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Compared with traditional cytotoxic drugs, antibody-drug conjugate (ADC) has the advantages of strong targeting and less toxic side effects, and has broad application prospects in the field of tumor treatment. As time goes by, ADC has received more and more attention. As an important class of drugs in the clinical research of cancer treatment, ADCs are combined with monoclonal antibodies through chemical bonds (linkers) through powerful small-molecule cytotoxins, which have a more complex structure than antibodies.
Fig. 1. Structure of antibody drug conjugates (ADCs).
The clinical efficacy of ADC mainly depends on the linker, drug and conjugation site (lysine, interchain cysteine, Fc glycan). It is therefore particularly important to understand the physicochemical properties of ADCs and to select appropriate analytical techniques for their evaluation and quality monitoring during manufacturing and storage. However, ADC is composed of highly specific and high-affinity antibodies, highly stable linkers and highly effective small molecule cytotoxic drugs. Its molecular properties are relatively complex, so that its characterization is more difficult and requires multiple analytical methods. characterization.
DAR is a unique derivative of ADC drugs, which is defined as drug/antibody ratio, that is, the average amount of drug carried by an antibody. DAR is the result of balancing efficacy and safety, and is one of the most important quality attributes of ADCs. DAR determines the amount of payload that can be delivered to the tumor, directly affecting the safety and efficacy of the ADC.
Fig. 2. ADC conjugation site and its mechanism of action.
In simple terms, the effect of ADC is directly related to DAR. However, the value of DAR is not the higher the better. Although the high DAR has a larger drug loading, it is also easier to be locked by the immune system, and thus be removed from the body as a foreign body, reducing the effectiveness of ADC. Therefore, the DAR of most ADC drugs is limited to 2-4. How to make the DAR value both curative and effective is still a question that is still under consideration.
The conjugation method is an important factor affecting DAR. Only by choosing a suitable conjugation technique can the toxin of the ADC be connected to the antibody uniformly and stably. There are many methods that can be used to measure the DAR value, and different measurement methods need to be selected according to different drug properties and structures of binding sites and linkers. Common DAR characterization methods include hydrophobic interaction chromatography (HIC), reversed-phase liquid chromatography (RPLC), liquid chromatography-mass spectrometry (LC-MS), ultraviolet-visible (UV/Vis) spectroscopy, capillary electrophoresis-dodecane sodium disulfate (CE-SDS) and other methods, among which chromatography and mass spectrometry are two commonly used methods to detect DAR values.
Ultraviolet-visible spectroscopy (UV-Visible) is currently a relatively simple DAR determination method. This method requires that the spectra of the drug and the antibody have different maximum absorption wavelengths. Calculate the concentration of the drug and antibody separately to obtain the DAR value of the ADC, which is applicable to a variety of ADCs. UV/Vis is based on protein concentration measurement technology. Using the absorbance of the ADC at the appropriate wavelength and the extinction coefficients of the antibody and payload, the average DAR can be determined. This technique is simple and easy to perform as no complex sample preparation and establishment of HPLC or MS conditions are required. However, it requires that the UV-vis absorbance maxima of the payload have an appropriate offset from that of the unconjugated antibody. Also, this method cannot determine the proportion of a single DAR species in a mixture.
Fig. 3. Example of ultraviolet-visible (UV/Vis) spectroscopic analysis of ADC (Br J Cancer. 2020, 123: 1502-1512).
Mass spectrometry (MS) is another commonly used technique for ADC characterization. Currently available mass spectrometers are typically based on electrospray ionization (ESI) and time-of-flight (TOF) detection, which can also be combined with the Orbitrap, which has an extended mass range. Mass spectrometry is suitable for the determination of DAR values of lysine conjugation ADCs, including liquid chromatography tandem mass spectrometry and MALDI-TOF-MS. Mass spectrometry can be divided into denaturing and non-denaturing electrospray mass spectrometry. The two different methods are based on the difference in the molecular weight of the substance for DAR analysis. After the antibody is conjugated with different amounts of cytotoxic small molecules, the ADC presents molecular heterogeneity. Different DAR substances have different mass-to-charge ratios (m/z) under the analysis of high-resolution mass spectrometry. The average DAR value is then calculated based on the corresponding DAR mass spectrum signal peak intensity or peak area.
Fig. 4. Analysis of ADCs by native mass spectrometry (Methods in Molecular Biology. 2020, vol 2078: 197-211).
The resolution of MS analysis is high enough to distinguish different types of heterosomes in ADC, which can be used to separate, identify and quantitatively detect different types of conjugated molecules. Therefore, the average DAR value can be calculated from the average weight of the detected heterosomes with different DAR values. ADC molecules introduce more heterogeneity due to coupling, so their MS analysis methods are much more complicated than traditional mAb MS analysis methods. In general, it is necessary to avoid the interference of antibody-related features as much as possible to obtain more ADC-specific structural information.
Since the development of ADC technology, it has experienced three generations of changes. With the continuous development of targets, linkers, cytotoxins and conjugation technologies, ADCs are becoming more and more mature. In recent years, a variety of DAR analysis and characterization techniques have been developed. Methods based on UV-visible spectroscopy, radiolabeling, chromatography, enzymatic hydrolysis, mass spectrometry and other technologies can be applied to the process control (IPC) or ADC sample characterization in ADC production.
BOC Sciences can measure the DAR and drug distribution of ADCs using a variety of techniques including high performance liquid chromatography (HPLC) and mass spectrometry (MS). We can also work with customers to develop custom analytical methods to meet your specific requirements such as sensitivity, accuracy and precision. If you are interested in our ADC Analysis and Characterization services, please contact us for more information.
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