Preface

Antibody drug conjugates (ADCs) are a type of therapeutic biological product that can accurately identify target cells through antibodies, enter target cells through endocytosis, and release conjugated small molecule drugs, thereby achieving precise killing of target cells and minimizing the killing of normal cells to the greatest extent possible. Due to the design principle of ADC, it is metaphorically referred to as a "biological missile" since the first ADC drug Mylotarg was developed in 2000 ® After gemtuzumab ozogamicin was approved by the FDA, as of June 2025, 19 ADCs have been approved for market worldwide. Since the global ADC sales exceeded 10 billion US dollars for the first time in 2023, the global ADC sales will continue to exceed 10 billion US dollars in 2024, maintaining a strong growth momentum.

Among the top ten ADCs in terms of global ADC sales in 2024, the second ranked one is KADCYLA produced by Roche. The drug was approved by FDA for marketing in 2013 and used for the treatment of HER2 positive breast cancer. Its global sales will be about 2.3 billion dollars in 2024 [2]. Enmetuximab is an ADC targeting HER2, containing humanized anti-HER2 IgG1 trastuzumab. The antibody covalently binds to microtubule inhibitory drug DM1 (a derivative of metoprolol) through a stable thioether linker MCC (4- [N-Maleimidomethyl] cyclohexane-1-carboxylate), with a molecular weight distribution ranging from 147 to 158 kDa. Its drug to antibody ratio (DAR) ranges from 0 to 8, with an average DAR of approximately 3.5 [3]. Due to the complexity of its structure, characterizing it becomes quite challenging. In this study, we used the newly released material at this year's American Society for Mass Spectrometry (ASMS) conferenceNew generation 2-in-1 ultra-high resolution mass spectrometer Exceeding Pro BiopharmaThe comprehensive characterization of KADCYLA has demonstrated its excellent analytical capabilities in the field of biopharmaceuticals, especially its electron transfer/higher energy collision dissociation (EThcD) function, which helps to accurately identify drug coupling sites and post translational modification (PTM) identification.

Application Highlights

As mentioned earlier, the new generation of 2-in-1 ultra-high resolution mass spectrometer Excesion Pro Biopharma, which was released this year, was used for in-depth characterization of KADCYLA. The highlights of this instrument include but are not limited to expanding the mass detection range to m/z=12000 Da (requiring BioPharma option), which can meet the molecular weight determination of monoclonal antibody monomers/dimers/trimers, as well as other high molecular weight protein complexes under non denaturing conditions; The optional ETD function can achieve fast and sensitive ETD/EThcD fragmentation, and when used in combination with High energy Collisional Dissection (HCD), it can obtain comprehensive and rich protein sequence coverage as well as PTM identification information. The structure of the Exceeding Pro Biopharma instrument is shown in Figure 1, and Figure 2 illustrates the process of in-depth characterization of KADCYLA in this experiment.

Figure 1. Structure diagram of Exceeding Pro BioPharma, with the hardware updates highlighted in blue compared to the previous generation product. The optional BioPharma Edition can expand the quality testing range to m/z=12000 Da (green box), and the optional ETD can achieve ETD/EThcD fragmentation (red box). (Click to view large image)

Figure 2. KADCYLA characterization flowchart.

(Click to view large image)

Slide down to view all content

Determination of complete molecular weight of KADCYLA under non denaturing conditions

Complete molecular weight determination is an essential part of the characterization of biopharmaceutical products. For ADCs, in addition to complete molecular weight, the average DAR value and drug load distribution (DLD) are also key attributes for evaluating drug quality. In this experiment, we conducted a complete molecular weight determination of KADCYLA under non denaturing conditions. Under non denaturing conditions, a neutral buffer salt system is used. Compared with denaturing conditions, protein molecules are closer to their natural state, and under non denaturing conditions, proteins have less charge and less overlap in mass spectrometry peaks between adjacent charge states. This can reduce mutual interference between mass spectrometry signals and help obtain more accurate and complete molecular weight determination results. Figure 3 shows the results of complete molecular weight determination of KADCYLA under non denaturing conditions. In order to make the sample closer to its natural state, we did not perform sugar removal treatment on it. As shown in Figure 3A, at the level of the original spectrum, the mass spectrometry peaks coupled with different numbers of drugs and different sugar components basically achieved baseline separation. After deconvolution, the drug coupling distribution of 0-8 can be clearly observed (Figure 3B). The BioPharma Finder software can automatically calculate the average DAR value, and the average DAR value of this ADC is 3.47 (Figure 3C), which is consistent with the reported 3.5 in the literature.

