Analysis Method for Drug-to-Antibody Ratio (DAR) of Antibody-drug Conjugates

The Drug-to-Antibody Ratio (DAR) is the most important quality attribute of ADC drugs because it determines the “payload” that can be delivered to tumors, directly impacting safety and efficacy. Here, we summarize some detection methods.

Ultraviolet-visible spectrophotometry (UV)

Ultraviolet-visible spectrophotometry is the simplest and most convenient method for determining the DAR value. By measuring the absorbance of ADC at different wavelengths and the extinction coefficients of the naked antibody and drug, the average antibody conjugation ratio can be determined. The theoretical basis for quantitative analysis using ultraviolet-visible spectrophotometry is the Beer-Lambert law, which states that absorbance is directly proportional to concentration:

A is the absorbance,

ε is the molar absorptivity (a physical constant related to the amino acid composition of the substance),

l is the path length of the cuvette (usually 1 cm or 1 mm),

c is the concentration.

When there are multiple components in the system, each with its own absorption spectrum and no interference between them, the absorbance of each component can be summed up.

By known absorbance and extinction coefficients and by solving simultaneous equations, the concentrations of various components in the sample can be obtained. This method requires that the drug has chromophoric groups in the ultraviolet/visible region; the antibody and drug exhibit distinct maximum absorbance values in their UV/visible spectra; the presence of the drug does not affect the optical absorption characteristics of the antibody portion in the ADC sample, and likewise, the antibody does not affect the optical absorption characteristics of the drug. Since proteins containing aromatic amino acid or histidine residues typically exhibit maximum absorption at 280 nm, it is required that the cytotoxic drug shows maximum absorption significantly different from 280 nm. For example, toxins exhibit maximum absorption at 252 nm, while antibodies exhibit maximum absorption at 280 nm. However, the linker should not have significant absorption at both 252 nm and 280 nm. If these conditions are met, the ADC can be regarded as a two-component mixture, and the concentrations of the antibody and drug can be separately determined using the Beer-Lambert law. The average DAR value (the ratio of molar concentrations) can then be calculated. Of course, if the linker absorbs significantly at both 252 nm and 280 nm but has different maximum absorption compared to the antibody and drug, then the ADC sample can be treated as a three-component system to quantitatively determine the DAR value. Therefore, when using ultraviolet-visible spectrophotometry to determine the average DAR value, the influences of the drug, linker, and antibody must be considered.

a. UV spectrum of toxin DM1, b. UV spectrum of trastuzumab, c. UV spectrum of trastuzumab ADC

The implementation steps for determining the average DAR value of ADC using UV-visible spectrophotometry (for antibody-drug dual-component systems) are as follows:

Determine the maximum absorption wavelength (λ(D)) of the drug: Measure the maximum absorption wavelength of the drug. Generally, toxins are hydrophobic and are usually dissolved in organic solvents (such as dimethyl sulfoxide, methanol) at appropriate concentrations, avoiding saturation. The maximum absorption wavelength for the antibody is usually selected at 280 nm.

Measure the extinction coefficient (ε) of the antibody and drug at 280 nm and the maximum absorption wavelength (λ(D)) respectively: Using known concentrations of the antibody and drug (with high sample purity recommended), determine the extinction coefficients of the antibody and drug at both wavelengths using the Beer-Lambert law.

Measure the absorbance of the ADC sample at 280 nm and the maximum absorption wavelength (λ(D)).

Calculate the average DAR value of the ADC.

Through the second step mentioned above, the extinction coefficients of the antibody and toxin at the two wavelengths have already been determined.

Through the aforementioned step 3, the absorbance values of the ADC at 280 nm and λ(D) have been measured. Using the Beer-Lambert law, the two absorbance equations can be set up as follows:

The above two equations can be used to obtain the concentration of antibody and drug respectively.

The calculated result of the ADC’s DAR value is as follows:

Note: c represents molar concentration.

