ADC Analysis and Characterization

Jeon et al. synthesized and evaluated antibody-drug conjugates (ADCs) with high drug-to-antibody ratios (DARs) using bismaleimide-DM1 as the linker-payload. They combined the highly effective compound DM1 with trastuzumab. To achieve high DARs, various bismaleimide linkers of different lengths were designed and synthesized to link DM1 to reduced cysteine via simple conjugation. LC and MS analyses showed that the resulting trastuzumab ADCs exhibited significant consistency. The experimental process is outlined below:

Linker-Payload Design and Synthesis

To prepare linkers with bismaleimide functional groups (Fig. 1a), they selected an approach involving the simplest alkyl diamine chains. It was expected that the yield of the interaction between the linker-payload and the antibody would fluctuate depending on the length of the alkyl chain, so bismaleimide functional groups were selected with the simplest carbon chain lengths ranging from 2 to 6. Using techniques outlined by Lorenzini et al. in 2014, they generated maleimides from diamines and successfully synthesized high-yield bismaleimide linkers. Subsequently, DM1 was conjugated to the bismaleimide linker through 1,4-addition, obtaining the final linker-payload (Fig. 1b). After completing a total of 2 steps, linkers with varying lengths carrying DM1 were obtained, which were then conjugated to trastuzumab (Fig. 1c).

Fig. 1. Structures of the linker, linker-payload, and ADCs (Bioorganic Chemistry. 149 (2024) 107504).

ADC Generation and Characterization

To generate ADCs using the established linker-payloads, trastuzumab was fully reduced with tris(2-carboxyethyl)phosphine (TCEP), yielding 8 available thiols. The linker-payloads, previously dissolved in DMSO, were then mixed and conjugated at appropriate temperatures, resulting in ADC formation. Hydrophobic interaction chromatography (HIC) column results showed that most trastuzumab molecules participated in the conjugation process, with no unreacted trastuzumab remaining. The homogeneity of the ADCs was found to vary with the alkyl chain length of the linker, while following the same conjugation conditions. Moreover, for ADC 2, BOC Sciences® analysis confirmed that using 20 equivalents of linker-payload resulted in a DAR of 8 (Table 1). They then doubled the amount of trastuzumab, obtaining a DAR close to 8. SDS-PAGE and LC-MS data confirmed that the DAR of the linker-payload was close to 8. Thus, they determined the optimal alkyl chain length to be 3 to 5 carbon atoms. Analysis of aggregates using a size exclusion chromatography (SEC) column indicated that aggregation rates increased with longer linker carbon chains.

In Vitro Testing, Binding Assay, Serum Stability, and Pharmacokinetics Testing

In addition, in vitro testing and binding assays compared the binding affinity between ADCs and T-Dxd. Through experimental studies, the authors concluded that ADC 2 exhibited good binding affinity compared to T-Dxd. Serum stability testing showed satisfactory results in mouse, rat, and human serum compared to T-Dxd. Pharmacokinetics (PK) profile studies confirmed the view that linker length significantly affects PK. The results on efficiency, stability, and PK profiles are crucial for PK and in vivo studies in animal tumor models in future research projects. Moreover, combining the current experimental results, there is sufficient evidence to suggest that dosing intervals may be increased before starting clinical trials.

ADC analysis methods mainly include chromatography, mass spectrometry, fluorescence microscopy, flow cytometry, and bioassays.

• Chromatography is used to separate and quantify the different components of molecules. High-performance liquid chromatography (HPLC) is commonly used to analyze the purity and stability of ADCs, as well as to monitor binding efficiency and DAR. Size exclusion chromatography (SEC) is another important technique to evaluate the size distribution and aggregation of ADCs, which affects their pharmacokinetics and efficacy.
• Mass spectrometry provides detailed information about a molecule's molecular weight, structure, and composition. Liquid chromatography-mass spectrometry (LC-MS) is commonly used to analyze intact ADCs and their subunits, as well as to identify and quantify drug payloads attached to antibodies. Tandem mass spectrometry (MS/MS) can be used to sequence the peptide chains of antibodies and verify site-specific conjugation of drugs.
• Fluorescence microscopy and flow cytometry enable the visualization and quantification of binding and internalization of molecules in cancer cells. These technologies can provide insights into the mechanisms of action of ADCs and help optimize their design to improve targeting and efficacy.
• Additionally, bioassays are important analytical methods for assessing the potency and efficacy of ADCs, measuring their ability to kill cancer cells or inhibit tumor growth. Cell-based assays, such as cytotoxicity assays and proliferation assays, are commonly used to assess the in vitro antitumor activity of ADCs. In vivo efficacy studies can also be performed using xenograft models to evaluate the therapeutic potential of ADCs in preclinical settings.

For ADC drugs, different coupling sites and coupling processes will lead to heterogeneity in loading and distribution of ADC molecules (Fig. 1), thus affecting drug efficacy and safety. Currently, there are a variety of analytical methods that can measure the drug-antibody ratio (DAR) at the complete quality level to achieve characterization analysis of the overall drug load and distribution of ADC, ensuring the safety and effectiveness of ADC drugs and supporting process optimization during development and production. In-depth characterization of the exact site where coupling occurs and the load distribution of the coupling site requires the use of peptide mapping through the LC-MS/MS platform.

Fig. 1. ADC drug conjugation methods.

LC-MS/MS peptide mapping analysis

As an important analytical tool, LC-MS/MS peptide map analysis is widely used in macromolecule drug characterization research to obtain primary and secondary structure information of molecules. The enzymatically hydrolyzed peptide sample is separated by liquid chromatography and then entered into high-resolution mass spectrometry for analysis. Regular secondary ions are obtained through a specific ion dissociation method (Fig. 2). The known sequence database and the collected mass spectrometry data are matched through data processing software to confirm the peptide sequence information. At the same time, for possible modifications on the peptide, taking into account the mass difference caused by the modification, by comparing the primary and secondary mass spectrometry data of the natural peptide and the modified peptide, the occurrence and location of the modification can be identified.

Fig. 2. Specific dissociation modes of peptides under different dissociation technologies.

For ADC drugs, the conjugated drug-linker can also be regarded as a variable modification, refer to the analysis strategy of PTM in characterization analysis. However, because drug-linker has a larger molecular weight and more complex structure than common PTM modifications (oxidation, deamidation). Therefore, in the current common HCD/CID dissociation mode, the drug-linker will fragment and cannot remain intact on the peptide fragment. At the same time, it may also affect the dissociation efficiency of amide bonds in the peptide sequence. Therefore, during data analysis, it is usually necessary to combine software search with manual confirmation strategies to ensure the identification of coupled peptides. In summary, the general process of coupling site analysis based on LC-MS/MS is shown in Fig. 3.

Fig. 3. General procedure for ADC coupling site analysis.