Analysis Of Antibody-drug Conjugates Research Articles

Multiepitope recognition technology promotes the in-depth analysis of antibody‒drug conjugates

The dynamic tracking of antibody‒drug conjugates (ADCs) in serum is crucial. However, a versatile bioanalytical platform is lacking due to serious matrix interferences, the heterogeneity and complex biotransformation of ADCs, and the recognition deficiencies of traditional affinity technologies. To overcome this, a multiepitope recognition technology (MERT) was developed by simultaneously immobilizing CDR and non-CDR ligands onto MOF@AuNPs. MERT's excellent specificity, ultrahigh ligand density, and potential synergistic recognition ability enable it to target the different key regions of ADCs to overcome the deficiencies of traditional technologies. The binding capacity of MERT for antibodies is ten to hundred times higher than that of the mono-epitope or Fc-specific affinity technologies. Since MERT can efficiently capture target ADCs from serum, a novel bioanalytical platform based on MERT and RPLC‒QTOF-MS has been developed to monitor the dynamic changes of ADCs in serum, including the fast changes of drug-to-antibody ratio from 3.67 to 0.22, the loss of payloads (maytansinol), and the unexpected hydrolysis of the succinimide ring of the linker, which will contribute to clarify the fate of ADCs and provide a theoretical basis for future design. In summary, the MERT-based versatile platform will open a new avenue for in-depth studies of ADCs in biological fluids.

Automated high-throughput buffer exchange platform enhances rapid flow analysis of antibody drug conjugates by high resolution mass spectrometry

Antibody drug conjugates (ADCs) are an increasingly important therapeutic class of molecules for the treatment of cancer. Average drug-to-antibody ratio (DAR) and drug-load distribution are critical quality attributes of ADCs with the potential to impact efficacy and toxicity of the molecule and need to be analytically characterized and understood. Several platform methods including hydrophobic interaction chromatography (HIC) and native size-exclusion chromatography-mass spectrometry (nSEC-MS) have been developed for that purpose; however, each presents some limitations. In this work, we assessed a new sample preparation and buffer exchange platform coupled with high-resolution mass spectrometry for characterizing the drug-load and distribution of several cysteine-linked ADCs conjugated with a variety of chemotypes. Several criteria were evaluated during the optimization of the buffer exchange-mass spectrometry system performance and the data generated with the system were compared with results from nSEC-MS and HIC. The results indicated that the platform enables automated and high throughput quantitative DAR characterization for antibody-drug conjugates with high reproducibility and offers several key advantages over existing approaches that are used for chemotype-agnostic ADC characterization.

Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. I − Optimization of separation conditions

Antibody-drug-conjugates (ADCs) manufacturing leads to a mixture of species which needs to be characterized during development and for further quality control. The coupling of on-line HIC x RPLC to high resolution mass spectrometry can be considered as a very efficient analytical method, providing extensive information on ADC sample, within a reduced time scale. Our intention in this first paper is to present the approach used to rationally optimize the numerous conditions that can affect the quality of the 2D-separation. HIC and RPLC conditions were therefore optimized to prevent salt precipitation due to solvent mixing and to enhance sensitivity, while limiting the total analysis time. We demonstrated that adding salt in the sample solvent before HIC injection allows a significant peak shape improvement. The gradient profile was also carefully optimized in both dimensions, leading to a two-step gradient in HIC and bracketed gradient in RPLC. This study shows that on-line HIC x RPLC hyphenated to high resolution mass spectrometry is a useful method to obtain rapid and extensive structural information on the peaks observed in the first HIC dimension, thereby leading, in a single step requiring 75min, to the precise determination of the average drug-to-antibody ratio (DAR) by HIC as well as the knowledge of the drug load distribution for a particular DAR. The structural characterization of ADC fragments by RPLC-QTOF will be discussed in the second part of this two-part series.

Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. II- Identification of sub-units for the characterization of even and odd load drug species.

This paper is the second part of a two-part series dedicated to the development of an on-line comprehensive HICxRPLC-UV/MS method for the characterization of a commercial inter-chain cysteine-linked ADC (brentuximab vedotin, Adcetris(®)). The first part focused on the optimization of the chromatographic conditions. In the second part of this series of papers, the structural characterization of the Brentuximab Vedotin was extensively discussed. With the combination of HIC and RPLC-MS data, the average DAR was easily measured in HIC and, at the same time, the predominant positional isomers were identified in RPLC-MS in one single injection. It was also demonstrated that the retention data obtained in the first and second dimensions was particularly useful to assist ADC characterization through the identification of sub-units. Using this methodology, the presence of odd DARs (1, 3 and 5) and their relative abundance was assessed by a systematic evaluation of HIC x RPLC-UV/MS data for both commercial and stressed ADC samples. Finally, once the exhaustive characterization of ADC was completed, MS could be conveniently replaced by UV detection to quickly assess the conformity of different ADCs batches.