In summary, the determination of the extinction coefficients of antibodies and drugs at two wavelengths is performed, and then the simultaneous equations of absorbance for ADC at two wavelengths are solved by quadratic equations.

Considerations for UV Analysis of DAR

Although the method of determining the average DAR of ADC using UV-visible spectrophotometry is simple and does not require high laboratory equipment conditions, there are still many influencing factors throughout the entire operation process, leading to significant errors in the measurement results.

1. Use quartz cuvettes instead of disposable cuvettes. Disposable cuvettes are mostly made of plastic material, and the limited wavelength range may not be suitable for certain ADCs. Additionally, keep the cuvettes clean.
2. Antibodies or drugs may have different extinction coefficients in different systems, which should be taken seriously.
3. The clarity of the sample is also important. If the sample is turbid, it will cause significant interference. At the same time, the sample needs to have an appropriate concentration, that is, a reasonable range of absorbance.
4. Pay attention to the influence of free toxins (small molecules).
5. Specific analysis should be conducted for ADCs with high DAR values or coupled with multiple small molecules, depending on the situation.

Reversed-phase High-performance Liquid Chromatography (RP-HPLC)

For cysteine-linked conjugates, the average DAR can be calculated by measuring the weighted peak area percentage of each coupled heavy chain and light chain drug using RP-HPLC. This method separates substances based on their polarity, and the DAR obtained by this method has a good correlation with spectrophotometric methods based on drug and antibody ultraviolet absorption.

By calculating the percentage of peak areas of each light and heavy chain and combining the number of coupled small molecule drugs for each peak, the weighted average DAR value is calculated using the following equation:

Formula for calculating average coupling rate: DAR = 2 × (ΣLC Weighted peak area + ΣHC Weighted peak area) / 100.

Considerations of RP Analysis DAR

1. Ensure that the sample is in a reduced state. For samples without cross-linking, ensure that only the L and H components are present.
2. Ensure effective separation of each component, especially for components with similar hydrophobicity.
3. RP-MS should be used to identify each peak because it is not possible to determine the components of each peak solely based on retention time. For some ADCs, the heavy chain may have a greater polarity than the light chain coupled with a small molecule and may be eluted first.

Hydrophobic Interaction Chromatography (HIC)

Hydrophobic interaction chromatography (HIC) is a traditional technique for protein separation, purification, and characterization. With the continuous development of antibody-drug conjugates (ADCs), HIC is becoming increasingly widely used in the characterization and analysis of ADCs. Unlike other techniques, HIC allows proteins to be analyzed under mild non-denaturing conditions while maintaining their natural structure and activity.

The method of HIC was first proposed by Tiselius, who referred to this technique as “protein salting out.” However, the actual term HIC was later coined by Hjerten, who described the process as the binding and elution of proteins on a solid matrix, leading to protein separation. The non-polar nature of the stationary phase in hydrophobic interaction chromatography is relatively weak, and the mobile phase often consists of a gradient elution with high-concentration salt buffers. The gentle separation conditions can avoid the irreversible adsorption and denaturation of proteins caused by the strong hydrophobicity of the stationary phase and organic mobile phase in reverse-phase chromatography, making it particularly suitable for the separation and purification of active substances. The surface of the stationary phase in hydrophobic interaction chromatography is made up of weakly hydrophobic groups, with hydrophobicity tens to hundreds of times lower than that of the stationary phase used in reverse-phase chromatography, while the mobile phase is a high-concentration salt solution. Protein molecules are distributed in such a stationary phase and mobile phase, retained by the interaction between the hydrophobic groups on the protein molecules and the hydrophobic groups on the stationary phase. As the ionic strength of the mobile phase gradually decreases during elution, the elution capability increases. By using eluents with decreasing ionic strength from high to low, components with gradually increasing hydrophobic interactions can be separated. Protein molecules are eluted according to their hydrophobicity, with less hydrophobic molecules eluting first. In such high-salt solutions, proteins do not deactivate. The strong interaction between high-concentration salt and water molecules reduces the surrounding water molecules forming cavities around hydrophobic molecules, promoting the interaction between hydrophobic molecules and hydrophobic ligands in the medium. The magnitude of this hydrophobic interaction depends on the polarity of the stationary phase and solute, the composition, and concentration of the mobile phase. Because the polarities of the amino acid residues on various protein surfaces differ, it is possible to separate proteins by changing the polarity of the stationary phase and the composition of the mobile phase.

