Antibody-drug conjugate (ADC) is a new type of biological targeting drug for the cancer treatment, which perfectly combines the high specificity of antibody and the strong lethal power of cytotoxin. With the targeting ability of antibodies, cytotoxins can be accurately delivered to the target cells, effectively increasing the drug concentration in tumor tissues, and greatly reducing drug exposure to other tissues and organs, to achieve synergism and reduce toxicity, thereby achieving “accurate treatment” at the cellular level.
Drug to antibody ratio (DAR) is an important attribute that affects the efficacy and safety of ADC. Low drug loading will weaken the efficacy of ADC, while high drug loading will have a negative effect on the pharmacokinetics and toxicity of ADC.
At present, there are many methods to determine the DAR value of ADC, and the commonly used ones are ultraviolet spectrophotometry (UV), hydrophobic interaction chromatography (HIC), liquid chromatography-mass spectrometry (LC-MS), reversed-phase high performance liquid chromatography (RP), hydrophilic interaction chromatography (HILIC), cathepsin B (Cathepsin B) digestion, and so on.
Ultraviolet spectrophotometry (UV)
Ultraviolet spectrophotometry is the simplest method to determine DAR based on Lambert Beer’s law.
Three conditions must be met to adopt this method.
- Small molecular drugs have chromogenic groups in the ultraviolet/visible region.
- Small molecular drugs and antibodies show obvious and different maximum absorption values in their UV/visible spectra.
- The presence of small molecular drugs do not affect the optical absorption properties of the antibody part of the ADC sample, and vice versa.
If these conditions are met, the ADC sample can be used as a two-component mixture, for Lambert’s law to determine the concentrations of antibodies and small molecular drugs respectively, and calculate the average DAR value accordingly.
The advantage of ultraviolet spectrophotometry is simple and does not need to be separated, but the shortage is that it is not accurate enough, and only the average coupling rate can be obtained, not including the information of small molecular drug distribution.
Hydrophobic interaction chromatography (HIC)
Hydrophobic interaction chromatography is the mostly used method for DAR value analysis.
According to the difference in hydrophobicity of small molecular drugs, the antibodies linked to different numbers of small molecular drugs can be separated through the increase of hydrophobicity. The antibodies of unconjugated small molecular drugs are the weakest in hydrophobicity and are eluted first. The more the number of small molecular drugs, the stronger the hydrophobicity. The weighted average DAR value is calculated by the percentage of chromatographic peak area and the number of coupling drugs.
The percentage of peak area represents the relative distribution of different quantities of small molecular drug antibodies. The attribution of each peak can be determined by referring to the peak distribution of LC-MS-Ms. After collecting the peaks in the HIC map, the specific peak can be identified by determining the molecular weight of each peak. Hydrophobic interaction chromatography can be used to analyze not only the DAR, but also the distribution of small molecular drugs. The advantage of HIC is that the conditions for elution are mild, which ensures that the biomolecules remain undenatured.
Liquid chromatography-mass spectrometry (LC-MS)
LC-MS can not only calculate the DAR value, but also give the distribution information of different numbers of small molecular drugs and the distribution of air-linked joints of by-products in the reaction process, which is very important for identifying different drug loading forms of ADC. The results can be verified by hydrophobic interaction chromatography.
The premise of determining DAR by LC-MS is that all substances have the same ionization efficiency as uncoupled antibodies regardless of drug loading. Therefore, its application is based on the assumption that all substances have the same recovery and ionization. But this is not always the case, because the coupling of drugs with positively charged amines will lead to changes in charge and hydrophobicity.
Reversed-phase high performance liquid chromatography (RP)
RP functions according to the polarity of the substance to be tested, which generally needs to dissociate the light chain and heavy chain of antibody by reduction reaction, and then separate the light chain and heavy chain containing different numbers of small molecular drugs by high performance liquid chromatography. By calculating the peak area percentage of each light and heavy chain, combined with the number of small molecular drugs coupled with each peak, the weighted average DAR value is calculated.
These two methods can not only calculate the DAR value, but also analyze the distribution of small molecular drugs at the light chain and heavy chain level, and the structure information of ADC is more abundant. For some ADCs, whose heavy chains having larger polarity than that of light chains coupled with one small molecule are more likely to be eluted, LC-MS is recommended for peak identification.
Hydrophilic interaction chromatography (HILIC)
Hydrophilic interaction chromatography is the mostly used method for the determination of antibody N-sugar spectrum, which functions according to the hydrophilicity of the substances to be tested.
In recent years, some scholars used hydrophilic interaction chromatography to analyze ADC. The pretreatment was the same as reversed-phase high performance liquid chromatography. ADC was digested by IdeS and then reduced to get Fc/2 and light chain L0, the light chains L1 conjugating one small molecule drug, Fd, and Fd1-Fd3 conjugating one to three small molecule drugs, which were analyzed by hydrophilic interaction chromatography.
Hydrophilic interaction chromatography can not only analyze the DAR value and drug distribution of ADC, but also analyze the distribution of N-sugar. The identification of each peak is recommended to use LC-MS. This method is confirmed by reversed-phase high performance liquid chromatography (RP-HPLC).
Cathepsin B enzyme digestion method (Cathepsin B）
Cathepsin B is a lysosomal cysteine proteolytic enzyme whose catalysis is realized by Cys and His. It is easily inhibited by sulfhydryl reagents, which also called sulfhydryl enzyme and belongs to the papain family. In the Cathepsin B digestion method, the ADC was cut under the hinge region with IdeS enzyme, and then reduced to make the small molecular drugs fully exposed. This pretreatment allows Cathepsin B to enter the cleavage site without restriction, ensuring adequate restriction endonuclease digestion. The pyrolyzed drug is separated from the protein component by reversed-phase high performance liquid chromatography (RP-HPLC), and its concentration is determined according to UV absorption.
Cathepsin B digestion method is suitable for ADCs with cleavable linker and Cathepsin B digestion site. This method overcomes the limitations in the analysis of ADC by UV spectrophotometry, hydrophobic interaction chromatography, and LC-MS. For example, ultraviolet spectrophotometry needs to meet three basic conditions, hydrophobic interaction chromatography requires different hydrophobicity of different coupling components, LC-MS requires that the ionization efficiency of each component should be the same, and so on. The Cathepsin B enzyme digestion method can complement the above methods which is reliable, accurate, and general, and can be used to measure the DAR value of any ADC with similar chemical properties.
In addition to the above methods, there are capillary electrophoresis, radiation measurement, iso-point focusing capillary electrophoresis, and so on. In practical use, appropriate analysis methods should be selected according to the ADC coupling characteristics.