（A）

（B）

（C）

Figure 3. Complete molecular weight determination results of KADCYLA under non denaturing conditions. A, Original spectrogram and local magnified image. B, Deconvolutional spectrogram. C. The BioPharma Finder software can automatically select relevant components and calculate the average DAR value. (Click to view large image)

Slide down to view all content

Data dependent EThcD secondary fragmentation is used for peptide mapping analysis,

Realize coupling site localization and PTM identification

Peptide mapping analysis based on liquid chromatography-mass spectrometry is one of the commonly used analytical methods in the characterization of biological therapeutic products, which can achieve sequence coverage, localization of various chemical modification sites, and qualitative and quantitative analysis of common post-translational modifications. Among various mass spectrometry fragmentation techniques, HCD fragmentation is widely used in peptide mapping analysis due to its ability to break peptide bonds and generate abundant b/y fragment ions for peptide identification. However, for some modified peptide segments, such as glycopeptides and linker drug coupled peptide segments, HCD fragmentation cannot accurately locate the modification sites for peptide segments with multiple potential modification sites due to the fragmentation of the sugar chain/linker drug coupling by HCD; In addition, for some peptide segments containing isomerized amino acids, such as leucine/isoleucine, aspartic acid/aspartic acid isomers, it is impossible to distinguish them due to the same mass of b/y ions produced by HCD. However, due to different reaction mechanisms, ETD can not only fragment the peptide backbone while fully preserving side chain modifications such as sugar chains and linker drug coupling, but also generate characteristic diagnostic ions for isomerized amino acids, thereby achieving identification and differentiation of isomerized amino acids. On the basis of ETD fragmentation, adding HCD assisted fragmentation, i.e. EThcD, can simultaneously obtain b/y and c/z ions in the same secondary spectrum, obtaining more comprehensive and rich information to characterize peptide segments, especially modified/isomerized peptide segments. After equipping the Exceeding Pro 2-in-1 ultra-high resolution mass spectrometer with the ETD option, rapid and sensitive ETD/ETchD fragmentation can be achieved. The data dependent EThcD secondary fragmentation (dd EThcD MS2) data acquisition mode can be set to complement the data dependent HCD secondary fragmentation (dd HCD MS2) to obtain comprehensive peptide analysis information.

In this study, we used Trypsin and AspN proteases to denature KADCYLA. Subsequently, dd HCD MS2 and dd EThcD MS2 data were collected from samples hydrolyzed by two different proteases to obtain information on sequence coverage, coupling site distribution, and post-translational modifications. Samples digested with dd HCD MS2, Trypsin, and AspN enzymes can achieve 100% sequence coverage with one injection (data not shown). Figure 4 shows the base peak chromatograms (BPC) and sequence coverage maps of peptide analysis samples using dd EThcD MS2, Trypsin, and AspN enzymatic hydrolysis, which can also achieve 100% sequence coverage for one injection. This proves that the ETD/ETchD fragmentation of the Exceeding Pro 2-in-1 ultra-high resolution mass spectrometer is compatible with conventional flow rate liquid chromatography, obtaining high-quality secondary spectra for peptide analysis.

Figure 4. KADCYLA peptide plot analysis based on peak spectrum and sequence coverage, both in dd ETchD MS2 data acquisition mode. Left, Trypsin enzymatic hydrolysis sample. Right, AspN enzymatic hydrolysis sample. For samples hydrolyzed by different proteases, single needle injection can achieve 100% sequence coverage. (Click to view large image)

As mentioned earlier, the linker drug (MCC-DM1) of KADCYLA is covalently coupled to lysine, and the N-terminus of trastuzumab also undergoes coupling. Therefore, for the entire KADCYLA molecule, there are a total of 92 potential coupling sites, including 46 non repeating sites (Figure 5). Due to the steric hindrance caused by covalent coupling of lysine, trypsin cleavage occurs, resulting in the production of linker drug coupled peptide segments containing multiple lysine residues. However, HCD fragmentation simultaneously breaks down the peptide backbone and the coupled drug, making it difficult to accurately determine the drug coupling site solely based on HCD secondary spectra. Due to different fragmentation mechanisms, EThcD can fragment the peptide backbone while retaining drug coupling groups. The optimized HCD assisted fragmentation energy can make the peptide fragmentation more complete, while maximizing the retention of fully coupled drugs. Table 1 shows the summary of drug coupling site information obtained by secondary fragmentation of Trypsin enzymatic samples using HCD and EThcD, respectively. Among the 46 potential modification sites, 43 were identified to have drug modifications, indicating that EThcD fragmentation can accurately locate the coupling sites for peptides containing multiple potential coupling sites. Combined with HCD results, comprehensive and detailed coupling site information can be obtained.