The cytotoxic payload of ADCs must traverse the lipid membrane to inhibit DNA replication, protein synthesis, enzyme activity, or cell division. To penetrate the membrane, the payload must be lipophilic and hence hydrophobic in nature. The conjugation of the payload to the antibody increases its hydrophobicity, a change detectable by HIC. The number of payloads correlates directly with hydrophobicity and retention time. Moreover, the more hydrophobic payloads present, the longer the retention time. Separating different drug types based on hydrophobic differences and quantitatively analyzing each drug type is the basis of HIC analysis of ADCs.

HIC can be combined with RP, MS, and other methods to analyze the conjugation status of ADCs. HIC must be coupled with MS to confirm the drug-to-antibody ratio (DAR) because changes in peak retention times do not directly indicate the extent of drug conjugation. Specific peak identification requires collecting peaks from HIC chromatograms and determining the molecular weight of each component. Once the conjugation status of each peak is determined, HIC can quickly and accurately determine the ratio of each drug. Therefore, HIC is the primary method for quality assessment and reaction monitoring, and due to the gentle nature of the separation process, it can be used to analyze ADCs with acid-labile linkages.

Using Tris-HCl-based buffers and linear gradients to elute proteins from the chromatographic column facilitates the separation of different hydrophobic substances. DAR is determined by comparing the relative amounts of each peak and the degree of payload conjugation (calculated by weighted average DAR through peak area percentages and the number of conjugated drugs). Compared to other techniques used for ADC analysis and characterization, HIC provides a unique, rapid analysis method with several advantages. For example, techniques such as mass spectrometry, reverse phase, and hydrophilic interaction chromatography (HILIC) rely on organic solvents as mobile phases, low or high pH values, high temperatures, etc., all of which may denature ADCs. Moreover, optimizing these methods for ADCs to address these issues is time-consuming. ADC components separated by HIC retain their activity and can be used for efficacy studies. HIC cannot be applied to some hydrophilic payloads. Additionally, highly hydrophobic payloads may require extensive method development. For hydrophobicity ranking of ADCs, HIC analysis is most suitable because column binding is driven by the hydrophobicity of proteins, which is directly proportional to retention time. There are no universal rules for optimizing HIC methods; different methods are required for different samples. The method described in this paper utilizes common ammonium sulfate buffer systems and butyl columns for characterizing, sorting, and analyzing ADCs, laying the foundation for developing optimized HIC methods.

The weighted average DAR value is calculated using the percentage of chromatographic peak area and the number of conjugated drugs. Calculation formula: Average conjugation rate calculation formula: DAR = Σ(Weighted peak area) / 100.

