Rapid Native HIC–MS Using Ammonium Tartrate for Robust Drug-to-Antibody Ratio Characterization of Antibody–Drug Conjugates

Antibody–drug conjugates (ADCs) require precise control of drug-to-antibody ratio (DAR) and drug load distribution (DLD) to ensure efficacy and safety, making robust analytical characterization essential. Hydrophobic interaction chromatography (HIC) is the standard technique for intact ADC DAR analysis, but coupling HIC to mass spectrometry (MS) is challenging due to the high salt concentrations needed for effective separation.

Recent studies conducted at Genentech (South San Francisco, California) presented a simple, rapid, and robust native HIC–MS method that overcomes these limitations by using ammonium tartrate, a thermally decomposable salt with kosmotropic strength comparable to ammonium sulfate (1). An online size exclusion chromatography (SEC) desalting step and elevated ionization temperature enable efficient MS detection without compromising chromatographic resolution. The 22-min method enables high-quality DAR characterization of both engineered and interchain cysteine-conjugated ADCs, offering an accessible alternative to complex multidimensional workflows (1). Furthermore, they developed a rapid 10-min, multiattribute HIC method using ammonium tartrate based mobile phase as a process analytical technology (PAT) for real-time bioconjugation reaction monitoring. This multiattribute HIC PAT method was used to simultaneously track DAR, DLD, and drug-linker (DL) concentration within complex reaction mixtures. (2)

LCGC International spoke to Bingchaun Wei, corresponding author of the papers, about the HIC and HIC-MS method they developed, as well as the key takeaways from the studies.

How does HIC separate ADC species, and why is it particularly suited for DAR ratio and DLD determination under nondenaturing conditions?

HIC separates analytes based on reversible interactions between hydrophobic patches on the protein surface and hydrophobic ligands (such as butyl or phenyl groups) on the stationary phase, mediated by a high-salt mobile phase that promotes salting-out/adsorption. For ADCs, HIC is the gold standard because payloads are generally hydrophobic. Conjugation of these payloads sequentially increases the overall hydrophobicity of the antibody. Therefore, ADC species elute in a predictable order of increasing drug load on the HIC column for DAR and DLD determination.HIC operates under non-denaturing conditions, preserving the non-covalent interactions that maintain the structural integrity of interchain cysteine-conjugated ADCs. Reversed-phase liquid chromatography (RPLC)also separates the analytes based on the hydrophobicity, but under denatured conditions with organic solvent, acidic modifier and high temperature. Non-denatured conditions disrupt the non-covalent interactions, dissociating the ADC into fragments and losing the intact DLD information.

What are the limitations of conventional HIC mobile phases in coupling with MS, and how does ammonium tartrate improve HIC-MS compatibility?

Conventional HIC methods rely on high concentrations (1.5–2.0 M) of non-volatile salts like ammonium sulfate to drive hydrophobic adsorption. These salts are incompatible with MS, suppressing electrospray ionization (ESI), clogging ion sources, and forming extensive adducts. Ammonium tartrate addresses this by serving as a "chameleon" salt. It possesses high kosmotropic strength comparable to sulfate, enabling necessary retention and resolution of ADC species. However, unlike sulfate, ammonium tartrate is thermally decomposable. Upon entering the heated ESI source, it breaks down into volatile components, preventing source clogging and minimizing adduct formation. This enables direct, online coupling of HIC to native MS with high sensitivity, eliminating complex online multi-dimensional HPLC setup, solvent modulation or offline fractionation (1).

Can you explain the principle of native MS and its application in analyzing intact ADCs?

Native MS analyzes biomolecules in their folded, physiologically relevant state using "soft" ionization and aqueous, volatile buffers at a neutral pH. This preserves non-covalent interactions destroyed in denaturing MS. Spectra exhibit lower charge states (higher mass-to-charge ratio [m/z]) and narrower charge distributions. For interchain cysteine conjugated ADCs, where heavy and light chains are held together only by non-covalent forces after reduction, native MS is essential. It measures the DAR and DLD at intact levels and detects impurities such as non-covalent aggregates.

How does ion mobility–MS (IM-MS) complement conventional MS in the structural characterization of ADCs?

IM-MS adds an orthogonal separation dimension based on the size and shape (collision cross-section [CCS]) of gas-phase ions. Although conventional MS separates based on m/z, IM-MS separates based on drift time. This resolves isomeric/isobaric species with the same mass but different conformations or higher order structures. For ADCs, IM-MS can detect drug-induced conformational changes and separate singly charged chemical noise from multiply charged protein ions, enhancing signal-to-noise (S/N) for low-abundance variants. Ehkirch and associates did great work back in 2017 by coupling HIC, size exclusion chromatography (SEC), and IM-MS together for an online four-dimensional (4D) ADC characterization (3).

What are the advantages of multidimensional LC–MS approaches over single-dimension LC or MS techniques for DAR profiling?

Multidimensional LC-MS (mD-LC–MS) not only provides two or more orthogonal separation mechanisms online, increasing peak capacity, but the technique also offers ways of modulating the solvent composition for different detectors. In the past, HIC separates DAR species, but it is salt-incompatible with MS. HIC x SEC-MS (Native HIC-MS) separates intact DAR species by hydrophobicity in the first dimension, then "heart-cuts" peaks to a second-dimension SEC column for rapid online desalting before native MS. This process allows precise mass identification of species eluting under each HIC peak without offline collection, providing a comprehensive fingerprint (retention time and mass) in a single run.

How does hydrophilic interaction chromatography (HILIC) differ from HIC in terms of ADC analysis, and when would you prefer one over the other?

