The key quality attributes of ADC drugs

Posted by beauty33 on November 30th, 2021

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 weightdetermined 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.

Unconjugated naked antibody

The naked antibody competes with the ADC for the target antigen, ultimately reducing the amount of drug delivered to the target cell. Therefore, the naked antibody level is a key parameter that needs to be monitored during process control and the entire validity period, because it directly affects the effectiveness of the drug.

Mass spectrometry is the most critical technical method for naked antibody analysis (as shown in the figure below, D0 is naked antibody). The percentage of naked resistance can be calculated based on the area under the curve and the total area. In general, LC-MS is the best method to completely characterize ADC.

Imaging capillary electrophoresis (iCIEF) has also been used to analyze ADC drug load distribution in recent years, but it faces a series of problems in general. The linker without drug coupling will also cause the charge to shift to the acidic end, resulting in a higher analysis value of the drug load distribution than the actual value. In addition, iCIEF cannot distinguish conjugates, process intermediates and impurities, such as distinguishing antibodies with only linker and antibodies with linker/drug conjugates. In addition, iCIEF is not suitable for some couplings, such as cysteine, polysaccharides or site-specific coupling chemicals, because they do not cause significant changes in charge and net pI.

Charge variant

The charge variation of protein therapy is an important quality characteristic and has a potential impact on stability and biological activity. In protein expression, drug conjugation, purification process, various post-translational modifications, including enzymatic or chemical degradation, various forms of charge-related variants can be observed. IEX and capillary electrophoresis (CIEF and iCIEF) are commonly used detection methods, but for lysine-conjugated ADC drugs, neither work ideally.

Molecular weight variants

Variations in molecular weight (polymers, particles, fragments) can directly affect the effectiveness or safety of protein treatments. When two or more monomers aggregate, aggregates with sizes ranging from tens of nanometers to hundreds of microns are formed. Aggregation is the most common way of physical degradation of proteins. Due to the presence of drugs/linkers, ADCs aggregate more easily than parent antibodies, and they have more hydrophobicity around the coupling site. In addition, drug coupling may also have a direct impact on the total surface charge and the thermal stability of the protein.

Fragmentation is the result of the cleavage of peptide bonds produced by chemical or enzymatic reactions. Although the protein backbone has relatively high stability, some sites may be easily broken, and fragmentation may occur at the same time during the formation of aggregates.

Molecular weight exclusion chromatography and CE-SDS are commonly used to analyze molecular weight variants and aggregates.

Summary

In the past two years, multiple ADC drugs have been successfully approved, making them an important part of the pipeline in more and more drug R&D companies.ADC combines the specificity of antibody drugs and the advantages of cytotoxicity of small molecule drugs, increasing the therapeutic window. But the coupling of the two also changes the physical and chemical properties of each other, causing changes in structure and charge. Compared with monoclonal antibody drugs, changes in quality attributes caused by coupling, such as DAR, drug load, naked antibody ratio, and easy aggregationneed to be considered in the development of ADC drugs.

References:

1. Chen Y. Drug-to-antibody ratio (DAR) by UV/Vis spectroscopy. Methods Mol Biol. 2013; 1045:267–73.

2. Adem YT, et al, Antibody Drug Conjugate Physical Instability and the Role of Drug Payload. Bioconjugate Chemistry. 2014; 25:656–64.

3. Anil Wagh et al, Challenges and new frontiers in analytical characterization of antibody-drug conjugates, MABS 2018, VOL. 10, NO. 2, 222–243

4. Jinsha Liu et al, Therapeutic Advances in Oncology, Int. J. Mol. Sci. 2021, 22, 2008.

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