Antibody Drug Conjugate Production Workflow

In this case study we demonstrate Sygnature Discovery’s ability to provide a complete, end‑to‑end Antibody Drug Conjugate (ADC) workflow to support client projects. In particular, we highlight the results of site specific and stochastic conjugation methods that were used.

At Sygnature Discovery, we can combine our in-house protein production expertise with specialist conjugation chemistries, and robust analytical characterisation to deliver high quality ADCs for clients.

In a recent project we generated a panel of trastuzumab based ADCs incorporating several novel linker payload constructs. The project delivered five high‑quality ADCs using a combination of site specific and stochastic conjugation chemistries, we established new capabilities, workflows and analytical methods that enhance our ability to provide the entire ADC production workflow in house.

Establishing Safe Handling of Cytotoxic Payloads

Prior to starting work with cytotoxic payloads, the team implemented a dedicated cytotoxic‑handling system which is an essential requirement for ADC payload chemistry. This includes:

• A new Class II biosafety cabinet with safe‑change filtration
• A dedicated cytotoxic waste stream
• Strict PPE requirements, including double gloves with DMSO‑resistant outers
• Use of screw‑cap tubes only and full secondary containment during transfers

This infrastructure now supports all future ADC projects at Sygnature Discovery.

Stochastic Conjugation

A common conjugation technique in ADC production, is stochastic (random) conjugation. Stochastic conjugation is widely used because it requires no antibody engineering and can be readily scaled. In this project, payloads were attached via reduced interchain disulfides (Figure 1). A major part of the project involved DAR calibration, varying TCEP concentration, payload excess, temperature and reaction time to achieve the desired ADC DAR species.

Conditions identified in the initial calibration served as a starting point for client specific payloads. By varying TCEP and payload equivalents, we achieved DAR4 and DAR8 ADCs with minimal additional optimisation. The method transferred well, enabling accurate detection of conjugation and rapid progression to scale up within days.

Site-Specific Conjugation

Site‑specific conjugation via microbial transglutaminase (tGase) requires access to a specific glutamine residue (Q295 in trastuzumab). However, the native glycan at N297 sterically blocks the enzyme. To resolve this, we optimised PNGase F deglycosylation of trastuzumab to ensure a single homogeneous species was produced (Figure 2).

Intact mass analysis of untreated trastuzumab (Pre‑PNGase F treatment, top panel) reveals multiple glycoforms, producing a heterogeneous cluster of peaks between ~147,900 –148,800 Da. Following enzymatic deglycosylation with PNGase F (Post‑PNGase F treatment, bottom panel) the heterogeneous glycoform pattern is reduced into a single species at ~145,169 Da. This shift reflects successful removal of glycans and yields a more homogeneous antibody population with a defined mass, suitable for site specific conjugation.

Using the optimised deglycosylation of trastuzumab protocol, tGase conjugation proceeded efficiently, producing two uniform DAR2 ADCs with excellent analytical profiles. Mass spectrometry confirmed the expected single heavy‑chain modification pattern characteristic of tGase‑mediated site‑specific conjugation (Figure 3).

Intact mass analysis of the light chain (top panel) shows a single peak at 23,442.7 Da, corresponding to the unmodified light chain (LC). This confirms that no payload is attached at the LC chain, consistent with the expected tGase mediated site‑specific conjugation chemistry, which targets a glutamine residue located on the heavy chain only.

The heavy chain spectrum (bottom panel) displays one species at 49,525.1 Da, representing a +1 payload addition. This demonstrates efficient, site‑specific incorporation of a single linker payload molecule per heavy chain (HC), giving the characteristic DAR2 ADC (one payload on each of the two HC subunits). Together, these spectra confirm successful site‑specific conjugation, high reaction fidelity, and the expected uniform DAR2 species for the engineered ADC.

Quality Control (QC)

To confirm ADC integrity, QC for all ADCs (Figure 4) produced includes:

• SEC-HPLC
• Mass spectrometry
• SDS-PAGE
• A280/BCA concentration determination

A. SEC‑HPLC chromatograms confirm monodisperse ADCs with minimal aggregation.
B. Intact MS outputs verify expected LC/HC masses and correct DAR assignment.
C. SDS‑PAGE shows the ADC under both reduced and non‑reduced conditions.
D. BCA measurements provide accurate concentration assessment. Collectively, these QC data confirm the identity, purity and suitability of ADCs for downstream biological studies.

Delivering High Quality ADCs for Client Research

Across all five ADCs generated in this project, our team delivered high purity ADCs suitable for downstream biological assays, supported by comprehensive analytical characterisation including accurate DAR determination, SEC‑HPLC, mass spectrometry, SDS‑PAGE, and A280/BCA concentration measurements. These ADCs were produced at milligram scale quantities, enabling clients to progress confidently into early discovery studies.

This work also demonstrated several core strengths within Sygnature’s Protein Science team:

• An accurate and robust mass spectrometry workflow for robust DAR assignment
• Expertise in both stochastic and site specific conjugation
• End‑to‑end ADC production, from in house antibody expression through to conjugation and purification
• Troubleshooting of challenging payload chemistries
• SEC purification of ADCs for complete free payload removal

Together, these capabilities now complement and enhance our established strengths in protein expression, purification, chemical biology and bioassay support, creating a comprehensive platform to accelerate client ADC programmes. In addition, through Sygnature Discovery’s fully integrated discovery model, spanning medicinal chemistry for linker and payload design, bioscience, DMPK and in vivo pharmacology, we can deliver the entire ADC workflow in house, enabling seamless, cross disciplinary project execution and supporting complex, multi‑component ADC programmes from concept to candidate.

Conclusion

ADCs represent some of the most complex biologics in development today, sitting at the interface of protein science, synthetic chemistry and analytical characterisation. This project showcased the breadth of Sygnature Discovery’s integrated capabilities, taking a therapeutic antibody from in house production through two conjugation strategies, multiple novel payloads, and full QC.

Through this work, Sygnature Discovery is now able to offer a complete, end‑to‑end ADC workflow, fully customised to each client’s payload chemistry, conjugation strategy and target DAR species. By optimising our intact mass spectrometry workflow, we can now resolve and quantify DAR species with minimal sample requirements, rapid turnaround times and high reproducibility, enabling confident decision making throughout ADC optimisation. Moreover, we can achieve a broad range of DAR species via both stochastic and site-specific conjugation, applying diverse linker payload systems to meet programme specific needs.

Whether clients require early proof‑of‑concept ADCs, high DAR exploration, method development or tailored conjugation strategies, our multidisciplinary team is equipped to accelerate discovery programmes and support the development of next‑generation ADC therapeutics.