Biologics – Formulation Development

Subvisible particles are a key quality metric for biologic drugs

Monitoring protein stability in biologic drug development is crucial because aggregates can limit a product’s shelf life and is a leading indicator of a therapy’s potential immunogenic threat. As a result, the FDA recommends that “strategies to minimize aggregate formation should be developed as early as feasible in product development”1 and “an assessment should be made of the range and levels of subvisible particles (2–10 μm) present in therapeutic protein products initially and over the course of the shelf life.”1 USP<787> and USP<788> provide specific regulatory guidance for subvisible particle counts for parenteral administration.

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.

Methods such as light obscuration (LO) and flow imaging (FI) can measure subvisible aggregates but require hundreds of microliters to milliliters of sample for analysis. In biologics development where formulations are being developed at increasingly high concentrations, this amount of material can be prohibitive and only available at late stages.

Low Volume, High Throughput Subvisible Aggregate Analysis

Today, formulations development is increasingly important as many drugs are administered subcutaneously at high concentrations (>100 g L-1) and low volumes. At this stage a candidate will be assessed for solubility in various pH- and salt-conditions, excipients, stability in platform formulations under representative stress conditions, such as increased temperature, shaking, and freeze/thaw, as well as compatibility with primary container. Ideally as much information as possible can be obtained with maximum throughput and minimal material consumption.

The HORIZON system’s 96-well automated format is highly suited to DoE approach to formulations development. Analysis takes about 1 minute per sample, so that several parameters can be tested in a short amount of time with no dilution or sample preparation required. Perform an excipient screen, pH screen, or test buffer conditions in a matter of hours. Since only 25 μL volume is needed per test, analysis does not consume large amounts of material even with replicates.

Test many conditions using 96-well HORIZON membrane plates. Each well requires only 25 μL sample and analysis is automated, taking about 1 minute per well.

Screen several conditions using only a small amount of sample. Polysorbate versus mechanical stress of an IgG molecule shows stabilizing effect. This experiment was run in about 30 minutes.

Measurements on the HORIZON system are more sensitive than flow-based methods

Despite its predominance, Light Obscuration routinely undercounts and under sizes particle measurements in bioformulations due to the low refractive index contrast of protein aggregates in aqueous solution. Flow imaging methods must contend with imaging microscopic translucent particles in liquid, which provides minimal refractive index contrast and can also be susceptible to undercounting and under sizing protein aggregates2.

The refractive index difference between protein aggregates in aqueous solution (flow imaging) and air (HORIZON).

Impact of Solution Refractive Index on particle counts: A comparison of Flow Imaging with the HORIZON system. While trends are similar, HORIZON is more sensitive due to its higher refractive index contrast and therefore counts and measures particles with higher accuracy.

The HORIZON system by contrast 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. Translucent particles can be clearly imaged, leading to more accurate particle counts.

Higher refractive index contrast also provides better quality imaging, for better accuracy and characterization of size and morphology of particles.

Comparison of images from HORIZON versus flow imaging. Polysorbate particles imaged on Horizon membrane are better resolved due to high refractive index contrast compared to solution measurements. From Helbig, C. et al. Backgrounded Membrane Imaging (BMI) for High-Throughput Characterization of Subvisible Particles During Biopharmaceutical Drug Product Development. J Pharm Sci. 2019 Mar 23. pii: S0022-3549(19)30197-2

Why use the HORIZON system for biologics formulations development:

  • 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 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
  • 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
  • Ability to differentiate different types of particles

References

  1. Bee JS, et al. Production of particles of therapeutic proteins at the air– water interface during compression/dilation cycles. Soft Matter. 2012 Sep 26;8(40):10329–35.
  2. Zölls et .al Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments. AAPS J. 2013;15:1200–11.