유희진 (한국기초과학지원연구원)

조건 (한국기초과학지원연구원)

유희진 (한국기초과학지원연구원)

Abhik Mojumdar (한국기초과학지원연구원)

김득현 (한국기초과학지원연구원)

최선희 (한국기초과학지원연구원)

현주용 (한국기초과학지원연구원)

조건 (한국기초과학지원연구원)

Antibody-drug conjugates (ADCs) are a novel class of anticancer drugs that link a targeting antibody with a potent cytotoxic drug through a specific chemical bond. ADC combines the specific targeting capability of antibodies with the strong cytotoxicity of small molecule drugs and has the advantage of delivering cytotoxic drugs directly to lesional tissues while limiting toxicity to non-target tissues. Additionally, ADCs are widely recognized as potential cancer treatments due to their lower toxicity and long half-life compared to small molecule drugs.

In this study, we evaluated various assays to determine the antibody-drug ratio (DAR) using cysteine ​​linker ADC. Cysteine-​​linked ADC undergoes partial reduction of the disulfide bond of the antibody to form a free cysteine ​​residue, and then connects this residue with a small molecule drug to form an antibody-drug conjugate mixture of 0 to 8 drugs [1]. Drug-to-antibody ratio (DAR), which refers to the average number of drugs linked to each antibody, is an important metric for ADC quality, as it can have a significant impact on the efficacy of the ADC. This is because low DAR values ​​may result in lower effectiveness, while relatively high DAR values ​​may have a negative impact on safety.

This study employed liquid chromatography (LC) coupled with Q-TOF mass spectrometry (Cyclic Ion mobility MS) using SEC and reversed-phase LC columns. Both the non-deglycosylated native analysis and the deglycosylated and reduced ADC were analyzed using different methods, and each component was separated by chromatography and confirmed using a mass spectrometer and a diode array detector. Additionally, the weighted average drug-to-antibody ratio (DAR) of the ADC was calculated by integrating the area percentage of each light chain and heavy chain peak in the UV absorption spectrum and correlating it with the number of drug conjugates associated with each peak. The results were obtained by calculating the mass spectral deconvolution data at averaging time using the DAR Calculator.

As a result, for ADCs with low average DAR, it is recommended to combine two or more complementary techniques to determine DAR values accurately. Additionally, to accurately measure the DAR of disulfide bond-reduced ADC samples, it is beneficial to apply additional technologies (such as HIC) to various ADC samples and conduct comparative analysis. As there are more technologies proven to aid in ADC analysis, the field continues to evolve, and many challenges remain.