Regulatory Considerations When Developing Assays for the Characterization and Quality Control of Antibody-Drug Conjugates

Therapeutic monoclonal antibodies can be conjugated with a variety of molecules including small-molecule drugs, radionuclides, peptides, other proteins (protein toxins, enzymes, cytokines), and polyethylene glycol. Other than polyethylene glycol, the conjugated moiety provides a specific mechanism of action (MOA) resulting in the death of the target cell. For these conjugates, the monoclonal antibody (mAb) acts as a drug delivery system as it targets the conjugated moiety specifically to a cell expressing the antigen recognized by the mAb. However, the mAb provides additional functions to the conjugate, such as a typical antibody half-life of several days to several weeks and, in some cases, antibody effector function, or signal transduction triggering cell death.

There are currently four antibody-conjugates on the market: certolizumab pegol is a pegylated anti-TNF Fab, where the pegylation extends half-life of the molecule; ibritumomab tiuxetan and Iodine 1-131 tositumomab are therapeutic anti-CD20 radioimmunoconjugates; and brentuximab vedotin, approved in 2011, is an antibody-drug conjugate (ADC) targeting CD30. Gemtuzumab ozogamicin was the first approved ADC, but was withdrawn from the market in 2010 after postmarketing clinical studies did not confirm the efficacy that was the basis for the original accelerated approval in 2000. However, recent clinical studies suggest favorable clinical results, indicating a possible commercial future for this ADC.1

Other than certolizumab pegol, these commercial mAb conjugates have oncology indications. This article will focus on ADCs, which to date are mostly under development or approved for oncology indications.

Antibody-drug conjugates: Background and components

Figure 1 – Antibody-drug conjugate IND submissions in five-year cohorts. IND submissions from 2008 through June of 2012 are double the number of INDs submitted between 1993 and 2007.

The first investigational new drug (IND) for an ADC was submitted in the early 1990s. In the past 4.5 years, the number of ADC IND submissions has more than doubled the number seen in the previous 15 years (Figure 1). The growth in ADC IND submissions is mainly due to advances in mAb development and methods for characterization; identification of cytotoxic small drugs; and, in particular, advances in linker and conjugation chemistry.

The three components of an ADC include the mAb, the cytotoxic small drug molecule, and the linker. Upon binding the target cell, the ADC is internalized where the drug is released and kills the cell by the mechanism of the small drug. The most frequently used small drugs to date include maytansinoids and auristatins, which disrupt the tubulin network, and calicheamicin and doxorubicin, which intercalate into the minor groove of DNA, but disrupt DNA by different mechanisms.

While the linker does not provide activity to the ADC, the design of the linker, its stability, and the conjugation chemistry are crucial for the success of an ADC. The linker should be stable in serum so that the cytotoxic drug is not released into circulation prior to reaching the target cell, but allow cleavage once internalized to permit the cytotoxic drug to reach its target.

Since ADCs are comprised of both drug and biological molecules, the characterization and quality assurance of an ADC need to be relevant both for the small drug and linker components as well as the mAb. Figure 2 depicts a schematic diagram of an ADC. Diagrams of ADCs frequently emphasize the linker–drug component as the drug provides the primary MOA (Figure 2a). In reality, the molecular weight of an IgG is >100-fold larger than calicheamicin, the largest of the drugs used in ADCs to date (Figure 2b). Once conjugated, the majority of analytical methods for characterization and quality control of the ADC are based on methods used for mAbs, in addition to those that control the drug loading ratio. Thus, the characterization and quality assurance of the small drug and linker components prior to conjugation provide important information for the overall understanding of the ADC.

Figure 2 – Schematic diagrams of antibody-drug conjugates. a) For purposes of illustration, ADCs are often depicted with the drug and linker components relative to the size of the antibody. b) In reality, the mass of the antibody is over 100-fold greater than the small drug and linker components. Therefore the characterization of the antibody-drug conjugate is largely based on methods used for monoclonal antibodies. However, methods assessing the drug loading and drug/antibody ratio should be included. Particular attention to the characterization and quality assurance of the drug and linker components prior to conjugation is necessary since typical methods for this cannot be performed on the ADC.

Drug and linker starting materials and intermediates

The drug/linker intermediates may be derived by fermentation, chemical synthesis, or semisyntheses process (chemically modified fermentation product). They may be either a small chemical compound, large complex molecule, or peptide. The stage of clinical development determines the extent of the description and characterization of these intermediates. A discussion regarding the designation of starting materials from which the drug/linker is produced should be included as part of an End-of-Phase 2 meeting with the Agency.

The extent of the characterization and quality control of the drug/linker intermediates is the same as if they were developed as a drug substance, since a complete characterization is difficult after conjugations. Characterization includes the chemical structure as well as the impurity profile. The impurity profile should include drug/linker-related impurities, process impurities, and a structural characterization of the impurities present at levels that are greater than the ICH Q3A-recommended identification threshold (typically higher than 0.1%).

Quality control testing and specifications for the drug/linker intermediates should include appearance, identity, assay (HPLC), and impurities. Stability testing should be performed under the long-term (real-time) and accelerated storage conditions to support the intended storage conditions of the drug/linker intermediates.

Monoclonal antibody intermediate

The extent of the characterization and quality control of the mAb intermediate is the same as if it was developed as a drug substance. This includes adventitious agent safety testing of cell banks and unprocessed bulk and endotoxin and bioburden testing of the purified bulk mAb.

