Author: Dr. Richard Easton Date: 25th March 2026
Antibody Drug Conjugates (ADCs) are antibody-based anti-cancer therapeutics consisting of monoclonal antibodies attached to a cytotoxic drug via a linker of some kind. ADCs are flourishing in the biopharmaceutical industry at present as companies explore different conjugation chemistries to link their non-proteinaceous drug to the antibody vector of choice. These highly targeted delivery systems offer the promise of lower drug levels in the patient and, as a result, potentially significantly reduced drug side-effects.
Careful analysis of conjugation products needs to be carried out to ensure that the conjugation itself has proceeded as expected. This includes determining that the antibody has not been adversely affected through, for example, oxidation, deamidation, non-specific drug binding or any other side reactions that are chemically feasible under the conjugation conditions used. Whilst we tend to think in terms of analytical assessment at the primary structure level, it’s important to remember that the attachment of drug groups with their own characteristics of size, charge distribution and hydrophobicity/hydrophilicity at various points could also influence secondary and tertiary structure, as well as creating the potential for aggregation.
For these reason, it’s important that these areas of Higher Order Structure (HOS) are also investigated to fully understand the impact of the conjugation process on the monoclonal antibody (mAb) and the resultant ADC. It should be pointed out that alteration of the HOS is not necessarily in itself problematic, after all the ADC is not being marketed as a biosimilar to the mAb. Rather, the data are generated to give a better understanding of the product following conjugation.
Alteration of the HOS of an ADC when compared to its native mAb may lead to questions regarding potential immunogenicity or could be a cause of increased aggregation, if observed. Furthermore, from a functional point of view, HOS changes may impact mAb target binding and therefore affect the ability of the mAb to deliver the drug to the site of action.
Secondary and tertiary structural techniques that are used routinely in protein analysis such as Circular Dichroism (CD), Fourier Transform-Infra-Red (FT-IR), Microfluidic Modulation Spectroscopy (MMS), Fluorescence analysis and Nuclear Magnetic Resonance (NMR; both 1D (1H) and 2D (1H-13C)) are equally applicable to ADCs (these techniques have been covered in a separate blog).
Since these techniques are used to assess the HOS of a molecule, it is critical to analyze the unconjugated antibody alongside the ADC to demonstrate what to expect from the data from the mAb’s native state.
The idea of orthogonality in HOS analysis is just as applicable for ADCs as it is for other proteins (see the blog mentioned above for a discussion of orthogonality), since each individual technique will have its own sensitivities for different structural features such as alpha helices or beta sheets. Any perturbation of the HOS in the ADC may disrupt these motifs to a greater or lesser degree both locally to the site(s) of conjugation or more globally across the molecule, therefore techniques need to be as all-encompassing as possible.
| mAb | 1 | 11 | 33 | 19 | 36 |
| ADC | 0 | 10 | 34 | 20 | 37 |
Far-UV CD data obtained from analysis of a native mAb (green) and an ADC (blue) of a monoclonal antibody. The results obtained from assessment of the raw data using the protein structure database and the CDSSTR algorithm are shown in tabulated form.
One consideration that does need to be borne in mind is the spectroscopic profile of the drug conjugated to the mAb. Depending on the drug, it may have absorption characteristics in the spectral range of some of the instrumentation (CD in particular). A spectroscopic profile of the native drug is therefore absolutely necessary when assessing HOS data from an ADC to help assess any impact the drug is having on the overall ADC profile. Again, the use of orthogonal techniques means that any interferences from the drug in a particular analysis can be circumvented by data from other procedures and a clearer understanding of the ADC HOS is obtained.
The chemical processes used to produce ADCs could result in enhanced aggregation as a result of structural disruption of the mAb and this needs to be investigated. Again, a sample of the native mAb should be analyzed alongside the ADCs to provide baseline aggregate levels. As with HOS analysis, aggregation of non-conjugated proteins has been discussed in a separate blog.
Just as with other HOS analyses, orthogonality is very important in aggregation studies. This comes out of the general principle of the value of orthogonal investigations, as recognized by the regulatory agencies, stemming from the fact that different aggregation techniques will give different values for the level of aggregation due to the nature of the techniques themselves. At BioPharmaSpec, we use a combination of sedimentation velocity ultra-centrifugation (SV-AUC) and size exclusion chromatography with multi-angle laserlight scattering (SEC-MALS) for aggregation studies and these have been shown to be applicable to ADCs.
SEC-MALS of a fusion protein (left) and SV-AUV of a mAb (right), including an expanded view to compare dimer and trimer components.
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In summary, HOS techniques are not only applicable but necessary for a full structural understanding of an ADC. When they are applied in an orthogonal manner a strong picture of ADC HOS emerges. Orthogonality of protein analytical aggregation techniques can also be successfully applied to ADCs with the data being used to either confirm satisfactory levels of aggregation or suggest possible modifications to process/ purification procedures if levels are outside an expected or acceptable range.
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