Small Molecule Impurities Represent a Key Challenge When Designing Control Systems for ADC Therapeutic Manufacture
Antibody-drug conjugates (ADCs) harness the specificity of an antibody to the cytotoxic potency of a small molecule drug to achieve targeted cell death. An important, emerging class of biopharmaceuticals, ADCs have proven efficacy in cancer treatment. Within the past decade, four ADC drugs have been granted approval for oncology indications, and many more such therapeutics are currently in pre-clinical or clinical development1. The ADC market is predicted to be worth approximately $10 billion by 20252.
Although ICH guidelines clearly define acceptable levels of drug-related impurities for small molecule new chemical entities (NCEs), there are currently no established rules for ADCs. Composed of both antibody and small molecule elements, ADCs have a complex structure from which many different impurities can arise. Drawing on existing ICH guidelines, a recent publication co-authored by AbbVie suggests a science-based approach that can be applied to the design of an effective control strategy for small molecule impurities in ADC therapeutics3.
ADCs are highly complex
ADCs typically consist of three main structural units - a monoclonal antibody (mAb), a small molecule drug, and a low molecular weight linker covalently attaching the two together4. It is common practice to combine the drug and the linker by chemical synthesis before conjugating this intermediate directly to the mAb, however a two-step process may also be employed. During the two-step approach, the linker is first attached to the mAb, then the drug is attached to the linker. Whichever method is chosen, conjugation of the linker to the mAb usually exploits lysine or cysteine residues, as illustrated.
Common conjugation chemistries for ADCs. The linker is typically attached between the drug and the mAb by a covalent bond through a cysteine or lysine.
The origin of small molecule impurities
Impurities can arise during any stage of ADC manufacture. Depending on their point of origin and their molecular weight, they are classified as being either mAb-related or small molecule impurities. Of these, the latter may be of particular concern since many of the small molecules used in ADC production are highly cytotoxic.
Small molecule impurities typically originate during manufacture of the drug, the linker, or the linker-drug intermediate. They may also form during manufacture or storage of the ADC drug substance (DS - mainly composed of the API) or drug product (DP - the formulated mixture of the API and any excipients). To ensure both ADC efficacy and patient safety, it is essential to evaluate levels of these impurities throughout the entire ADC manufacturing process, and to fully understand any associated risk.
Key assumptions in assessing the risk of small molecule impurities
In suggesting an effective control strategy for small molecule impurities in ADCs, the authors of the aforementioned publication adopted several assumptions3:
- The focus of the control strategy is on small molecule impurities only; mAb-related impurities are not covered
- The small molecule drug would be toxic to patients if present at high enough doses
- The worst-case assumption, that all small molecule impurities containing structural elements of the drug will have a similar level of toxicity as the drug itself, is used
- The ADC is intended for use in treating cancer
- The linker-drug intermediate is synthesized first, and is then conjugated to the mAb
- Data is available to demonstrate that the small molecule drug does not need to be considered unusually potent as defined in ICH Q3A (i.e. the drug is not pharmacologically active or toxic at 1mg/day patient exposure)
Impurity control in the linker-drug intermediate
The majority of small molecule impurities which occur in the DS or DP originate from the linker-drug intermediate. These include residual solvents, reagents and their by-products, elemental impurities, and contaminants which are structurally-related to the linker-drug intermediate or its individual components. Since impurity control is best achieved close to the point of introduction, or at a step where the impurity can be more easily removed, manufacture of the linker-drug intermediate is a key stage in the ADC manufacturing process at which to incorporate suitable controls.
Fortunately, many of the small molecule impurities which arise during manufacture of the linker-drug intermediate lack the capacity to conjugate to the mAb. These can therefore be removed during downstream purification steps. By demonstrating effective process control, ADC manufacturers can minimize the number of tests required to show that these contaminants are no longer present.
A different approach is necessary for linker-drug impurities which are able to conjugate to the mAb. Because purification steps do not typically afford separation of the resulting conjugation products from other proteinaceous materials, it is instead important to understand how the quantity of these impurities in the linker-drug intermediate relates to their levels in the DS and DP. Molecular weight is a key factor in calculating the associated impurity dose.
To put this into context, the linker and the drug have an average molecular weight of just 1000 Da each, meaning that they make up only a small proportion of the mass of the ADC. For example, an ADC with a drug-to-antibody (DAR) ratio of four will have an approximate molecular weight of 160,000 Da. This means that <5% of its mass is comprised of the small molecule components. Using the following equation, it is possible to calculate how impurity doses vary as a function of their level in the linker-drug intermediate prior to the conjugation reaction:
Calculated daily impurity dose. This is equivalent to the ADC dose multiplied by the amount of impurity in the linker-drug intermediate, multiplied by the DAR, and then multiplied by the ratio of the molecular weight of the linker-drug impurity relative to the molecular weight of the ADC; dividing by the dose frequency provides an indication of the daily impurity dose.
