conjugates for Synthesis, Process Optimization, and Quality Control.

ABSTRACT: Drug-antibody ratio (DAR) of antibody-drug conjugates (ADCs) is important for their therapeutic efficacy and pharmacokinetics, therefore control on DAR in synthesis process is a key for ADC quality control. Although various analytical methods were reported, the real-time monitoring on DAR is still a challenge because time-consuming sample preparation is usually needed during the analysis. Antibody deglycosylation of ADC simplifies DAR measurement, however long-time PNGaseF digestion for deglycosylation hampers the real-time detection. Here, we report a rapid DAR analysis within 15?min by robust deglycosylation treatment and LC-MS detection that enables real-time DAR monitoring for optimization on ADC synthetic process. With this approach, we were able to screen suitable conjugation conditions efficiently and afford the ADCs with expected DARs. To the best of our knowledge, this is the first report on real-time DAR analysis of ADCs for conjugation optimization and quality control, compatible with random lysine-linked ADCs, glycosite-specific ADCs, and the complicated dual-payload ADCs.

SUBMITTER: Tang Y

PROVIDER: S-EPMC5552727 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Real-Time Analysis on Drug-Antibody Ratio of Antibody-Drug Conjugates for Synthesis, Process Optimization, and Quality Control.

Tang Yubo Y Tang Feng F Yang Yang Y Zhao Lei L Zhou Hu H Dong Jinhua J Huang Wei W

Scientific reports 20170810 1

Drug-antibody ratio (DAR) of antibody-drug conjugates (ADCs) is important for their therapeutic efficacy and pharmacokinetics, therefore control on DAR in synthesis process is a key for ADC quality control. Although various analytical methods were reported, the real-time monitoring on DAR is still a challenge because time-consuming sample preparation is usually needed during the analysis. Antibody deglycosylation of ADC simplifies DAR measurement, however long-time PNGaseF digestion for deglycos ...[more]

PMID: 28798339

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Project description:Photoimmunotherapy (PIT) is an emerging low side effect cancer therapy based on a monoclonal antibody (mAb) conjugated with a near-infrared (NIR) phthalocyanine dye IRDye 700DX. IR700 is fluorescent, can be used as an imaging agent, and also is phototoxic. It induces rapid cell death after exposure to NIR light. PIT induces highly selective cancer cell death, while leaving most of tumor blood vessels unharmed, leading to an effect called super-enhanced permeability and retention (SUPR). SUPR significantly improves the effectiveness of the anticancer drug. Currently, the therapeutic effects of PIT are monitored using the IR700 fluorescent signal based on macroscopic fluorescence reflectance imagery. This technique, however, lacks the resolution and depth information to reveal the intratumor heterogeneity of mAb-IR700 distribution. We applied a minimally invasive two-channel fluorescence fiber imaging system by combining the traditional fluorescence imaging microscope with two imaging fiber bundles (~0.85mm). This method monitored mAb-IR700 distribution and therapeutic effects during PIT at different intratumor locations (e.g., tumor surface vs. deep tumor) in situ and in real time simultaneously. This enabled evaluation of the therapeutic effects in vivo and treatment regimens. The average IR700 fluorescence intensity recovery after PIT to the tumor surface is 91.50%, while it is 100.63% in deep tumors. To verify the results, two-photon microscopy combined with a microprism was also used to record the mAb-IR700 distribution and fluorescence intensity of green fluorescent protein (GFP) at different tumor depths during PIT. After PIT treatment, there was significantly higher IR700 fluorescence recovery in deep tumor than in the tumor surface. This phenomenon can be explained by increased vascular permeability immediately after NIR-PIT. Fluorescence intensity of GFP at the tumor surface decreased significantly more compared to that of deep tumor and in controls (no PIT).

Dataset Information

Real-Time Analysis on Drug-Antibody Ratio of Antibody-Drug Conjugates for Synthesis, Process Optimization, and Quality Control.

Publications

Real-Time Analysis on Drug-Antibody Ratio of Antibody-Drug Conjugates for Synthesis, Process Optimization, and Quality Control.

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