Separating biomolecules with hydrophobic interaction chromatography (HIC)

In contrast to small molecules, biopharmaceuticals (biologics) are large and highly complex, requiring rigorous characterization and analytical protocols to progress through the development and manufacturing pipeline. Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) have become popular biopharmaceutical candidates over the years, due to their high potency and degree of specificity. However, because of their size and complexity, they are notoriously difficult to separate and analyze. This difficulty highlights the requirement for advanced separation techniques to help progress biopharmaceuticals through quality by design (QbD) implementation.

Characterizing biopharmaceuticals

A critical first step in implementing QbD involves identifying the critical quality attributes (CQAs) appropriate to biopharmaceuticals. As defined by the International Conference on Harmonization (ICH) Q8 (R2), CQAs are a “physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality.” The complexity of biomolecules like mAbs and ADCs makes the identification of CQAs particularly challenging. This means it requires the use of a range of advanced analytical procedures.

Liquid chromatography (LC) and high performance liquid chromatography (HPLC) are longstanding powerful techniques for characterizing biopharmaceuticals. Size exclusion chromatography (SEC), ion-exchange chromatography (IEX), and reversed-phase LC are popular options for characterizing proteins. Reverse phase conditions, however, can lead to protein denaturing, which impacts analysis.

Hydrophobic interaction chromatography (HIC) can overcome this hurdle, by maintaining the protein’s biological activity through intact analytical methods. Unlike reversed-phase chromatography, which is performed under denaturing conditions, HIC is performed in aqueous eluents containing just salt and buffer, which keeps the protein in its native state.

Innovative LC technology

Agilent has developed a new HIC column in its AdvanceBio range to address the separation of particularly challenging molecules, such as mAbs, ADCs, and other recombinant proteins. Novel hydrophobic interaction chemistry has been optimized to ensure the best selectivity and robustness, using ZORBAX fully porous silica particles for high purity, strength, and uniformity. Run-to-run reproducibility is important for consistent performance, and the AdvanceBio HIC columns provide excellent reproducibility for over 400 injections. Batch-to-batch quality control (QC) is also ensured, as every batch of media is tested with NIST mAb standard for quality and dependability.

By using HIC rather than reversed-phase chromatography, CQAs can be resolved from parent molecules with only small differences in hydrophobicity, without compromising speed or accuracy. Other key benefits of the AdvanceBio HIC columns include:

• Single chemistry: proprietary bonding technology eliminates the need for multiple column screenings. Optimized chemistry provides better peak shape with lower backpressure.
• Enhanced robustness: improved column lifetime.
• Great productivity: faster analysis without compromising performance, and every batch of columns is tested with NIST mAb standard for quality and dependability.
• Two column lengths available: 100 mm for routine use (separation in 24 mins) and 30 mm for a three-fold increase in speed (separation in 8 mins).

Advance your CQA monitoring

Developing an effective ADC is a complicated endeavor. First, antibodies must be engineered with the proper affinity and specificity, which must then be conjugated to an effective drug/cross-linker combination. Then, the optimal number of drugs per antibody must be determined, making up the drug-to-antibody ratio (DAR), which is a CQA of ADCs. It needs to be carefully monitored during the development process to ensure that the ADC produces the desired potency, minimizes off-target effects, and exhibits good pharmacokinetic properties.

Determining and monitoring ADC DAR values throughout the drug discovery process informs the choice of ADC candidate to move forward with. The first step in ADC analysis is separation by LC. Due to the diverse chemical nature of ADCs, Agilent offers many columns, such as the AdvanceBio HIC column, so users can select the one that provides the best performance for a specific ADC of interest.

The faster run times and longer column lifetime of the Agilent AdvanceBio HIC columns will allow you to achieve higher throughput, with less downtime and lower costs. Ultimately, helping you to progress your biomolecule through to completion sooner.

To find out how Agilent can help you advance your biopharmaceutical characterization, click here.