The linker payload is integral to the effectiveness of an antibody drug conjugate (ADC)—the linker controls where and how the drug is released to destroy cancerous cells, while the payload determines potency and therapeutic potential.
Due to the potent toxin involved in linker payloads, many of the challenges surrounding these compounds parallel those that scientists and engineers encounter in high potency active pharmaceutical ingredient (HPAPI) development.
For this reason, organisations with HPAPI expertise are inherently well-positioned to support the synthesis of these complex molecules.
Both processes necessitate stringent containment measures and specialised training to mitigate safety risks, thorough purification approaches, and a focus on reproducibility and scalability in a contained environment.
However, there are important differences between the two, particularly when it comes to purification, which demand specialised solutions. In this blog, we’ll take a closer look at some of the purification challenges associated with linker payloads, and discuss what is needed to effectively address them.
Common challenges with purification in linker payload synthesis
Careful purification is crucial in linker payload synthesis to ensure therapeutic efficacy, effective targeting, stability, compliance and safety.
However, several factors make these conjugation intermediates particularly challenging to purify relative to small molecule APIs. These include:
Linker payloads involve multiple diverse components to achieve precise conjugation and targeting when used to develop an ADC.
Scientists must ensure that each of these components remain compatible with downstream conjugation processes, as structural inconsistencies could impact targeting ability or the payload’s release mechanism.
Payload components are typically non-polar, limiting their solubility. To address this, scientists often introduce hydrophilic groups or soluble linkers.
Though this improves solubility, it also increases molecular complexity by mixing polarity and charge, creating added challenges for purification.
Drug linker synthesis is generally carried out at a small scale due to its potency and structural complexity.
Any product loss is magnified at smaller scales, creating added pressure to maximise yield while still achieving strict purity targets.
The biggest challenge associated with purification is instability. Linker payloads are labile compounds, and they are intentionally designed to cleave once they reach their intended target.
This makes them prone to degradation during synthesis, unwanted side reactions, or breakdown under harsh solvents, temperatures, and pH levels.
Many purification techniques rely on these harsh conditions; recrystallisation, for example, uses solvent mixtures and heating to leave impurities behind. The labile nature of linker payloads, therefore, rules out many common purification methods.
So it is important to look towards alternative purification methods that don’t risk compromising the end product.
Achieving purity targets for linker payloads
Although more complex than other purification methods, chromatography remains the most favourable approach for the purification of these ADC intermediates. Its ability to effectively separate complex molecules when paired with an appropriate phase system makes it integral to achieving the high purity required in ADC development.
Both normal-phase (NP) and reverse-phase (RP) chromatographic techniques can be used in drug linker purification. Normal-phase chromatography uses a polar stationary phase and non-polar mobile phase, so it is effective in separating polar compounds. Reverse-phase chromatography uses a non-polar stationary phase and polar solvents, making it better suited in separating non-polar linker payloads.
While RP chromatography is useful for separating linker payloads and removing minor impurities, it also leaves large volumes of remaining solvent to be removed. Lyophilising large volumes of material is not only inefficient, but can also put the compound at risk of degradation.
To address these challenges with RP chromatography, a specialised solid-phase extraction (SPE) technique has emerged. Sometimes, this approach is referred to as a “catch and release” purification. Let’s take a closer look at this process:
- Chromatography: After reverse-phase chromatography, the purified linker payload is contained in a large volume of polar solvent.
- Solvent removal: The solvent containing the payload is eluted through a reverse-stationary phase in the SPE system, and the payload adheres to the stationary phase.
- Non-polar solvent: Once the sample is loaded, the stationary phase is washed with a volatile, non-polar mobile phase, flushing the purified payload from the stationary phase.
- Lyophilisation: The non-polar solvent is much easier to remove than the original polar solvent, and lyophilisation becomes far more efficient and less risky.
This approach significantly reduces the volume of material to be lyophilised, cutting down processing time while preserving the integrity of the final product.
By combining chromatographic separation with an effective solvent removal strategy, scientists can more efficiently and effectively achieve the stringent purity and stability requirements that linker payloads, and ultimately ADCs, necessitate.
Supporting complex linker payload synthesis and purification at Sterling
Successfully synthesising and purifying linker payloads requires more than just technical skill.
It demands significant expertise in safety and containment, proficiency across a variety of analytical and purification techniques, and an understanding of the full ADC lifecycle, from exploratory medicinal chemistry to manufacturing.
At Sterling, we bring all of these elements together. Our advanced infrastructure supports a variety of processes within configurable containment measures.
By combining the extensive high-potency API expertise at our Wisconsin, US facility with the comprehensive ADC knowledge of our Deeside team, we can support even the most complex ADC projects from discovery onward, including challenging linker payload synthesis and purification processes.
Our robust approach to purification ensures efficiency, quality, and compliance, while setting ADC projects up for successful conjugation.
If you’d like to learn more about how we can support your linker payload synthesis requirements or other ADC project needs, speak to a member of our team.
