The Key Quality Attributes of ADC Drugs

  • Antibody-drug conjugates (ADCs) are conjugates of cytotoxic small-molecule drugs with targeted monoclonal antibodies through a linker. The specificity of antibodies reduces the toxicity of small-molecule drugs and increases the therapeutic window. In the past two years, ADC drugs have made breakthroughs, and several drugs have been approved for marketing.

    Key quality attributes

    ADC drugs mainly have three components: 1. Specific targeting antibodies; 2. High-titer cytotoxic drugs; 3. Linkers. ADC drugs have a complex structure and strong heterogeneity, and contain a variety of product-related impurities, so it is important to determine their key quality attributes.

    DAR (drug-to-antibody ratio)

    DAR represents the average number of cytotoxic drugs coupled to the antibody, and it is an important quality attribute. Low drug load (low DAR) will reduce the efficacy of ADC, while high drug load (high DAR) will change the pharmacokinetics and toxicity of ADC molecules. Controlling the coupling reaction, especially the concentration of reactants, will cause changes in DAR (especially in the case of random coupling), which is the most critical step in ADC development. Processing/treatment control after production is beneficial to reduce unconjugated naked antibody and low/high DAR products, and control DAR within the target range. Therefore, drug loading and drug distribution are CQAs that should be controlled in ADC production. Spectroscopy, radiation, chromatography, mass spectrometry, etc. are commonly used analytical methods.

    UV-Vis Spectroscopy

    Ultraviolet visible spectroscopy is a simple and most commonly used method for DAR measurement. The prerequisites of this method are: 1) the drug should contain an ultraviolet absorbing group; 2) the drug and the antibody show obvious independent maximum absorption peaks in the ultraviolet-visible spectrum; 3) the presence of the drug should not affect the light absorption characteristics of the antibody in the ADC sample, and vice versa.

    According to the measured absorbance and extinction coefficient, the concentration of protein and drug can be calculated according to the Beer-Lambert principle, and the average DAR can be calculated accordingly.

    Mass spectrometry

    Mass spectrometry is another commonly used method to analyze DAR. Currently, electrospray ionization mass spectrometry (ESI), time-of-flight mass spectrometry (TOF) or Orbitrap are used to distinguish the heterogeneous molecular species of ADCs. The average DAR can be calculated based on the molecular weight determined by mass spectrometry and the corresponding peak area.

    Drug load distribution

    The overall drug load distribution can be characterized by a complete mass analysis, and the less heterogeneous ADC produced by cysteine or other site-specific coupling can also be analyzed by hydrophobic interaction chromatography. In order to determine the drug load distribution at each coupling site, a more comprehensive analysis, such as peptide maps, is required.

    If good chromatographic separation is achieved, liquid chromatography can be used to obtain accurate information about the occupation of the coupling site. It should be pointed out that due to steric hindrance and conformational changes, conjugated compounds may affect the efficiency of protein hydrolysis. Therefore, it is necessary to use a second enzyme to hydrolyze ADC drugs in continuous or parallel processing.

    For ADCs with random coupling, if the absorption peak of the conjugated drug and the absorption peak of the naked antibody do not overlap, the peptide map of the ADC can be used to compare and locate the peptide coupled to the drug. LC-MS analysis can determine the location of the peptide and the coupling site coupled to the drug molecule.

    The detection limit of this method is usually below 1%. When the MS signal is used to quantify the coupled peptide, the impact of additional compounds on the ionization efficiency due to the loss of primary amine groups and the introduction of hydrophobic moieties should be considered to achieve accurate quantification. However, the effect of coupling on ionization efficiency may not be so obvious for the intact protein. In addition, trypsin/Lys-C usually used for peptide mapping does not cleave modified lysine, so it is difficult to measure the occupancy rate of this part.

    Another difficulty in ADC peptide mapping analysis is that the coupled peptide will precipitate and adsorb on the surface of the analysis bottle, which can sometimes be alleviated by adding acetonitrile or isopropanol to the sample solution.