Considerations for HIC Analysis of DAR

1. Pay attention to the storage and expiration date of ammonium sulfate, which can generally be stored for one month under closed conditions at room temperature. When using it, filter through a 0.2μm membrane filter and observe if there is any crystallization.
2. The payload is inherently hydrophobic. The stronger the hydrophobicity, the longer the retention time. Very hydrophobic payloads may affect the shape and elution of ADC peaks and may require organic modifiers for complete column elution. For payloads with very strong hydrophobicity, when optimizing HIC methods, consider chromatographic columns with non-branched and shorter side chains.
3. Increasing the pH of the mobile phase buffer helps improve the binding of antibodies and ADCs to the column, while decreasing the pH helps elute proteins. The isoelectric point of most antibodies is between 7 and 9. Therefore, when the pH of the buffer is close to the pI, it reduces the net surface charge and may promote interaction with the column. Conversely, lowering the pH of the buffer, away from the pI, increases the net surface charge and may help elute proteins. However, the effect of pH changes on ADC binding and elution is unpredictable and depends on the resin type, buffer salt, and sample.
4. Samples that do not bind to the HIC column will pass through, eluting with the solvent peak, leading to an increase in solvent peak intensity. Increasing the concentration of ammonium sulfate to 2 M and adjusting the pH closer to the ADC pI position is a strategy to improve binding. However, for some ADCs, chromatographic columns may require longer chains or more non-polar groups.
5. Generally, there is no problem with poor recovery rates in HIC. Samples that are not injected into the HIC column are often mistaken for not eluting from the column, when in fact the ADCs did not enter the column from the beginning. Therefore, it is important to verify whether ADCs are adsorbed on the column and if the instrument is working properly. Also, ensure that the intensity of the solvent peak does not increase. When ADCs cannot be eluted, organic modifiers such as isopropanol or acetonitrile (15%, v/v) can be added to the mobile phase B to facilitate the release of ADCs from the column matrix. However, protein salting out or denaturation should be avoided. Organic modifiers should not be used in mobile phase A. These modifiers compete directly with the column for binding and help elute proteins by increasing the surface tension of the mobile phase, thus reducing hydrophobic interactions. Therefore, adding modifiers to mobile phase A increases the likelihood of proteins not binding to the column. If using other chromatographic columns, first ensure that the uncoupled antibody binds and is eluted before the gradient reaches 25%.
6. The general method for classical conjugated ADCs usually cannot achieve baseline separation of peaks; whereas for more uniformly site-specific conjugated ADCs, better results have been obtained, as seen in Figures 3 and 4 respectively. Optimization is required to achieve good peak separation, considering factors such as flow rate, gradient, HIC column, and mobile phase composition. Due to the complexity of molecules, optimizing HIC methods often differ from unconjugated ADCs.
7. Peak separation is another important parameter to check in data analysis. The expected number of peaks should be reflected in the chromatogram. Missing peaks or additional peaks can affect DAR calculations. Therefore, mass spectrometric analysis should highlight the number of conjugated drugs and the drug load of each peak to determine the adequacy of peak separation methods. When considering this, improving peak separation can be achieved by extending the gradient.
8. Gradient optimization can improve peak shape and separation, enhancing data accuracy. In general, increasing the length of the gradient can improve peak separation and shape. Due to the complexity of ADCs, gradient optimization parameters must be determined experimentally. Gradient selection depends on the mobile phase, flow rate, and chromatographic column. Elution gradients with ammonium sulfate buffers are shorter, but must be extended when using other buffer salts.
9. Butyl resin is the most widely used resin for HIC columns due to its long resin chain and moderate binding tendency. Typical HIC columns have linear alkyl chains or simple aromatic groups and can be further modified by branching. HIC columns from different manufacturers may have differences, affecting binding, peak separation, and elution. Using chromatographic columns with longer or more branched chains can enhance sample binding and retention. The binding capacity of the resin depends on the length of the chain, with shorter chains having weaker binding capacity and vice versa. Some common resins, arranged by chain length from low to high and binding ability, are: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl.