The characterization of the mAb includes methods that assess primary, secondary, and higher-order structure; size and charge variants; glycosylation; and other post-translational modifications such as deamidated and oxidized amino acid residues. Antibody function should also be assessed, including antigen binding, signal transduction that may lead to cell death or growth inhibition, antibody effector function, and binding to FcγR and FcRn. Even though cytotoxicity of the small drug component is intended as the major MOA, several ADCs are reported to have additional MOAs. The best known example of this is T-DM1, a maytansinoid derivative conjugated to trastuzumab for the treatment of HER2 positive breast cancer.2 The MOA of trastuzumab includes antibody-dependent cellular cytotoxicity and inhibition of cell proliferation by different paths, including prevention of HER2 receptor dimerization, increased endocytic destruction of the receptor, and inhibition of shedding of the extracellular domain.3

Product-related impurities can include charge and size variants, which should be identified in order to understand the impact they may have on the in vivo behavior of the ADC. Removal of process-related impurities should be assessed during the mAb manufacture. It may be acceptable to provide risk assessments early in development for some small-molecule process-related impurities. Removal of process-related impurities can be validated and the data provided with the Biologics License Application (BLA).

The potency assay for the mAb intermediate typically measures antigen binding. If the mAb provides additional MOA, more than one potency assay should be considered. It may be acceptable for this additional method to be performed on the mAb intermediate; however, there may be specific products where the additional method should be performed on the ADC.

Characterization of the antibody-drug conjugate

Once conjugated, a structural characterization assessing drug loading is crucial. This characterization includes determining the molar absorption coefficient, the drug load distribution, individual drug load variants, and drug/antibody ratio. The ability to characterize individual drug load variants depends on the conjugation chemistry. For example, when conjugation is to cysteine residues of partially reduced mAbs, there will be a small number of available cysteine residues available for conjugation commonly resulting in ~4 drug molecules per antibody. Conjugation to lysine residues is more complex, since there are greater than 50 lysine residues in an mAb, which may lead to greater variation in the drug loading distribution. Mass spectrometry, hydrophobic interaction chromatography, and reversed-phase HPLC are used to characterize the distribution of drug load variants. Peptide mapping methods enable the identification of the conjugated lysines, but it is difficult to identify all the drug load variants.

The impurity profile of free drug-related substances, quenching agents, residual solvents, and other process-related impurities can be characterized and controlled. A risk-based approach may be applied to support control of conjugatable versus nonconjugatable drug-related substances.

The impact of conjugation chemistry on the mAb should be determined for important biological functions of the mAb, including binding to antigen and effector function or signal transduction, if relevant. In addition, a comparison of product-related variants of the ADC should be compared to the unconjugated mAb to determine if the conjugation leads to novel mAb-related variants other than the specific conjugation sites.

A comparability study should be performed on the ADC drug substance when manufacturing changes are introduced to either the intermediates or the ADC drug substance processes.

Quality control testing and specifications of the antibody-drug conjugate drug substance and drug product

For quality control related to the cytotoxic drug, specifications should be established for identity and total drug content (UV absorption) and purity (free drug-related substances, quenching agents, residual solvents, and heavy metals).

To initiate a Phase I clinical trial, the free drug-related impurities in the clinical lot can be qualified relative to data from toxicology studies along with comparable drug/antibody load ratios between the toxicology and clinical lots. The characterization of the impurity profile of the drug/linker intermediates is the same as for drug substance impurities prior to initiation of pivotal clinical trials.

The identity and purity release methods for the ADC often include the same methods used for the mAb intermediate. Size variants (aggregates and fragments) should be controlled. Levels of free antibody should be determined.

The potency assay for an ADC should demonstrate cytotoxicity specific for an antigen positive tumor cell line. For Phase I clinical trials, the release specification for the potency assay should not be broader than the dose escalation scheme.

Stability testing should include an assessment of free drug and stability indicating methods related to the mAb. One lot of mAb intermediate and one lot of the drug/linker intermediates may be used to manufacture multiple lots of an ADC drug substance. Therefore, different combinations of the intermediate lots should be used to manufacture the ADC drug substance for the pivotal clinical trials and the qualification lots in order to maximize the data to establish release specifications for commercialization.

In summary, ADCs are both drug and biological molecules. The characterization, manufacturing process controls, comparability, release, and stability assays need to be appropriate for the entire molecule.


  1. Castigne, S.; Pautas, C. et al. Effect of gemtuzumab ozogamicin on survival of adult patients with de-novo acute myeloid leukaemia (ALFA-0701): a randomized, open-label, phase 3 study. The Lancet2012, 379, 1508–16.
  2. LoRusso, P.M.; Weiss, D. et al. Trastuzumab emtansine: a unique antibody-drug conjugate in development for human epidermal growth factor receptor 2-positive cancer. Clin. Canc. Res.2011, 17, 6437–47.
  3. Hudis, C.A. Trastuzumab—mechanism of action and use in clinical practice. N. Eng. J. Med.2007, 357(1), 39–51.

Marjorie A. Shapiro, Ph.D., reviews monoclonal antibody products in the Division of Monoclonal Antibodies, Office of Biotechnology Products (OBP). Xiao-Hong Chen, Ph.D., reviews small drug oncology products in the Division of New Drug Quality Assessment 1, Office of New Drug Quality Assessment (ONDQA). Both OBP and ONDQA are within the Office of Pharmaceutical Science, Center for Drug Evaluation and Research, U.S. Food and Drug Administration (FDA), Silver Spring, MD, U.S.A.; tel.: 888-463-6332; e-mail: [email protected]. Disclaimer: This article does not represent official FDA policy or guidance. It is the opinion of the authors based on their experience reviewing antibody-drug conjugates.