Solubility of ADCs: influencing factors and optimization tips

The solubility of ADCs is a critical factor not only for clinical efficacy and safety, but also for manufacturability. Here, we will discuss the factors that affect the solubility of ADCs and how to optimize their solubility.

Therapeutic mAbs and the development of ADCs

Clinical success rates of therapeutic antibodies have surpassed expectations, which has led to significant industry investments in biologics research and development in addition to traditional small molecules. However, unconjugated antibodies mainly target the soluble and cell-surface antigens. Recent advancements in the fields of protein engineering, formulation, and delivery devices, have pushed the limits of biologics even further. One notable development is the evolution of ADCs, that combine both extracellular and intracellular targeting, maximizing the therapeutic window of antibody drugs.5,7

What are ADCs?

Antibody-drug conjugates are a new class of highly targeted biopharmaceuticals designed to deliver highly potent small molecule drugs utilizing the target specificity and lengthy half-life of an antibody. They comprise three components: a selective monoclonal antibody (mAb), the payload or the cytotoxic agent, and the linker.

Currently, ADCs are used primarily for the management or treatment of cancer5 (READ MORE: ADC for therapy). When it comes to clinical use, safety and efficacy are of central importance. ADCs must be formulated to remain stable in systemic circulation until it reaches the target site, to prevent off-target killing and toxicity5,6.

Hence, finding the right target protein, linker, and payload for design and appropriate excipients for the formulation is crucial to clinical success. One of the key attributes that determine ADC stability and clinical effectiveness is solubility. 

Click here to find drug aliquots of the latest ADCs for research. 

Factors affecting Solubility

As already mentioned above, the solubility of ADCs is a critical factor for clinical efficacy and safety as well as for the manufacturability. Poor solubility can lead to aggregation and precipitation of the ADC, which can reduce its efficacy and increase its toxicity.

Compared to unconjugated mAb drugs, ADCs have low solubility and are marketed at low concentrations (<20 mg/mL) owing to their high selectivity and tumor killing efficacy. Moreover, lower concentrations also reduce the risk of aggregation.

In the following, we will take a closer look at the influencing factors that affect the solubility of ADCs and the optimization of their solubility. 

Cytotoxic Payloads

The payload selection is based on solubility, conjugation, and stability7. The current generations ADCs internalize and release active cytotoxic molecules inside tumor cells. An ideal payload for these molecules should have invitro subnanomolar IC50 values toward tumor cell lines and functional groups (such as a disulfide, alkene, or epoxides) with sufficient solubility in aqueous solutions for conjugation reactions with the antibody and for solubility of the resulting ADC product5. Moreover, it is estimated that a drug-to-antibody ratio (DAR) above 4 can diminish their solubility7.

However, to reduce bystander cytotoxicity, the payload’s solubility must be carefully chosen. Due to greater hydrophilicity, amatoxin-based ADCs have higher solubility and uniformity than other cytotoxic payloads5.


The low solubility of numerous candidate payloads can be compensated for by hydrophilic linkers, such as those containing sulfonates or poly (ethylene glycol), enabling higher drug-to-antibody ratio (DAR) than hydrophobic linkers, such as Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)7.

Strategies for optimizing protease-sensitive linkers involve the use of β-glucuronide linkers, which are recognized and hydrolyzed by β-glucuronidase for payload release5. The lysosomal enzyme β-glucuronidase is overexpressed in certain tumor types. Thus, a glucuronic acid-based linker has the potential to improve the stability of the ADC in the bloodstream6. Additionally, the hydrophilic nature of this linker can improve the solubility of the intact ADC compared to the dipeptide-based alternative. When compared to the self-immolating p-aminobenzyl carbamate dipeptide ADC, those with glucuronic acid-based linkers showed improved solubility of the intact ADC5.

Bioconjugation methods

The choice of antibody (mAb) and bioconjugation technology also affects the ADC stability. Typically, endogenous mAbs have interchain disulphide bridges containing amino acids such as lysine, histidine, tyrosine and cysteines, that are utilized for bioconjugation. However, site and residue-specific modifications are also employed for enhancing physicochemical and functional properties. A two-step conjugation strategy involving binding of cationic organometallic Pt (II)-based linker to histidine residues was shown to improve the water solubility of the linker-payload conjugate and minimize antibody aggregation3. Similarly, Thiomab Technology, a cysteine conjugation and re-bridging technique, is known to maintain mAb stability, improve binding affinity and minimize ADC aggregation5.

