Biologics – Developability Assessment

Sending drug candidates selected during early discovery stage with suboptimal properties into late-stage development can increase failures or lead to expensive and time-consuming formulation and development efforts. Early developability assessment using an array of different techniques can help identify inherent stability risks early in a molecule’s development, so that any liabilities can be engineered out or managed early on.

The HORIZON® system is the industry’s first analytical system designed specifically to address the demand for quantitative subvisible particle analysis in high throughput even when limited sample material is available – as early as candidate selection or during development of higher concentration formulations. Based on the USP method of membrane microscopy, image analysis on the HORIZON system is automated and requires very little sample. The HORIZON system is an ideal tool for incorporating subvisible aggregate analysis into the biologics workflow as an orthogonal technique from discovery phase all the way downstream.

Sub-visible particles fall into a unique size range: too big for DLS, too big for SEC, and too small for visual analysis by eye.

Because microscopic particles are a key quality attribute of a protein drug product and an indicator of pharmaceutical stability, there is a need to characterize subvisible protein aggregates throughout the entire development workflow, starting at the discovery stage, through formulations, and bioprocessing. Yet to this day, subvisible aggregate particle analysis is a neglected component of early development programs.

Subvisible particle analysis can reveal stability issues when other techniques lack sensitivity

During the candidate selection phase, information about properties such as aggregation behavior, hydrophobicity, and stability is needed.  Techniques used to predict protein stability during developability assessment or pre-formulations typically measure smaller, soluble aggregates and protein unfolding/Tm. However, relying on information from such methods as SEC, DLS, MALS, or iso-electric focusing is limiting and can mean potentially missing critical information on aggregate particles. Measuring in the subvisible range can reveal issues that would otherwise remain undetected.

mAb samples subjected to mechanical stress show low counts of soluble aggregates as measured by SEC. However, when aggregates in the subvisible range are measured by flow microscopy (FM), the same samples show high aggregate counts. Adapted from Southall, et al. “Particle analysis as a formulation development tool”, Amer. Pharm Rev. (2011).

mAb samples subjected to freeze thaw show low counts of soluble aggregates as measured by SEC or icIEF. However, when aggregates in the subvisible range are measured by flow microscopy (FM), the same samples show high aggregate counts. Adapted from Southall, et al. “Particle analysis as a formulation development tool”, Amer. Pharm Rev. (2011).

The HORIZON measures aggregates that are not visible with SEC

SEC analysis of a particular mAb sample shows the sample is 97.1% monomer. Bowers, K. (June 11, 2019) “Seeking the Unseen: Sub-Visible Particle Analysis as a Core Analytical Technique” [Cambridge Healthtech Institute Webinar].

HORIZON image for the same mAb sample shows the presence of subvisible particles missed by SEC analysis. Bowers, K. (June 11, 2019) “Seeking the Unseen: Sub-Visible Particle Analysis as a Core Analytical Technique” [Cambridge Healthtech Institute Webinar]

HORIZON measures changes in stability that are missed with DLS

Dynamic Light Scattering measurement in assessing long-term stability. At later time points the hydrodynamic radius is relatively constant, suggesting that the sample is stable. Bowers, K. (June 11, 2019) “Seeking the Unseen: Sub-Visible Particle Analysis as a Core Analytical Technique” [Cambridge Healthtech Institute Webinar]

HORIZON measurement in assessing long-term stability. At later time points the subvisible particle count has increased substantially, indicating stability issue is missed by looking at hydrodynamic radius alone. Bowers, K. (June 11, 2019) “Seeking the Unseen: Sub-Visible Particle Analysis as a Core Analytical Technique” [Cambridge Healthtech Institute Webinar]

Automated analysis with the HORIZON system is fast and requires as little as 25 μL per sample, making detecting presence of larger, insoluble aggregates accessible early on for use in developability assessment and pre-formulations studies. Measurement of subvisible particles helps to complete the stability picture – which can minimize risk and enable more informed decisions about which candidates to move forward into development.

Why use the HORIZON system for developability assessment of biologics:

  • Low sample consumption (as little as 25 μL per test) so that multiple measurements can be made and averaged
  • Higher throughput for testing lots of candidates or conditions
  • Provides detailed information on aggregate particles – size, morphology, counts, distribution
  • Rapid analysis time
  • Wide working range: Measures particles from 2 μm to 4 mm with high reproducibility
  • Particles are imaged without the interference of buffer or matrix for higher sensitivity
  • User-friendly software interface
  • Quality images of particles
  • Quantitation of non-homogeneous particle populations (size, density, morphology)
  • Sensitivity to detect changes as a function of stress and solution conditions