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Antibody-drug conjugate (ADC) is a complex that conjugates cytotoxin to monoclonal antibodies through a constructed linker and selectively delivers cytotoxins to tumors. With rich experience and extensive professional knowledge, BOC Sciences is responsible for ADCs quality research, including ADC drug identification, specification, content, purity, safety and quality analysis, method development, validation, and testing.
ADCs are a new class of biopharmaceuticals synthesized by conjugating monoclonal antibodies and small-molecule drugs of potent cytotoxicity through biologically active linkers. The heterogeneity of these unconjugated naked antibodies and small molecules as well as the coupling drug may affect the efficacy and safety of ADC drugs overall. Common problems involve in the preparation of antibody-conjugated drugs include: (i) certain antibodies maybe not couple with small-molecule drugs, (ii) multiple binding sites (Cys, Lys residues, etc.) of antibody, and the difference of binding sites and binding amount can lead to heterogeneity, (iii) high hydrophobicity small molecule drugs may lead changes to the overall ADC drug properties.
Based on monoclonal antibody drugs, the conjugation of antibody and small molecule drugs can lead to changes in the physical and chemical properties, such as structure and charge changes. Moreover, different drug-to-antibody ratios (DAR), binding sites and occupancy levels can also bring about a high degree of heterogeneity between ADC drugs. As a result, a higher structurally complex ADC requires more complicated quality control. As a leading service supplier in ADC development, BOC Sciences retains professional technologies and equipment for ADC drugs quantitative analysis to support our customers better screen ADC drugs.
| Projects | Analysis Services |
| Antibody analysis | Antibody affinity, specificity, binding rate, binding position, stability, cross-reactivity, etc. |
| Primary structure | Sequence, molecular weight, post-translational modifications, disulfide bonds, coupling sites, etc. |
| Advanced structure | Secondary structure, tertiary structure, thermal stability, etc. |
| ADC analysis | Total antibody, conjugated antibody, free cytotoxins, conjugated cytotoxins, etc. |
| Purity and impurities | Aggregates, fragments, charge variants, free drugs, solvent residues, storage conditions, etc. |
| DAR analysis | DAR average, drug distribution, heterogeneity, etc. |
| Content and potency | ADC concentration, binding activity, cell killing activity, target affinity, cytotoxicity and pharmacokinetic analysis. |
| Chemical analysis | Ph, osmotic pressure, appearance/color, visible foreign matter, solubility, insoluble particles, PS80/20, etc. |
In comparison with monoclonal antibodies, the production process of ADC is much more complicated. Therefore, to ensure the safety and efficiency of ADCs drugs, their quanlity control must be monitored exclusively. Before ADC filing, a comprehensive evaluation of the ADC drug structure, total antibodies, conjugated antibodies, free load and related metabolites is crucial for later development. Thus, BOC Sciences has compliance advanced chromatographies and mass spectrometry instruments operated by our professional bioinformatics analysis team to quickly and accurately provide you with a professional system of antibody-conjugated drug analysis and evaluation services.
The measurement of biological activity is an important quality control indicator to ensure the effectiveness of ADC. The biological activity of ADC drugs is based on the targeting properties of antibodies and the killing properties of toxin drugs. At the molecular level, ELISA, FACS, Biacore and other methods can be used to identify the binding ability, binding kinetic changes and specificity of ADC drugs to the corresponding antigens, and the changes in binding activity before and after antibody coupling can also be evaluated. At the cellular level, cell proliferation inhibition assays or cell killing assays can be used to analyze the ability of ADC drugs to bind to their receptors, release drugs to target cells, and kill target cells. BOC Sciences provides a one-stop system for analyzing and determining antibody-conjugated drugs. Our biological testing services include cell health testing, drug efficacy testing, cell testing, internalization, inhibition of cell proliferation, cytotoxicity, cytokine secretion ability, antibody-dependent cell-mediated cytotoxicity (ADCC), and complement dependent cytotoxicity (CDC). BOC Sciences will provide you with a detailed technical report that includes: experimental steps, related MS parameters, original data, and ADCs analysis results.
In vitro analysis is a critical step in ADC development because it allows researchers to evaluate the binding specificity of the antibody components as well as the potency and selectivity of the cytotoxic payload. BOC Sciences offers a range of in vitro assays to evaluate the pharmacokinetics, pharmacodynamics and efficacy of ADCs. These assays include cell-based assays such as cell proliferation and cytotoxicity assays, as well as biochemical assays that measure drug release and stability.