Excipients and formulation

Antibody drugs are stable over narrow pH ranges. Lower pH may induce protein cleavage and a higher pH can cause aggregation. Electrolytes used in buffers for formulation can affect conformational stability, equilibrium solubility, intermolecular repulsion, and rate of aggregation12. It has been shown that electrostatic shielding in 0.9 % NaCl solution can enhance protein-protein interactions, resulting in reduced solubility of high DAR species or an increased aggregation tendency. The addition of certain amino acids as excipients (for example arginine and proline) increases mAb solubility and can prevent aggregation and unfolding12.

External factors

Apart from the intrinsic factors (i.e., molecular structure and formulation), external factors such as storage and transportation can also affect the stability of the drug. ADCs are complex molecules and are more sensitive to external stressors like temperature variations and agitation when compared to the non-conjugated parent antibody. For this reason, most ADCs for clinical use are manufactured as lyophilized powder for improved shelf-life. In the next section, we focus on the effect of temperature variation on ADC stability.

Effect of temperature on ADC stability

ADCs are usually stored at 2-8oC. Frequent freeze-thaw cycles and elevated temperatures are known to induce aggregation of ADCs12. The same problem can be a result of the freezing process, since certain formulation components can induce instability. For example, sodium chloride, used as a tonicity agent, increases the osmotic strength of the remaining solution during freezing, which can cause aggregation1. Likewise, if any component of pH buffers such as sodium phosphate buffer, precipitates, a pH shift can occur leading to the same outcome1. To improve stability during freezing, surfactants, commonly polysorbates, are used as protective agents. Also, introducing four N-glycosylation sites to protein drugs is known to prevent aggregation induced by either heat stress or freeze-thaw cycles12.

Here are a few experiments that demonstrate ADC stability under thermal stress:

  1. In an experiment, an ADC where the antibody was linked to the val-citn Monomethyl Auristatin E (vc-MMAE) linker drug through the reduction of interchain disulfides, was shown to rapidly form high molecular weight species (HMWS) at 40 °C5. The ADC had an average DAR of 3.5 and was formulated at 30mM buffer, adjusted to pH 5.5, and concentrated to 10 mg/ml. The HWMS was formed due to the increased sensitivity of the tertiary structure to thermal stress, presumably due to the presence of conjugated drugs at the inter-chain cysteines close to the CH2 domain.
  2. A study was designed to monitor the stability of a specific ADC coupled to MMAE toxin by one step or two-step technology9. Three rounds of freeze–thaw cycles and shaking did not significantly alter the integrity of the molecular structures of the ADC and the DAR was not affected.
  3. Stability assessment of Trastuzumab versus Trastuzumab emtansine (Kadcyla®) demonstrated the ADC is more prone to instability under stressors such as agitation and elevated temperatures (25oC and higher), compared to the parent mAb8. However, repeated freeze-thaw cycles did not show any aggregation in both products.

High DAR ADC vs. stability: The example of Sacituzumab govitecan (IMMU-132)

The aggregation of ADCs is determined by their sequence/structure, linker, payload, conjugation chemistry and DAR. Although a DAR of 4 has been considered optimal, Sacituzumab govitecan is a paradigm shift in ADC development with a DAR of 7.6 and a linker that is moderately stable4.

Sacituzumab govitecan or IMMU-132 incorporates a humanized monoclonal IgG directed against Trop-2 (hRS7 IgG1κ), conjugated to the highly toxic active metabolite of irinotecan, SN-384,11. ADCs conjugated to such ultratoxic drugs require high stability in the bloodstream with activity only possible at the target site4.

Trodelvy® (Sacituzumab govitecan-hziy) was recently approved by the US-FDA and received marketing authorization by EMA for the treatment of patients with metastatic triple-negative breast cancer (mTNBC). Here are some of the drug characteristics with respect to solubility and stability.

  • Payload: SN-38 with an IC50 of ~1.0–6.0 nM is highly hydrophobic, with a limited number of coupling sites. A dipiperidino side chain was added at the hydroxyl on the C10 position to improve water solubility4.
  • Linker: The linker designated CL2A is pegylated with a lysine residue in the linker system that improved solubility and minimized aggregation4,11. In-vitro serum stability studies show that the linker is moderately stable and did not affect the rate of SN-38 release.
  • DAR: The linker conjugates to 7-8 molecules of SN-38 without affecting its binding and pharmacokinetics. The low stability and high DAR was demonstrated to improve toxicity profile and bystander effect.
  • Formulation: Trodelvy delivers 180 mg sacituzumab govitecan-hziy, the API. Excipients include MES, polysorbate 80 and trehalose dihydrate. Reconstitution with 20 mL of 0.9% Sodium Chloride Injection, USP, results in a concentration of 10 mg/mL with a pH of 6.53.
  • Freeze-thaw cycles: As per the FDA stability data (, Trodelvy was shown to be stable for up to 3 freeze/thaw cycles at -20°C/ ice-water bath, for 4 cycles at -70°C/ ice-water bath, stable for 6 hours in ice-water bath (benchtop). In addition, after 4 freeze-thaw cycles at -70°C/ ice-water bath resulted in 9.7% free SN-38. And, 19.4% free SN-38 after 6 hours in ice-water bath (benchtop)3.

