Hydrophobic interaction chromatography (HIC) is widely used to characterize antibody-drug conjugates, but its role in real-time process monitoring has remained limited. However, by replacing conventional sulfate salts with ammonium tartrate, researchers at Genentech have developed an approach that preserves HIC separation performance while enabling compatibility with mass spectrometry – effectively transforming HIC into a real-time tool for ADC reaction monitoring.
Here, Bingchuan Wei, a scientist in Genentech’s Synthetic Molecule Pharmaceutical Sciences group, who led the work, explains how years of iteration culminated in a practical real-time monitoring strategy – showcased in their most recent study – and why he believes extending the role of HIC beyond end-point characterization will continue to shape ADC process understanding and control in the years ahead.
What inspired you to explore new directions in hydrophobic interaction chromatography (HIC) for ADC characterization?
For years, HIC has been the gold standard for determining the drug-to-antibody ratio (DAR) and drug load distribution (DLD) of ADCs. However, identifying HIC peaks has historically been a bottleneck, typically requiring either complex multi-dimensional LC-MS setups, which often denature the ADC, undermining the purpose of native separation, or labor-intensive offline fraction collection. Furthermore, we recognized that traditional bioconjugation reaction acts like a “black box,” where reagents are added and the product is tested hours later, simply because standard HIC methods lack the speed required and capability in separating complex reaction matrix to serve as a Process Analytical Technology (PAT) method for bioconjugation. Our goal was to transform the traditional HIC method into a rapid, online “fingerprint” of the reaction mixture, enabling direct native HIC-MS coupling for real-time peak identification.
What initially drew your attention to ammonium tartrate as a mobile phase, and what made it a compelling candidate to investigate further?
My journey with this mobile phase began well before the pandemic, inspired by the pioneering work to couple HIC and MS by Bifan Chen from Ying Ge’s group at UW-Madison, Andy Alpert from PolyLC and scientists from Abbvie. This inspiration ultimately led to our 2019 Analytical Chemistry publication, where we successfully coupled isocratic HIC with MS using ammonium acetate – a volatile, MS-compatible salt – to analyze mAb oxidation and free thiol variants under fully native condition without organic solvent addition. However, during that study, we identified a critical limitation of ammonium acetate: it lacked the retention power necessary for separating more complex hydrophobic species.
Around that time, I began experimenting with ammonium tartrate, an idea sparked by an early Science paper from Fred McLafferty’s group. We observed that ammonium tartrate offered significantly stronger retention than acetate for the same mAbs while still yielding a high-quality native MS signal through directly native HIC-MS coupling at 100 mM tartrate concentration. Although we did not include these findings in the 2019 paper because the signal consistency was not yet perfected, and we don’t have an explanation for why tartrate, a non-volatile salt, works at such a high concentration, the potential of tartrate remained a compelling open question.
The pandemic forced a pause on my HIC-MS research as my focus shifted to RNAs and other novel modalities. Recently, however, we were able to pivot back to analytical development for ADCs. I was joined by Trevor Kempen, a talented co-op student from Dwight Stoll’s group, and Lance Cadang, an expert MS scientist, as well as many colleagues in our department. Together, we resumed the search for a “Goldilocks” salt – one with the strength to retain DAR0 species on the HIC column but without the risk of destroying your mass spectrometer. We confirmed that ammonium tartrate occupies a unique chemical niche: it possesses high kosmotropic strength comparable to sulfate, enabling excellent resolution of DAR species, yet it is thermally decomposable. We realized that you can intentionally raise the temperature of the electrospray source and break tartrate down into volatile gases rather than causing clogging, effectively “tricking” the instrument into accepting a salt that acts like a non-volatile during separation. This discovery allowed us to implement a simple online SEC desalting step immediately following HIC, achieving comprehensive native HIC-MS profiling of all DAR species in an interchain cysteine-conjugated ADC within 70 minutes.
Please tell us about your most recent work on HIC-MS
Regulatory agencies, particularly the FDA, have increasingly emphasized the adoption of PAT to enhance process understanding and control. For ADC manufacturing, the bioconjugation reaction is a critical unit operation where the critical quality attributes (CQA) are defined. Traditionally, this process was treated as a "black box," with samples taken only at the endpoint for offline analysis.