10. Sample purity is critical for successful HIC analysis. All samples should be filtered to remove free loads and aggregates to ensure accurate data. For small-scale conjugates, ADCs can be purified using buffer exchange columns; for large-scale preparations, ADCs can be purified using hydroxyapatite chromatography or tangential flow filtration to remove or minimize contaminants such as free loads and aggregates. Free loads, especially those that are very hydrophobic, can also bind to HIC columns, complicating data interpretation (as shown in Figure 6). Protein aggregates are often more hydrophobic and separate from monomers, forming separate peaks. Since peak identification is crucial for accurate data interpretation, every effort should be made to eliminate peaks caused by artifacts such as aggregates and free drugs.
11. Do not exceed an injection volume of 100 μL, otherwise ADCs may not bind to the column. This method aims to minimize sample preparation. If the injection volume is large, the salt concentration at the sample-buffer interface is too low, causing the ADCs not to be adsorbed onto the column. To overcome this problem, a volume of up to 50% of mobile phase A can be added to the sample. Also, avoid adding mobile phase A directly to the sample, which may cause protein precipitation.
12. Pay attention to the retention time of the solvent peak. Drifting retention time may be due to salt deposition blocking the pipeline, requiring equipment cleaning. Record the initial instrument back pressure after column installation. An increase in back pressure affects results. Before and after sample runs, flush the HPLC with 50 mL of water to remove residual salts from the instrument to prevent clogging or restricted flow. Ammonium salts may precipitate in the instrument, leading to poor sample binding and recovery rates, shifting retention times, incomplete sample elution, affecting method optimization, data interpretation, and troubleshooting.
13. Column temperature has minimal effect on sample adsorption and elution but affects peak shape. The column temperature should not exceed 30°C, as it can denature proteins. Column temperature optimization should be done last.
14. When monitoring elution peaks at A280nm, gradient elution with ammonium sulfate buffer may cause a slight baseline shift. However, any absorption wavelength that maximizes the signal-to-noise ratio can be used. In some cases, after additional payloads, conjugated peaks can be distinguished from non-conjugated peaks at a specific wavelength. For example, detection of pyrrolobenzodiazepine-conjugated ADCs at 330 nm.
15. Peak integration is a necessary condition for DAR calculation, and a good peak shape is of significant importance for accuracy in computation. The shape of a peak is mainly influenced by the chromatographic column and gradient. If a peak is too broad or too narrow, it can affect the accurate integration of the peak. Extending the gradient can improve the shape of narrow peaks, while shortening the gradient can aid in shaping broad peaks. Changes in peak shape should only be made to ensure integration accuracy. Altering the composition of the mobile phase, for example, using ammonium acetate buffer or different chromatographic columns, may improve peak shape.
16. ADCs with non-hydrophobic or hydrophilic payloads may not exhibit typical elution peaks. These ADCs can elute with shorter retention times or co-elute with the naked antibody. If the retention time of the ADC matches that of the unconjugated antibody, HIC cannot be used for analysis and characterization.
17. Conjugation sites affect the shape of peaks and the number of elution peaks. To date, all approved ADCs have used random conjugation methods with lysine or hinge cysteine. This conjugation method produces multiple peaks corresponding to the degree of drug loading, which can make analysis complex. Additionally, several ADCs in clinical trials are site-specifically conjugated to one or more engineered sites. These site-specific ADCs are generally homogeneous, producing a single peak when analyzed by HIC, and are less hydrophobic than randomly conjugated ADCs. Compared to randomly conjugated ADCs, some site-specific conjugation sites can minimize exposure to the payload, thereby reducing hydrophobicity and retention time.
18. Record the initial solvent peak intensity. An increase in solvent peak intensity indicates incomplete sample binding. Solvent peak intensity is very useful for excluding ADC binding issues. During method optimization, tracking solvent peak intensity when testing columns or mobile phases is very useful.
19. For hydrophobicity ranking of antibodies and ADCs, appropriate sample concentrations and buffer solutions should be used. Due to the relationship between hydrophobicity and off-target toxicity, hydrophobicity-based drug candidate ranking is crucial. Figure 7 shows HIC analysis of six antibodies targeting the same tumor-specific antigen, which are ADC candidates. In this case, mAb-A exhibits the lowest hydrophobicity. When analyzing hydrophobicity ranking of ADCs, factors such as antibody, conjugation site, conjugation method, and payload all influence retention time.

