Advanchrom HIC-Butyl FAQ: Linker Stability, Sample Handling, Column Treatment…

The following Q&A section features questions from attendees during our live webinar held on May 22nd. It covers essential questions on ADC analysis using Advanchrom HIC-Butyl columns, reflecting a range of practical interests and technical inquiries. To enhance clarity and readability, all questions and responses have been reformatted in a formal style.

Additionally, some answers have been expanded after the webinar to provide additional context and clarification; these extended responses are marked in red.

If you haven’t watched yet, here is the full recording of our live webinar:

Q1: Is this column used for cancer research?

A1: It is used for the analysis of ADC (antibody-drug conjugate) drugs. ADC drugs are developed for cancer treatment.

Q2: I think we should use an internal standard for such analysis to ensure no dissociation of the drug from the antibody.

A2: An internal standard (IS) doesn’t prevent drug-antibody dissociation. Yet, a well-chosen IS (like a stable isotope-labeled complex) may help monitor and partially correct for dissociation, but only if it behaves similarly to the actual complex. 

To minimize dissociation, optimize sample handling conditions (e.g., pH, temperature, etc.) and consider using orthogonal methods like SEC or native MS to assess complex stability.

Q3: What is the sample? Is it blood with the ADC drug?

A3: No. The initial sample would be the laboratory-synthesized ADC, which will then be compared to the standard of that particular ADC.

Q4: If I heard correctly, you said that excessive use of an organic mobile phase could break the linker between the antibody and payload. Is this applicable to all types of linkers? What is the recommended percentage of organic solvent—is it a maximum of 25%, as stated in your last slide?

A4: For cysteine and histidine linkers in ADCs, organic solvent content should not exceed 25%. However, other types of linkers—such as enzymatic and engineered linkers—are more robust and can tolerate up to 30% organic solvent. Since cysteine and histidine linkers are most commonly used, it is advisable to limit the organic modifier to a maximum of 25%.

Q5: Is this how therapeutic drug monitoring works?

A5: Yes. In a simplified sense, cancer drugs function through a lock-and-key mechanism. The antibody binds to a specific site on the cancer cell and delivers the drug inside the cell. The drug then inhibits cell growth and ultimately kills the cancer cell.

Q6: In what areas are these columns used? Pharmaceutical production, scientific, and R&D laboratories—anything else?

A6: The HIC columns are primarily applicable to the analyses of protein, antibody, and ADC. Applications in other fields have not been revealed yet.

Q7: Is there a way to determine whether the sample’s linker has degraded into a lower drug-to-antibody ratio (DAR), or if it remains intact?

A7: The conditions of linker breakdown is harsh: for example, the non-cleavable linker requires lysozyme treatment under low pH conditions (4.5–5.0) for cleavage. HIC operates under milder conditions, typically in a salt phase and at neutral pH around 7.0, preventing linkers from breakdown.

Furthermore, there are three ways to determine whether the linker has broken:

1. Combine LC-MS and HIC to assess whether the linker is intact or broken.
2. Compare the sample to both fresh (intact) and intentionally degraded controls to identify signs of linker breakdown.
3. Use multiple techniques, LC-MS, HIC, and immunoassays (e.g., ELISA, etc.) to get a full picture of linker integrity and DAR stability.

Q8: Would the gradient slope help in method development?

A8: Yes, the gradient slope can be helpful. Slowing the gradient—that is, reducing the rate at which salt concentration changes in the mobile phase—may cause peak broadening during elution. However, to some extent, it can improve resolution between analytes.

Q9: How should we treat the column to minimize unwanted interactions?

A9: Conventional silica-based columns often exhibit protein adsorption issues. However, the silica in our columns is coated with a protective polymer layer, which not only shields the silica substrate but also prevents nonspecific adsorption. 

On the other hand, newly manufactured columns may still contain a small number of sites capable of adsorbing proteins—such as on the frits or inner surfaces of the stainless-steel tubing. 

It is recommended to pre-condition the column by injecting a sufficient amount of protein to block these sites or to passivate the column using EDTA before use. This helps ensure the reliability and reproducibility of the results.

Q10: Will you introduce a preparative version of this column?

A10: A preparative version is not under development at this time, but will be introduced later based on market needs.