Biologics – Late Stage Formulation

Subvisible particles are a key quality metric for biologic drugs

Subvisible protein aggregates can form at any stage in manufacturing process or during transportation and storage – triggered by oxidation, mechanical stress, impurities, pH shifts, temperature changes, device interface, etc.

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.

Drug product formulation involves characterizing the processes for manufacture of the bulk formulation and final dosage form, stability and forced degradation studies, and interaction with primary packaging.  Particles in protein drug formulations are often characterized by two common techniques a) light obscuration (LO) and b) flow imaging (FI). These flow-based systems consume significant sample from purging and dead volume, and have low throughput due to the washing and priming required to keep the flow cell clean and prevent sample-to-sample carryover.

The HORIZON® system technology is based on <USP 788> method of membrane microscopy – but automated. The method is completely reinvented using modern robotics, image processing, and novel optics technology.  Since there are no fluidics on the HORIZON system, it is easy to use, with no washing or priming, or risk of cross-contamination. Translucent particles can be clearly imaged, leading to more accurate particle counts.

The 96-well plate format consumes very little sample, which can be crucial when testing high concentration protein drug formulations.

Measurements on the HORIZON system are accurate and robust

The HORIZON system measures by conducting image analysis on particles captured on a membrane surface. Because the imaging is done in air rather than liquid, the system is not subject to matrix effects or solution refractive index.

Accuracy: Direct comparison with SEM

Robustness: Linearity in dilution of IgG aggregates

Side Illumination can distinguish between different types of particles

The HORIZON system has a second mode of detection from a 465nm light source that illuminates samples from the side. Side illumination membrane imaging (SIMI) can distinguish between particles that are malleable and lay flat on the membrane surface, such as protein aggregates, versus particles that are rigid and have height such as glass, plastic, and metal. The SIMI function can provide added information to indicate whether a particle is intrinsic to the sample (protein) or an extrinsic contaminating particle.

Use of Side Illumination Mode (SIMI) to differentiate protein aggregates from other non-malleable particles 

IgG aggregates mixed with ETFE particles, viewed in brightfield and SIMI. Only ETFE particles light up in SIMI mode.

Crushed, powered glass spiked into a mAb sample, viewed in brightfield and SIMI modes.

Side illumination intensity of protein particles compared to metal, glass, and ETFE.

Why use the HORIZON system for downstream formulations:

  • Low sample consumption (as little as 25 μL per test)
  • Higher throughput for testing lots of formulations and conditions
  • Provides detailed information on particles – size, morphology, counts, distribution
  • Rapid analysis time
  • Wide working range: Measures particles from 2 μm to 4 mm with high reproducibility
  • 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
  • Ability to differentiate different types of particles
  • CFR Part 11 software available