Require Trodelvy for research? Click here .


Although ADCs are a great innovation in the category of biopharmaceutical drugs, stability is one of the main concerns during design, manufacturing, storage, and transportation. Instability can result in heterogeneity of the final product affecting the therapeutic efficacy. Going forward, the Food and Drug Administration (FDA) approval is more likely for homogeneous ADC products. Improved site-directed conjugation chemistry and antibody engineering methods are being developed to ensure homogeneity during manufacturing. Further technological advances in payload solubility, linker chemistry and site-directed conjugation can improve the physicochemical and pharmacokinetic stability of ADCs.

Clinical-grade ADCs for research & development

The active pharmaceutical ingredients (API) in reference drugs or biosimilars are called clinical-grade molecules. Their efficacy and safety in treating a particular disease in humans were demonstrated in clinical trials. When it comes to a known and proven function in humans, clinical-grade molecules are the only reference. For early-stage drug development assays, functional in vitro and in vivo experiments, analytical methods for molecular characterization or biocomparability studies, clinical-grade molecules are the preferred choice. the process of procuring those molecules from approved drugs can be time consuming, costly, and complex. 

The Evidentic GmbH provides clinical-garde molecules such as ADCs at their original concentration and formulation from the original pharmaceutical drugs that are approved by the European Medicines Agency (EMA). As a pharmaceutical wholeseller, Evidentic can procure any EU-licensed molecule. Choose from the ready-to-ship molecules or order the molecules you desire. 

Require reference ADC drugs for characterization, analytical studies, or your research program? Click here


  1.  Best Practices in Formulation and Lyophilization Development. [Accessed 10 Nov 2022]
  2. Beckley, N. S., Lazzareschi, K. P., Chih, H. W., Sharma, V. K., & Flores, H. L. (2013). Investigation into temperature-induced aggregation of an antibody drug conjugate. Bioconjugate chemistry24(10), 1674–1683.
  3. NDA/BLA Multi-disciplinary Review and Evaluation BLA 761115 TRODELVY, sacituzumab govitecan-hziy [Accessed 1 Dec 2022].
  4. Goldenberg, D. M., & Sharkey, R. M. (2019). Antibody-drug conjugates targeting TROP-2 and incorporating SN-38: A case study of anti-TROP-2 sacituzumab govitecan. mAbs, 11(6), 987–995.
  5. Kim, E. G., & Kim, K. M. (2015). Strategies and Advancement in Antibody-Drug Conjugate Optimization for Targeted Cancer Therapeutics. Biomolecules & therapeutics, 23(6), 493–509.
  6. Kostova, V., Désos, P., Starck, B., & Kotschy, A. (2021). The Chemistry Behind ADCs. Pharmaceuticals, 14(5).
  7. McCombs, J. R., & Owen, S. C. (2015). Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry. The AAPS Journal, 17(2), 339-351.
  8. Mohamed, H. E., Mohamed, A. A., Al-Ghobashy, M. A., Fathalla, F. A., & Abbas, S. S. (2018). Stability assessment of antibody-drug conjugate Trastuzumab emtansine in comparison to parent monoclonal antibody using orthogonal testing protocol. Journal of pharmaceutical and biomedical analysis, 150, 268–277.
  9. Nicolas Schneider, Morgan Mathieu, Christian Belmant, Agnès Represa, Sandra Savard-Chambard Gas, Angélique Boedec Herbette, Hélène Rispaud, Naouel Lovera, Delphine Bregeon and Courtney Noah. BioProcess International. [Accessed 10 Nov 2022]
  10. Single Use Support. Controlled freezing of ADCs. [Accessed 10 Nov 2022]
  11. Tong, J. T. W., Harris, P. W. R., Brimble, M. A., & Kavianinia, I. (2021). An Insight into FDA Approved Antibody-Drug Conjugates for Cancer Therapy. Molecules (Basel, Switzerland), 26(19), 5847.
  12. Yoann Le Basle, P. Chennell, Nicolas Tokhadze, Alain Astier, V. Sautou. Physicochemical Stability of Monoclonal Antibodies: A Review. Journal of Pharmaceutical Sciences, Wiley, 2020, ff10.1016/j.xphs.2019.08.009ff. ffhal-02385936f
Publishing Date:
Wednesday, 1 February, 2023
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