In vivo analysis is another critical component of ADC development as it provides valuable insights into pharmacokinetics, biodistribution, and therapeutic efficacy throughout the organism. BOC Sciences offers a range of in vivo studies to evaluate the safety and efficacy of ADCs in animal models, including pharmacokinetic studies, biodistribution studies and efficacy studies in xenograft models.
The absorption, distribution, metabolism and excretion (ADME) of ADC drugs in various tissues directly affects the efficacy and safety of ADC. ADCs are more complex and challenging than other drugs. Therefore, ADME of ADC is a very complex process that requires comprehensive consideration of ADC, total antibody, conjugated antibody, free load, and conjugated load. Based on this, BOC Sciences has established a comprehensive pharmacology analysis program to explore the distribution, metabolism, elimination, linkers, and exposure-effects of ADC drugs.
Drug metabolism and pharmacokinetic (DMPK) research on ADC drugs runs through all stages of ADC drug early discovery/screening, preclinical and clinical. There are different research focuses at different stages. Each stage typically includes quantitative analysis of total antibody, conjugated antibody (ADC), free payload, and possible metabolites. In the drug screening stage, in vitro stability, DAR and in vitro biotransformation identification are usually added; while in preclinical and clinical stage evaluation, immunogenicity assessment are usually included. BOC Sciences' ADC DMPK analysis usually requires the collaboration of multiple analysis platforms, combining classic small molecules or macromolecules and fusion analysis methods.
The drug-to-antibody ratio (DAR) is a unique and important quality attribute of ADC drugs. It represents the average number of antibody-conjugated small molecule toxic drugs. A lower DAR will reduce the effectiveness of the ADC, but an excessively high DAR will reduce the effectiveness of the ADC. DAR will affect the pharmacokinetics and toxicity of ADC analysis. It is generally believed that a DAR between 2 and 4 is the most preferred ADC drug. The degree of drug conjugation affects the stability and aggregation tendency of ADC drugs. Only by choosing the appropriate coupling technology can the ADC toxin be uniformly and stably connected to the antibody. Therefore, DAR value is a key quality attribute for ADC drug analysis and an important quality control link in the ADC drug development process.
The stability of ADC is usually divided into physical stability and chemical stability. The physical stability of ADC mainly depends on the properties of the monoclonal antibody (parent), but the conjugation of toxin molecules and monoclonal antibodies affects the properties of the monoclonal antibody to a certain extent. Different coupling methods will lead to a decrease in the colloidal stability of the protein, an increase in the hydrophobicity of the coupling site, and a change in the surface charge distribution of the protein. Coupling will also decrease the conformational stability of the protein, which may lead to protein aggregation. The chemical stability of ADC mainly refers to the stability of the linker and toxin molecules, and its chemical instability mainly comes from the pH-sensitive linker. pH will induce the hydrolysis of the linker, resulting in the release of toxin molecules outside the tumor cells, and the linker will also accelerate degradation due to the action of oxidants.
BOC Sciences is a trusted partner for researchers developing ADCs. We have a comprehensive suite of analytical, stability testing and process development services to support researchers developing high-quality ADCs that have the potential to revolutionize cancer treatment. Whether you are looking to develop a new ADC or optimize an existing formulation, BOC Sciences can provide the expertise and support you need to succeed.
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.
Currently, several methods are available for measuring the concentration of ADC in a sample. One commonly used method is the use of enzyme-linked immunosorbent assay (ELISA), which relies on the specific binding of antibodies to ADC molecules. In this assay, ADC-containing samples are immobilized on a solid surface and ADC-specific detection antibodies are added. The amount of ADC in the sample is then measured by the intensity of the signal produced by the antibody-ADC complex.
Another method of measuring ADC concentration is liquid chromatography coupled to mass spectrometry (LC-MS). The technique separates the components of a sample based on its chemical properties and then uses mass spectrometry to detect and quantify the ADC molecules.
A third method for measuring ADC concentration is high performance liquid chromatography (HPLC), which separates sample components based on their interaction with stationary and mobile phases. The concentration of ADC in the sample is then determined by measuring the peak area or height corresponding to the ADC molecule in the chromatogram. HPLC is a widely used method for analyzing the purity and concentration of pharmaceutical compounds, including ADCs.
In addition to these methods, there are other techniques that can be used to measure ADC concentration, such as capillary electrophoresis, fluorescence spectroscopy, and surface plasmon resonance. Each of these methods has its own advantages and limitations, and the choice of method depends on factors such as the required sensitivity and specificity, the complexity of the sample matrix, and available resources.
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.
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