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Simplify Your Antibody-Drug Conjugate Characterization Process with Confidence

Antibody drug conjugates (ADCs) are complex molecules composed of an antibody linked to a biologically active cytotoxic (anticancer) payload or drug. Antibody drug conjugates are examples of bio-conjugates and immune-conjugates which work to limit side effects and provide a wider therapeutic window than other chemotherapeutic agents.

It is critical to monitor the drug-to-antibody ratio (DAR). Agilent can help with a complete portfolio of end-to-end workflow solutions for analysis of antibody drug conjugates including the InfinityLab Bio LC portfolio, biocolumns, LC/MS, and software suites.

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Proven Methods to Simplify Your ADC Analysis

Complete Workflows, Proven Methods, and Tips to Help Optimize Your Analyses.

Learn how the Agilent AssayMAP Bravo can reduce time and labor required to purify ADCs from serum and prepare them for biotransformation analysis.

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Native Mode Analysis of mAbs and ADCs

Intact workflow solutions for characterizing antibody drug conjugates and other biomolecules by SEC and HIC chromatography techniques.

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ADC Analysis Consumables Ordering Guide

Your analysis made easier. Tips and recommended products to help you streamline and optimize your analysis.

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HPLC Column Selector Tool

Agilent AdvanceBio LC columns are designed and produced to provide results you can be sure of when analyzing highly complex biotherapeutic molecules and monitoring their purity, potency, and other critical quality attributes.

This online tool allows easy column selection by application, particle type, particle size, pore size, linear diameter, and length.



Featured Application Notes

A complete workflow to quantitatively determine brentuximab vedotin's protein and payload stability in monkey plasma was developed. This workflow uses the Agilent AssayMAP Bravo automated liquid handling platform to purify drug molecules from a biological matrix. This was then analyzed with the Agilent 1290 Infinity II Bio LC and Agilent 6495 triple quadrupole LC/MS systems to determine protein and payload concentration. Understanding the stability of different parts of the drug molecules over time is crucial for drug design and its clinical outcome. As a result, these data highlight the complete quantification workflow to support antibody drug conjugate research and development.

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Explore a novel 2D-LC approach for DAR determination which overcomes the tendency of ADCs to degrade under organic and acidic solvent conditions typical of analysis. This method uses HIC, multiple heart-cutting (MHC), and subsequent desalting and separation using size exclusion chromatography (SEC) online with native MS analysis. This workflow uses an Agilent 1290 Infinity II 2D-LC systemAgilent 6545XT AdvanceBio LC/Q-TOF  and Agilent MassHunter BioConfirm software. This native MS analysis method not only provides the accurate average DAR value for ADC samples, but also enables excellent chromatographic separation, preserves the intact native structures, and performs the accurate intact mass determination for all ADCs with various DARs.

  
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During manufacturing, it is important to monitor critical quality attributes (CQAs) of ADCs including drug-to-antibody ratio (DAR) and aggregation. Find a proven method using the Agilent Cary 3500 UV-Vis Multicell spectrophotometer for analysis of DAR and aggregation index of ADCs demonstrating the systems unique capability of analyzing samples at multiple temperatures simultaneously. With the custom equation function in the software, simple methodology, and accurate temperature control, the Cary 3500 UV-Vis allows for the determination of DAR and aggregation index with confidence and ease.

  

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The addition of organic modifiers like isopropanol in hydrophobic interaction chromatography (HIC) can be an important parameter to decrease the retention of hydrophobic antibody drug conjugates (ADCs) as well as to adjust selectivity. The drug-to-antibody (DAR) of brentuximab vedotin was determined using a ternary gradient with isopropanol as organic modifier in the third channel. Excellent reproducibility was found for this challenging combination of high-salt-containing buffer and organic solvent, using the Agilent 1260 Infinity II Prime Bio LC.

    

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Scientists developing antibody‑conjugated biotherapeutics need to obtain accurate and reliable DAR information without having to become experts in sample preparation, mass spectrometer operation, data interpretation, or manual DAR calculation. Agilent provides an integrated workflow that delivers reliable DAR values right to the user’s email, while requiring very little hands-on input. The Agilent AssayMAP Bravo Platform automates sample preparation, and Agilent MassHunter Walkup software ensures optimal MS data with little or no knowledge of instrument operation. Agilent BioConfirm software provides automatic calculation of the average DAR for all forms of the ADC, including intact and deglycosylated configurations, as well as reduced heavy and light chains. 

   

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Additional Resources

Liquid Chromatography (LC) Strategies

Liquid Chromatography- Mass Spectrometry (LC/MS) Strategies

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We’d like to share the latest information from Agilent to help optimize your antibody drug conjugates CQA methods including applications, educational events, webinars, products, and more. 

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