Unlocking the Key to ADC Drug Quality Control with Welch’s New HIC Columns!
Introduction
Antibody-drug conjugates (ADCs) play an important role in oncology treatments. ADCs are drug molecules formed by conjugating antibodies with cytotoxic payloads via linkers. Their ingenious design integrates the precise targeting capability of antibody drugs with the potent cytotoxic effects of small molecules, making ADCs a “biological missile” for targeted cancer therapy.
To date, 17 ADC drugs have been approved globally, with 83 new ADCs entering clinical trials in 2024 alone. The global ADC market is estimated to exceed $13 billion.
However, the structural complexity and inherent heterogeneity of ADCs pose significant challenges in quality control, requiring rigorous analytical methodologies. Among these, the drug-to-antibody ratio (DAR) is a critical quality attribute, as it directly reflects the payload quantity per antibody, impacting both therapeutic efficacy and safety.
Therefore, accurate determination and monitoring of DAR are essential throughout ADC development and commercial production.
Introducing Welch Advanchrom HIC-Butyl
Hydrophobic interaction chromatography (HIC) is a widely used chromatographic technique that separates molecules based on differences in surface hydrophobicity. It is an essential method for determination and characterization of DAR in ADCs.
To meet the growing demand for high-performance protein separation, Welch Materials introduces the Advanchrom HIC-Butyl column. This high-performance HIC column is designed using advanced large-pore silica microspheres combined with proprietary surface bonding technology, making it ideal for antibody and ADC characterization.
Product Features
- Unique chemical design offering excellent selectivity for ADC molecules
- Ultra-high purity large-pore silica microsphere matrix for superior column efficiency
- Low non-specific adsorption ensuring high recovery rates
- Excellent pressure tolerance and durability
- Outstanding batch-to-batch reproducibility
- Effective retention and separation using lower salt concentrations
Product Information
| Product Name | Advanchrom HIC-Butyl Column | Column Material | Stainless Steel |
| Bonded Phase | Butyl | Dimensions | 4.6×50 mm; 4.6×35 mm |
| Matrix | Ultra-high purity large-pore silica | Max Pressure | 6000 psi |
| Particle Size | 3 μm | Max Temperature | 60°C |
| Pore Size | 1000 Å | pH Range | 2 – 8 |
| Product Name | Advanchrom HIC-Butyl Column |
| Bonded Phase | Butyl |
| Matrix | Ultra-high purity large-pore silica |
| Particle Size | 3 μm |
| Pore Size | 1000 Å |
| Column Material | Stainless Steel |
| Dimensions | 4.6×50 mm; 4.6×35 mm |
| Max Pressure | 6000 psi |
| Max Temperature | 60°C |
| pH Range | 2 – 8 |
Application Cases of Advanchrom HIC-Butyl
HIC-UV Application: DAR Analysis of Bispecific ADC
| Sample | ADC | ||
| Column | Advanchrom HIC-Butyl | ||
| Specification | 3 μm, 4.6×50 mm | ||
| Mobile Phases | A: 100 mM PB Solution + 1.0 M (NH4)2SO4 , pH 7.0 B: 100 mM PB Solution, pH 7.0 / IPA = 80/20 |
||
| Flow Rate | 0.5 mL/min | ||
| Wavelength | 280 nm | ||
| Column Temperature | 25°C | ||
| Injection Volume | 10 μL | ||
| Elution Program | Time (min) | Phase A (%) | Phase B (%) |
| 0 | 90 | 10 | |
| 3 | 90 | 10 | |
| 35 | 0 | 100 | |
| 38 | 0 | 100 | |
| 39 | 90 | 10 | |
| 45 | 90 | 10 | |
Conclusion: Under ammonium sulfate conditions, ADC molecules with different DAR values show effective retention and separation on HIC columns. The unconjugated antibody, having the weakest hydrophobicity, elutes first, while ADC molecules with higher drug loading exhibit stronger hydrophobicity and elute later.
HIC-MS Applications: Analysis of DAR2 Monoclonal ADC & Intact Antibody (Rituximab)
Although ammonium sulfate is widely used for DAR determination, it is non-volatile and incompatible with mass spectrometry (MS). To address this, we also tested a volatile ammonium acetate mobile phase system.
Conditions:
| Sample | ADC / Rituximab | ||
| Column | Advanchrom HIC-Butyl | ||
| Specification | 3 μm, 4.6×50 mm | ||
| Mobile Phases | A: 1.0 M CH3COONH4 B: 25 mM CH3COONH4 / ACN = 70/30 |
||
| Flow Rate | 0.5 mL/min | ||
| Wavelength | 280 nm | ||
| Column Temperature | 30°C | ||
| Injection Volume | 10 μL | ||
| Elution Program | Time (min) | Phase A (%) | Phase B (%) |
| 0 | 100 | 0 | |
| 2 | 100 | 0 | |
| 15 | 0 | 100 | |
| 20 | 0 | 100 | |
| 22 | 100 | 0 | |
| 30 | 100 | 0 | |
Conclusion: Under ammonium acetate conditions, excellent retention and peak shape are achieved for both DAR2 ADC and rituximab, providing an MS-compatible solution for future DAR characterization.
Comparison with Competitor Columns: Separation of Standard Proteins
| Sample | (~1 mg/mL dissolved in Mobile Phase A) 1. RNase A; 2. Lysozyme; 3. α-Chymotrypsinogen |
||
| Column | Advanchrom HIC-Butyl | ||
| Specification | 3 μm, 4.6×50 mm; 3 μm, 4.6×35 mm | ||
| Mobile Phases | A: 100 mM PB Solution + 2.0 M (NH4)2SO4 , pH 7.0 B: 100 mM PB Solution, pH 7.0 / ACN = 90/10 |
||
| Flow Rate | 1.0 mL/min | ||
| Wavelength | 280 nm | ||
| Column Temperature | 30°C | ||
| Injection Volume | 10 μL | ||
| Elution Program | Time (min) | Phase A (%) | Phase B (%) |
| 0 | 100 | 0 | |
| 1 | 100 | 0 | |
| 15 | 0 | 100 | |
| 20 | 0 | 100 | |
| 22 | 100 | 0 | |
| 30 | 100 | 0 | |
Conclusion: Both Welch HIC columns provided excellent retention and separation of the three standard proteins, delivering superior peak shape and theoretical plates compared to competitor columns.
Mobile Phase Recommendation
Due to the non-volatile nature of conventional ammonium sulfate mobile phase, it is incompatible with mass spectrometry and thus can only be used in HIC-UV systems. For HIC-MS applications, volatile ammonium acetate should be used instead.
Quality Assurance
Each Advanchrom HIC-Butyl column is manufactured with a mature packing process and strict quality control to ensure superior separation performance.
Ordering Information
| Part No. | Product | Specification |
| 0001-01037 | Advanchrom HIC-Butyl | 3 μm, 4.6×50 mm |
| 0001-01036 | 3 μm, 4.6×35 mm |
For further information, please contact our sales team at info@welchmat.com , and we shall serve you wholeheartedly.