Since we solved the first part of the question of HIC peak identification with a tartrate mobile phase, we continued to work on the chromatographic method optimization. In the most recent publication, we developed a rapid 10 min, multi-attribute HIC method as a process analytical technology (PAT) for real-time bioconjugation reaction monitoring. The method not only monitors DAR and DLD, but also the free drug linker concentration in the reaction mixture.
The most critical insight in the bioconjugation reaction we investigated was: “reaction is fast, dissolution is slow.” We observed that the drug-linker was being consumed faster than it could dissolve from the slurry, effectively starving the reaction. This dynamic insight, which endpoint analysis would never have revealed, allowed us to realize that simply continuous stirring the reaction mixture could overcome this bottleneck, rather than adding more expensive drug-linker or antibody into the reaction mixture.
Were there any findings or observations that surprised you during this work?
I was surprised by just how much in-depth process knowledge and efficiency gain we observed from one rapid analytical method. Once we understood that the reaction was dissolution-rate limited and adjusted the mixing parameters accordingly, we saw the reaction time drop from an overnight process (approximately 16 hours) to just 90 minutes. Process understanding from advanced analytical methods can lead to significant efficiency gain, cost reduction, and better product quality. This study certainly highlighted how powerful real-time analytics is in the modern pharmaceutical industry.
What are the broader implications of this study – for ADC development, for process understanding, or for analytical workflows more generally?
Instead of manufacturing a batch and hoping it meets specifications, we now have the analytical eyes to steer the process into the correct lane in real-time. For ADC development specifically, it shines a light into the bioconjugation “black box” and provides in-depth understanding of bioconjugation chemistry. From an analytical development and characterization perspective, it proves that we can couple "unfriendly" chromatographic modes like HIC to high-resolution MS under native mode, opening new avenues for characterizing proteins and bioconjugates under native conditions.
For teams considering adopting a similar HIC strategy, what do you see as the most important practical or conceptual considerations?
While we have validated this platform across a diverse array of ADCs in the publication, varying in payload, linker, antibody, and conjugation chemistry, it is impossible to encompass every potential structural permutation in the study. Consequently, we advise teams to view this method as a robust framework that can be tweaked to match the specific physicochemical properties of their molecule and the reaction matrix. As an example, we adjusted the method by different column temperature for different ADC conjugation chemistry: lower (25 °C) for interchain cysteine conjugated ADCs to prevent thermal induced dissociation and high (40°C) for engineered cysteine conjugated ADCs to improve resolution.
Looking ahead, how do you envision real-time analytical tools shaping the future of ADC development and process control?
Aviv Regev, head of Genentech research and early development (gRED), has spoken about the “lab in the loop” as a vision that reimages the study of biological system; I believe the same “lab in the loop” concept can be applied in ADC biomanufacturing. Currently, we measure the product quality at the end of the reaction and humans make decisions to adjust the process. I hope we will be able to combine chromatography and mass spectrometry with orthogonal analytics such as multi-attribute Raman spectroscopy (MARS) as a comprehensive means of reaction monitoring. The aim would be to establish a real-time feedback loop while analytical data feeds directly into active, real-time process control, and real time release testing (RTRT).
More broadly, how do you see the role of analytical science evolving within the wider landscape of drug development and manufacturing?
Analytical science is moving from a service function, which often confirms what the process team made, to a co-pilot of innovation. Therapeutic modalities are increasingly complex. Therefore, analytical scientists are becoming architects of the product profile. We are no longer just doing the measurement; we have a critical role in defining them and influence our process and formulation colleagues on how process and formulation influence product quality dynamically.
As this work continues, what directions or questions are you most excited to explore next?
I am eager to expand this “real time” analytical philosophy to other complex therapeutic modalities. I also plan to further investigate the synergy of orthogonal technologies – integrating separation science, mass spectrometry and advanced spectroscopy – to construct a comprehensive view of the manufacturing process. My goal is to move beyond static characterization and achieve a dynamic, mechanistic understanding of processes, structure and function ensuring we can design quality into these next-generation medicines from the very start.
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