HIC separates based on hydrophobicity, whereas HILIC separates based on hydrophilicity and polarity. HIC is generally preferred for determining DAR and DLD for most ADCs because of the hydrophobic payloads. If you have ADCs with highly hydrophilic and polar linkers or payloads, or if you want to perform glycan analysis for ADCs, HILIC can be a viable option. In addition, HILIC metabolites.

Describe how PAT can be integrated with HIC-MS to provide real-time monitoring of ADC bioconjugation reactions.

Regulatory agencies, particularly the Food and Drug Administration (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.

In our study, we coupled a rapid HIC method online to monitor the reaction kinetics of a bioconjugation reaction. This setup simultaneously tracked unconjugated antibody, DAR species, average DAR, and free drug-linker concentration. Dynamic monitoring revealed that in slurry reactions, drug-linker dissolution was rate-limiting step for the reaction. Real-time feedback allowed adjustment of mixing speeds, enhancing dissolution and driving the reaction to completion in 90 min (vs. 16 h), optimizing the process for target DAR and minimizing over-conjugation (2).

What challenges arise in measuring ADC mixtures containing unconjugated antibodies, various DAR species, and residual drug-linkers using chromatography and MS?

Identifying and quantitating low abundance species requires good chromatographic separation and MS sensitivity. Heterogeneous mixtures including various DAR species, as well as protein and drug linker modifications, cause challenges in chromatographic separation. MS sensitivity can suffer from peak co-elution and salt background.

In addition, for reaction kinetics measurement, we need rapid analytical method to reduce the sampling cycle time, especially for fast reaction like maleimide-cysteine reactions. Therefore, there is a need in developing fast PAT methods for real-time reaction monitoring.

How can native HIC-MS be optimized to maintain ADC structural integrity while providing high-resolution, reproducible DAR measurements?

Multiple approaches have recently been developed to couple HIC with MS for native protein and ADC analysis (3–7). These approaches fall into two categories: 1) direct coupling using an MS-compatible HIC method, and 2) reducing the salt content prior to MS analysis.

In our recent studies (1,2), we developed a novel native HIC-MS workflow. The major optimization points were as follows:

1. Mobile phase salt: Using ammonium tartrate for high kosmotropic strength for HIC retention and resolution power and MS compatibility with thermal decomposition capability.
2. MS source conditions: High source temp (>300 °C) to decompose tartrate and desolvate protein without causing in-source fragmentation and source clogging.
3. Column Temperature: 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.
4. Online desalting: Incorporating a post-HIC SEC guard column to separate ADC and salt, remove the background and boost MS sensitivity.

Explain how capillary zone electrophoresis–MS (CZE-MS) can be used in ADC analysis and what advantages it offers for resolving complex drug-load distributions.

CZE separates based on electrophoretic mobility of analyte without a stationary phase. It offers ultra-high resolution for charge variants and ADC fragments often superior to ion exchange chromatography (IEX) or SEC. For lysine-conjugated ADCs, CZE-MS resolves DAR species based on charge shift because lysine contains +1 charge in typical formulation pH. For new payloads like oligonucleotides, CZE-MS can be a great way to monitor the oligonucleotide antibody ratio (OAR) of antibody oligonucleotide conjugates (AOCs). A recent study from Xu et al. showed a nice case study by using CZE-MS for ADCs and AOCs (8).

References

(1) Kempen, T.; Cadang, L.; Fan, Y. et al. Online Native Hydrophobic Interaction Chromatography-Mass Spectrometry of Antibody-Drug Conjugates. MAbs 2025, 17 (1), 2446304. DOI: 10.1080/19420862.2024.2446304

(2) Cadang, L.; Li, S.; Wang, J. et al. Real-Time Bioconjugation Reaction Monitoring of Antibody–Drug Conjugates with Multiattribute High-Throughput Hydrophobic Interaction Chromatography. Anal. Chem. 2025, 97 (48), 26411-26418. DOI: 10.1021/acs.analchem.5c03553

(3) Ehkirch, A.; D’Atri, V.; Rouviere, F. et al. An Online Four-Dimensional HIC×SEC-IM×MS Methodology for Proof-of-Concept Characterization of Antibody Drug Conjugates. Anal. Chem. 2018, 90 (3), 1578-1586. DOI: 10.1021/acs.analchem.7b02110

(4) Yan, Y.; Xing, T.; Wang, S. et al. Online Coupling of Analytical Hydrophobic Interaction Chromatography with Native Mass Spectrometry for the Characterization of Monoclonal Antibodies and Related Products. J. Pharmaceut. Biomed 2020, 186. DOI: 10.1016/j.jpba.2020.113313

(5) Wei, B.; Han, G.; Tang, J. et al. Native Hydrophobic Interaction Chromatography Hyphenated to Mass Spectrometry for Characterization of Monoclonal Antibody Minor Variants. Anal. Chem. 2019, 91 (24), 15360-15364. DOI: 10.1021/acs.analchem.9b04467

(6) Chen, B.; Lin, Z.; Alpert, A. J. et al. Online Hydrophobic Interaction Chromatography–Mass Spectrometry for the Analysis of Intact Monoclonal Antibodies. Anal. Chem. 2018, 90 (12), 7135-7138. DOI: 10.1021/acs.analchem.8b01865

(7) Chen, B.; Peng, Y.; Valeja, S. G. et al. Online Hydrophobic Interaction Chromatography–Mass Spectrometry for Top-Down Proteomics. Anal. Chem. 2016, 88 (3), 1885-1891. DOI: 10.1021/acs.analchem.5b04285

(8) Xu, T.; Zhang, F.; Chen, D. et al. Interrogating Heterogeneity of Cysteine-Engineered Antibody-Drug Conjugates and Antibody-Oligonucleotide Conjugates by Capillary Zone Electrophoresis-Mass Spectrometry. MAbs 2023, 15 (1), 2229102. DOI: 10.1080/19420862.2023.2229102