Daniel Waldera-Lupa | ProtaGene

Festival of Biologics Day 1 @ 15:00

Improving ADC development: high-throughput and in-depth characterization of ADCs by high-resolution mass spectrometry

Antibody-drug conjugates (ADCs) are targeted biopharmaceuticals that combine potent cytotoxic drugs with highly selective monoclonal antibodies to deliver chemotherapy specifically to cancer cells, reducing damage to healthy tissue and minimizing side effects. This allows for higher drug doses and increased efficacy. However, ADCs with lysine or cysteine conjugation sites are highly heterogeneous, making their characterization challenging. Determining conjugation sites accurately is key to effective targeting. The drug-to-antibody ratio (DAR) also impacts efficacy, safety, and therapeutic potential. High-resolution mass spectrometry is essential for characterizing ADCs, providing data on conjugation sites, molecular weight, stability, and impurities, and aiding in optimization and quality control.

In the present study, we have developed two analytical workflows for the characterization of ADCs:

1.    High-Throughput SEC-MS Workflow: This approach operates under native conditions, enabling rapid analysis of drug load distribution and DAR via fully automated data processing.

2.    High-Resolution RPLC-MS/MS Workflow: This method facilitates precise determination of conjugation sites, quantification of site occupancy, and analysis of other post-translational modifications.

In a forced degradation analysis, we demonstrated that exposure to elevated temperature stress significantly impairs both the DAR and the conjugation site, highlighting the vulnerability of ADCs under stress conditions. In addition to the detailed assessment of conjugation site occupancy, we were also able to identify and quantify several critical quality attributes (CQA) of the antibodies, including several deamidation and oxidation sites. These results provide valuable insights into the stability and integrity of ADCs and emphasize the importance of stringent quality control in the development process.

With our approach, we show that these analytical workflows are very well suited for both high-throughput and comprehensive characterization of lysine- and cysteine-conjugated ADCs and can be transferred to ADCs with other conjugation strategies. In general, this approach can be used in the development process of ADCs for the comparison of production batches, stability studies and forced degradation analyses for quality control and assurance.

Daniel Waldera-Lupa, ProtaGene

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Daniel Waldera-Lupa / Improving ADC development: high-throughput and in-depth characterization of ADCs by high-resolution mass spectrometry

Daniel Waldera-Lupa, ,
ProtaGene

Born in Bochum, Germany, in 1984, with a passion for science and a drive for discoveryStudied biochemistry at the Ruhr University Bochum, graduating with a Master of Science in Biochemistry in 2009Awarded a Ph.D. (summa cum laude) in Chemistry and Biochemistry from Heinrich-Heine University...

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