Figure 5. All potential modification sites of KADCYLA molecule are marked with red boxes in the protein sequence, with a total of 92 sites, including 46 non duplicated sites.

(Click to view large image)

Table 1. Summary of KADCYLA Identification Sites

(Click to view large image)

Red font, coupling site. ++", the coupling site can be confirmed by secondary fragment ions. There is a coupling on the peptide segment, but the specific coupling position cannot be confirmed by fragment ions. - ", coupling not identified.

Slide down to view all content

When using AspN for enzymatic hydrolysis of KADCYLA, the heavy chain produces a peptide segment D containing three lysine residuesK(216)K(217)VEPK(221)SC， And according to the peptide analysis results of trypsin hydrolyzed samples, it can be concluded that all three lysines may be coupled. The MCC-DM1 linker drug of KADCYLA contains an isomer center (Figure 6, top left), which results in the drug coupled peptide segments being eluted in the form of peaks on reverse phase chromatography. As shown in Figure 6, due to the presence of multiple potential coupling sites and MCC-DM1 isomerization centers, peptide segment DK(216)K(217)VEPK(221)The extraction ion current (XIC) of SC shows multiple sets of peaks. Thanks to the informative secondary spectrum generated by EThcD fragmentation, it is possible to accurately identify which site of peptide segments eluted at different retention times has undergone coupling (Figure 6, bottom), and the relative proportion of each modification can be calculated based on the XIC peak area (Figure 6, top right).

Figure 6. Identification of coupling sites in peptide segments containing multiple coupling sites using EThcD. (Click to view large image)

For ADC samples, in addition to drug coupling sites, various post-translational modifications related to drug quality also need to be characterized and monitored. Figure 7 shows the peptide FNWYViso based on the EThcD secondary fragmentation spectrumD(283)Identification results of aspartic acid isomerization of GVEVHNAK. Thanks to the high sensitivity and high-quality accuracy of the instrument platform, even the isomerized peptide segments with a relative content of only 0.18% unmodified peptide segments can still identify abundant c/z ions, as well as the characteristic c+57/z-57 ions produced by aspartic acid isomerization in ETD fragmentation, thus achieving the confirmation of aspartic acid isomerization. For other common post-translational modifications such as deamidation, oxidation, and glycosylation, relative quantitative results can be obtained (Figure 8).

Figure 7. Confirmation of aspartic acid isomerization using EThcD fragmentation. The magnified image below shows the c+57/z-57 ions. (Click to view large image)

Figure 8. Other common post-translational modifications relative quantitative results, all of which are the average values of three repeated injections of dd ETchD MS2. A, Methionine oxidation. B, Deamidation of asparagine. C, Asparagine succinylation. D, Tryptophan oxidation. E, Heavy chain N-glycosylation. (Click to view large image)

Slide down to view all content

summary

This article presents the experimental results of characterizing lysine coupled ADC KADCYLA using the new generation 2-in-1 ultra-high resolution mass spectrometer Excesion Pro BioPharma. Under non denaturing conditions, complete molecular weight determination of KADCYLA can be performed, and the distribution of drug coupling numbers from 0 to 8 can be measured without deglycosylation. After software processing, the average DAR value obtained is 3.47, which is consistent with the reported value of 3.5; The rapid and sensitive fragmentation of EThcD not only enables 100% sequence coverage for single needle injection, common post-translational modification identification and relative quantification, but also enables identification of coupling sites and isomerized amino acids, providing reliable results for the comprehensive characterization of complex biomolecules.

References:

[1] Fu, Z., Li, S., Han, S. et al. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Sig Transduct Target Ther 7, 93 (2022).

[2] https://assets.roche.com/f/176343/x/38d96ed8ec/fb24e.pdf

[3] Joubert, N., Beck, A., Dumontet, C., Denevault-Sabourin, C. Antibody–Drug Conjugates: The Last Decade. Pharmaceuticals 2020, 13, 245.

If you need to collaborate in reprinting this article, please leave a message at the end of the article.