Multi-Attribute Quantitation of Antibody-Drug Conjugates by LC-MALS

Significance of the topic

Liquid chromatography coupled with multi-angle light scattering (LC-MALS) provides a single-run, orthogonal approach to quantify multiple critical quality attributes of antibody-drug conjugates (ADCs) under near-native conditions. ADCs are inherently heterogeneous in drug load and prone to aggregation or fragmentation; robust, routine methods that simultaneously report aggregation state, absolute molar mass, extinction coefficients, and drug-antibody ratio (DAR) are therefore essential across discovery, process development, and quality control to ensure accurate dosing and product stability assessment.

Objectives and study overview

This work demonstrates a platform workflow using size-exclusion chromatography with MALS (SEC-MALS) plus dual-wavelength UV and differential refractive index (dRI) detection to: quantify monomer, dimer, and higher molecular weight (HMW) species; determine ADC extinction coefficients; and measure average DAR and DAR distribution. Orthogonal separations—hydrophobic interaction chromatography (HIC) and reverse-phase chromatography (RPC) coupled to MALS—are applied to resolve payload variants and refine DAR species assignments. Model ADCs evaluated were trastuzumab emtansine (T-DM1) and brentuximab vedotin (cAC10-vcMMAE).

Methods and experimental design

The platform combines SEC-MALS-UV-dRI for native characterization with HIC-MALS and RPC-MALS for resolving payload heterogeneity. Key experimental points include:

• SEC performed in physiologic PBS (and compared to 50 mM ammonium acetate) to assess aggregation under native vs MS-compatible conditions.
• Dual-wavelength UV (280 nm and drug-specific wavelengths: 252 nm for T-DM1, 248 nm for brentuximab vedotin) together with dRI permit direct measurement of protein and drug contributions and calculation of extinction coefficients for intact ADCs.
• HIC-MALS separates cysteine-conjugated ADC DAR species (non-denaturing gradient), while RPC-MALS (TFA/ACN gradient) is used to probe lysine-conjugated ADC heterogeneity and potential positional isomers.
• Data processing and component assignment performed using ASTRA software with blank baseline subtraction for gradient methods to reduce artifacts.

Used instrumentation

• ACQUITY Premier UPLC system with eλ PDA detector (Waters Corporation)
• microDAWN MALS photometer (Wyatt Technology)
• microOptilab differential refractive index detector (Wyatt)
• Columns: ACQUITY Premier Protein SEC (4.6 × 150 mm, 1.7 µm, 250 Å); ACQUITY Premier Protein BEH C4 (2.1 × 50 mm, 1.7 µm, 300 Å); ProteinPak Hi Res HIC (4.6 × 100 mm, 2.5 µm)
• Data analysis: ASTRA software (Wyatt)

Main results and discussion

• Unconjugated mAb controls (trastuzumab, brentuximab) measured by SEC-MALS were >99% monomer with measured protein molar masses ~145 kDa, matching sequence expectations. Measured extinction coefficients at 280 nm were determined (e.g., trastuzumab ε280 ≈ 1.423 mL mg−1 cm−1; brentuximab ε280 ≈ 1.544 mL mg−1 cm−1) and used for subsequent ADC quantitation.
• SEC-MALS quantified ADC species and determined DARs in a single native run. T-DM1 (lysine conjugate) presented primarily monomer (~96% by mass) with a monomer-average DAR ≈ 3.1 and overall average DAR ≈ 3.4 (RPC-MALS), consistent with known heterogeneity for lysine conjugation. cAC10-vcMMAE (cysteine conjugate) showed a predominant monomer (≈85.8% mass), with dimer ≈2.5% and HMW aggregates ≈8.9%; SEC-MALS reported monomer-average DAR ≈3.6 and overall DAR ≈4.0.
• ADC extinction coefficients measured by SEC-MALS-UV-dRI differ from unconjugated mAb coefficients; using the unconjugated mAb ε to quantify ADC concentration can produce substantial bias (example: trastuzumab ε would overestimate T-DM1 concentration by ≈10%).
• Mobile-phase selection affects observed aggregation: T-DM1 displayed a small dimer peak in PBS but that dimer was absent when SEC was done in 50 mM ammonium acetate, indicating buffer-dependent stability and/or altered detection of noncovalent species.
• SEC partially separated DAR species within monomer peaks: drug molar mass increased across T-DM1 monomer peak (leading edge DAR ≈1 to trailing edge DAR ≈5), suggesting hydrophobic interactions drive chromatographic discrimination of DAR variants.
• HIC-MALS of brentuximab vedotin resolved at least five major DAR-related peaks: free mAb (~3.8% mass), DAR2 (~23.8%), DAR4 (~34.6%), DAR6 (~4.3%), DAR8 (~12.6%), plus dimer/HMW co-eluting fraction (~15.7%). The HIC-derived average DAR (≈4.3) aligns with SEC-MALS totals, and HIC better resolves cysteine-conjugate DAR species than SEC.
• RPC-MALS of T-DM1 revealed two partially resolved populations; a secondary peak with narrow DAR distribution (≈2.9) likely represents positional isomers, while the main peak contains a continuous DAR distribution (average ≈3.6). RPC-MALS potentially enables integration with LC-MS because MALS is non-destructive.
• Limitations: gradient separations (HIC, RPC) introduce baseline drift and lower signal-to-noise for minor species, increasing uncertainty in molar mass and DAR for low-abundance peaks. HIC method used did not fully resolve dimer/HMW species.

Benefits and practical applications of the method

• SEC-MALS-UV-dRI is a practical, native-condition platform for routine QC-style monitoring of aggregation, monomer purity, and average DAR in a single injection.
• Direct measurement of intact ADC extinction coefficients supports accurate UV-based concentration determinations during development and clinical production stages, reducing bias compared to using unconjugated mAb coefficients.
• HIC-MALS provides orthogonal quantitation of DAR species for cysteine-conjugated ADCs, enabling more detailed assessment of conjugation distributions relevant to potency and stability.
• RPC-MALS aids characterization of lysine-conjugated ADC heterogeneity and can flag positional isomers for follow-up by LC-MS.
• Combined LC-MALS workflows complement LC-MS and ELISA, offering non-destructive molar mass information that can guide and validate mass-spectrometric analyses and stability assessments.

Future trends and potential uses

• Integration of LC-MALS with native MS (online or fraction-coupled) to combine absolute molar mass and sequence-level identification, improving characterization of low-abundance species and positional isomers.
• Method optimization for gradient MALS (improved baseline subtraction, higher-sensitivity detectors) to reduce uncertainty in HIC/RPC quantitation of minor DAR species.
• Wider adoption of intact extinction-coefficient measurement as a standard QC metric across ADC development pipelines to harmonize concentration reporting.
• Analytical advances tailored to site-specific conjugation chemistries as those approaches mature clinically, requiring higher resolution of positional and stoichiometric variants.
• Automation and standardized platform methods for regulatory submissions and lot release testing that combine SEC-MALS, HIC-MALS, and orthogonal MS methods into validated workflows.

Conclusions

SEC-MALS coupled with dual-wavelength UV and dRI detection is a versatile, easy-to-implement platform for simultaneous measurement of ADC aggregation, absolute molar mass, intact extinction coefficients, and average DAR under native conditions. Orthogonal HIC- and RPC-MALS analyses enhance resolution of DAR variants and positional isomers and provide complementary quantitation that aligns well with SEC-MALS totals. These LC-MALS methods strengthen routine ADC characterization and can be integrated with LC-MS for deeper structural insight, supporting development and QC of heterogeneous ADC modalities.

References

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