Defining a Scientifically Justified Agitation Stress Model for Biologics Formulation Stability

The development and manufacturing of high-concentration biologics necessitate rigorous testing to ensure product stability throughout the drug product lifecycle (1,2), according to Siddhant Sojitra, associate scientist II, Injectable Drug Product Development at Alexion Pharmaceuticals, Inc, who presented the poster “Defining a Scale-Down Agitation Model for Early-Stage High Concentration Biologics Formulation Development” at AAPS PharmSci 360, held Nov 9-12, 2025 in San Antonio, Texas (3).
Mechanical stresses that occur during manufacturing, transportation, storage, and administration can potentially lead to physical degradations, such as foaming, protein aggregation, or the formation of particles, Sojitra states. These outcomes can critically affect product quality, "thereby impacting product quality." To accurately simulate these stresses and identify stable formulations early in development, Sojitra and colleagues in the Injectable Drug Product Development department at Alexion—Huan Kang, PhD, scientist II; Jay Yang, PhD, director; and Vinay Radhakrishnan, PhD, executive director—systematically evaluated methods for defining appropriate agitation stress testing conditions and endpoints.

How do different agitation models influence mechanical stress outcomes?

To define a suitable standard test, three distinct physical agitation models were put under systematic evaluation: an orbital shaker, a multichannel vortexer, and a bench-top shipping simulator (VR5500). The study utilized high-concentration biologics formulations that are representative of clinical and commercial products. Initial screenings were conducted using an agitation-sensitive molecule within Type I 2R and 6R vials, assessing varying fill volumes and representative container closure systems (CCS).

Early findings from the CCS screen revealed that the orientation of the container closure system significantly altered the stress experienced by the sample. For instance, using the orbital shaker, horizontal placement subjected the samples to greater stress compared with vertical placement. Conversely, the vortexer generated a higher degree of stress when samples were placed vertically. Based on these initial findings, 4 mL of fill volume in 6R vials was chosen for the detailed comparison of the three agitation models.

When comparing the overall severity of the agitation models, both the orbital shaker (operating at 200 RPM) and the vortexer (operating at 1200 RPM) created mechanical stress conditions that were more severe than those produced by the VR5500 shipping simulator. This elevated stress level makes the shaker and vortexer suitable for developing robust stress conditions necessary for formulation studies. A crucial finding in the research was the qualitative difference in the degradation products generated by the various models. The orbital shaker was primarily found to promote the formation of high molecular weight species (HMWS), which were measured using size-exclusion ultra-performance liquid chromatography. In contrast, the vortexer was more likely to generate subvisible particles (SVP), which were measured using micro flow imaging. This distinction highlights the specific mechanical interactions occurring within the different testing apparatuses, "demonstrating different agitation models impact distinct QA," according to the study team. Samples were also analyzed for quality attributes, including turbidity, measured by optical density.

What are the optimal parameters for early-stage formulation assessment

Based on the overall findings, the orbital shaker was identified as the appropriate stress condition for detailed formulation development studies, specifically agitation for 24 hours at ambient temperature. Due to potential material constraints often encountered during early research phases, 2R vials were selected as the standard configuration for subsequent agitation testing.

The researchers then focused on determining the optimal fill volume within the 2R vials by systematically assessing volumes ranging from 0.5 mL to 2 mL. The extent of change in quality attributes, particularly the formation of HMWS or SVP, was directly dependent on the volume used. Ultimately, a minimum fill volume of 1 mL was determined to be optimal for use in early-stage agitation stress testing.

This comprehensive approach allowed the researchers to define a scientifically justified agitation model for the early-stage development of high-concentration biologics. The data support a defined testing approach that utilizes 2R vials with a minimum fill volume of 1 mL, placed horizontally, and subjected to orbital shaker agitation at ambient temperatures for up to 24 hours. This prescribed setup offers a reliable and consistent method to assess how mechanical stress affects product quality, which is vital for identifying stable formulations during the initial development phases.

References

1. Zarzar, J; Khan, T; Bhagawati, M; et al. High Concentration Formulation developability approaches and Considerations. MAbs. 2023;15(1):2211185.

2. European Medicines Agency. Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products. Accessed Nov 10, 2025.
3. Sojitra, S; Kang, H; Yang, J; Radhakrishnan, V. Defining a Scale-Down Agitation Model for Early-Stage High Concentration Biologics Formulation Development. AAPS PharmSci 360 Poster Presentation. Nov 10, 2025.