Based on this equation, a small molecule impurity with a molecular weight of 2000 Da, present at a level of 3wt% in the linker-drug intermediate, would result in a 0.15wt% impurity in the ADC. Dosing the ADC at 50mg every three weeks would result in an impurity dose of 75µg, equating to a daily impurity dose of 3.6µg/day. This is illustrated in the following table:
Impurity dose based on level of conjugatable impurities in the linker-drug intermediate.
It is important to note that a small molecule impurity level of 3wt% is an extreme example. Linker-drug manufacturing processes typically deliver intermediates with impurity levels well below this. Furthermore, many small molecule impurities lack the structural elements required for toxicity, making them a lower concern than those impurities with cytotoxic potential.
Impurity control in the DS
To produce the DS, the linker-drug intermediate is conjugated to the mAb. Impurities of particular concern at this stage of the ADC manufacturing process are conjugates formed of the linker-drug impurity and the mAb; and the linker-drug intermediate itself, plus its quenched derivatives or by-products.
Unwanted conjugates arise when any linker-drug impurities possessing the ability to attach to the mAb do so by forming a covalent bond. Since these unwanted conjugates may be present at higher levels than those small molecule impurities which lack the capacity to conjugate to the mAb, it is especially important to demonstrate their control.
The linker-drug intermediate may itself become an impurity in the DS because it is common practice to use a stoichiometric excess to drive the conjugation reaction to completion in a timely manner.Incorporation of a well-designed ultrafiltration/diafiltration (UF/DF) step should remove most linker-drug intermediate impurities, however it may be necessary to include additional purification steps such as column chromatography. A decision tree can be used to inform this decision.
Decision tree for assessment of impurities in linker-drug intermediates.
Stability of the ADC
A property unique to ADCs is their capacity to release free drug and drug-containing degradation products. These components can have a high associated risk of toxicity, while detachment of the drug from the mAb can also reduce product potency, making assessment of ADC stability an essential part of any ADC manufacturing process.
In considering a worst-case scenario, whereby all of the drug becomes detached from the ADC, the limits established in the ICH guidelines for NCEs can be applied. If this theoretical maximum amount of free drug falls below ICH limits, the safety concern is minimal. The maximum amount of free drug, expressed as wt/wt%, can be calculated using the following equation:
Calculated maximum amount of free drug.
In the more likely scenario that only a proportion of the drug leaves the ADC, the absolute amount of free drug can be calculated thus:
Calculated absolute amount of free drug.
During this assessment of ADC stability, the risk of free drug-related impurities may be found to be low, making it possible to remove tests for these impurities from DP release and stability assessment. Alternatively, if it is determined that the amount of free drug at the end of the desired shelf-life is excessive, several options are available:
- Reducing the shelf-life
- Changing the storage conditions
- Changing the formulation
- Changing the presentation (e.g. developing a lyophilized product)
- Adding purification steps (e.g. to remove degradants from DS prior to DP manufacture)
Small molecule impurities represent a key challenge which must be addressed when designing control systems for ADC therapeutic manufacture. Although the difference in molecular weight between the linker-drug intermediate and the ADC means that patient exposure to small molecule impurities is comparatively low, the potential cytotoxicity of these components makes it essential that effective measures are implemented to minimize their levels.
Many small molecule impurities are cleared by typical manufacturing processes, however the control of those small molecule impurities with the capacity for conjugation to the mAb is best implemented during manufacture of the linker-drug intermediate. It is wise to consider incorporating additional purification steps to remove linker-drug impurities from the DS, and it is also important to demonstrate stability of the ADC to ensure robust, safety-compliant ADC production.
Offering expertise in mAb manufacture, conjugation and fill-finish manufacturing, AbbVie is your partner of choice for ADC production. Contact AbbVie Contract Manufacturing to discuss your ADC manufacturing requirements.
- Antibody-Drug Conjugates: Pharmacokinetic/Pharmacodynamic Modeling, Preclinical Characterization, Clinical Studies, and Lessons Learned, Hedrich WD et al, Clin Pharmacokinet. 2018 Jun;57(6):687-703
- Control Strategy for Small Molecule Impurities in Antibody-Drug Conjugates, Gong HH et al, AAPS PharmSciTech. 2018 Apr;19(3):971-97
- Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry, McCombs JR and Owen SC, AAPS J. 2015 Mar;17(2):339-51