Liquid Chromatography-Mass Spectrometry (LC-MS)

Non-denaturing mass spectrometry has proven its powerful advantages for quantitative and qualitative analysis of antibody-drug conjugates (ADCs), especially when the ADC subunits involve non-covalent interactions (e.g., cysteine-conjugated ADCs). The application of non-denaturing mass spectrometry is widespread, such as in the determination of drug-to-antibody ratio (DAR) in antibody-drug conjugates (ADCs): ADC drugs are classified into two categories based on the conjugation sites of antibodies, namely lysine-conjugated and cysteine-conjugated ADCs. For lysine-conjugated drugs, determination is often based on traditional reversed-phase liquid chromatography/mass spectrometry (RPLC/MS) platforms, where proteins need to be denatured for DAR determination. However, this denaturation condition can lead to the separation of antibody heavy and light chains for cysteine-conjugated ADCs. Therefore, the detection of cysteine-conjugated ADCs can be aided by non-denaturing mass spectrometry.

Liquid chromatography-mass spectrometry analyzes based on molecular size, distinguishing peaks of antibodies conjugated with different numbers of small molecule drugs by increasing the mass number of the drugs. The weighted average DAR value is calculated by the percentage of area under each peak and the number of conjugated drugs, according to the formula: DAR = sum of weighted peak areas / 100. LC-MS can not only calculate DAR values but also provide information on the distribution of antibodies conjugated with different numbers of small molecule drugs, as well as the distribution of by-products such as free linkers in the reaction process.

Other Methods

There are several other methods for determining the drug-to-antibody ratio (DAR) of antibody-drug conjugates (ADCs), such as Hydrophilic Interaction Chromatography (HILIC) and Cathepsin B enzyme cleavage method.

Hydrophilic Interaction Chromatography (HILIC): This method separates substances based on their different hydrophilic properties and is the most commonly used method for determining antibody N-glycan spectra. Prior to HILIC analysis of ADC DAR values, the ADC is pretreated similarly to reversed-phase high-performance liquid chromatography (HPLC), where the ADC is cleaved with IdeS enzyme and reduced to obtain Fc/2, light chain L0, light chain L1 conjugated with one small molecule drug, Fd, and Fd1-Fd3 conjugated with 1-3 small molecule drugs. The analysis is then conducted using hydrophilic interaction chromatography.

To calculate the average conjugation rate, the following formula is used: DAR=2× (ΣLC Weighted peak area+ΣFd Weighted peak area)/100。

Hydrophilic Interaction Chromatography not only analyzes the DAR values and drug distribution of ADCs but also examines the distribution of N-glycans. Peaks are first identified using LC-MS. This method is complementary to reversed-phase high-performance liquid chromatography.

Cathepsin B Enzymatic Cleavage Method (Cathepsin B): Cathepsin B is a cysteine protease found in lysosomes. Its catalytic activity is facilitated by Cys and His residues and is easily inhibited by thiol reagents. Cathepsin B is known as a thiol protease and belongs to the papain-like cysteine protease family.

In the Cathepsin B enzymatic cleavage method, ADCs are first cleaved below the hinge region using IdeS enzyme and then reduced to expose the small molecule drugs fully. This pretreatment ensures unrestricted access of Cathepsin B to the cleavage sites, ensuring complete enzymatic cleavage. The drugs released after cleavage from the protein components are separated using reversed-phase high-performance liquid chromatography, and their concentrations are determined using UV absorbance.

The above is the commonly used method for determining the DAR value of antibody-drug conjugates (ADCs). There are also some less commonly used methods, such as CE-based DAR value measurement, and so on. With the development of analytical techniques, methods for determining DAR values will continue to evolve. However, the fundamental point is to ensure effective separation of different components so that DAR values can be accurately calculated. For the aforementioned methods of determining DAR values for ADCs, each method is not isolated. Multiple methods should be used to analyze DAR values, and different methods should corroborate each other to gain a deeper understanding of DAR value analysis and obtain more accurate results.

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