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Quantitation of therapeutic proteins on intact level not only preserves the whole molecule information but also helps visualization of high-level structure of target protein. With improvements in High Resolution Mass Spectrometry (HRMS) instrumentation and sample preparation strategy, recent literature has demonstrated the feasibility of quantitative analysis of monoclonal antibodies (mAbs) and antibody drug conjugates (ADCs) in complex biological matrices under denaturing conditions. However, it is often more desirable to study the emerging class of therapeutic proteins with non-covalent designs in their biological states with native mass spectrometry (nMS). There is a lack of literature on intact quantitation of therapeutic proteins in biological matrices with nMS, due to limited sensitivity caused by less efficient desolvation in nMS. Here we report a high-throughput method quantifying intact mAb and ADC in plasma utilizing nMS along with a sample preparation strategy with compatible buffer systems. To determine the linear dynamic range (LDR) of this workflow, calibration curve standards were prepared using NIST mAb spiked in blank rat plasma at concentrations of 0.5, 1, 2, 5, 10, 20, 50, 100 µg/mL respectively. 100 µL of each calibration standard was subjected to affinity enrichment using anti-human IgG capture on Agilent Bravo AssayMAP platform. The eluted samples post affinity purification were subjected to LCMS analysis using Dionex Ultimate 3000 UHPLC with PolyLC PolyHYDROXYETHYL A column coupled to Bruker maXis II qTOF MS. The LDR was determined as 1 to 50 µg/mL with an LLOQ of 1 µg/mL and LOD of 0.5 µg/mL. To further evaluate this workflow, samples of an ADC rat toxicity study were analyzed. The results corelated well with bottoms-up peptide quantitation data. Moreover, average DAR could also be calculated with nMS data at different time points, that can aid in determining the the in-vivo stability of this ADC.

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Yihan Li
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Yihan Li

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Presenter
Yihan Li
AbbVie
Email: yihan.li.dmpk@abbvie.com
Hetal Sarvaiya profile image

Hetal Sarvaiya

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Co-Author
Hetal Sarvaiya
AbbVie
Email: hetal.sarvaiya@abbvie.com