(M1030-11-60) Detecting and Identifying Contaminants for Root Cause Analysis: Distinguishing Free Fatty Acid Breakdown Products of Surfactants, Silicone Oil and Protein Aggregates

Purpose: Aging surfactants produce free fatty acids that can induce the formation of dangerous protein aggregates. Protein aggregates, however, can form as a result of other conditions, including handling and thermal or pH changes. These experiments are designed to detect and distinguish the degradation products of PS80 to identify a potential cause of protein aggregate formation.

Methods: Total Holographic Characterization (THC) is used to distinguish individual sub-visible contaminants in model biologic pharmaceuticals [1], ranging in size from 0.5 μm – 10 μm. All measurements are performed on xSight. Samples flow down a channel in a disposable microfluidic sample chip in concentrations and viscosities typical of biologic pharmaceutical formulations [2]. Protein aggregates are distinguished from other contaminants that are found in pharmaceutical manufacturing, including silicon oil emulsion droplets [3], and breakdown products of industry standard surfactants [1]. Total Holographic Characterization requires 25-35 mL of sample per measurement and measurements can be performed in a matter of minutes. In addition, xSight meets all of the requirements for 21 CFR Part 11.

Results: Aging surfactants produce free fatty acids that can be monitored and identified using THC. Control samples of a variety of free fatty acids including oleic acid, and linoleic acid are compared with accelerated aging of polysorbate 80 (PS80) to identify the breakdown products of the surfactant using THC. Experiments in which PS80 is degraded by base hydrolysis and enzymatic hydrolysis demonstrate that the degradation of PS80 produces dominantly oleic acid. THC uses Lorenz-Mie Theory to analyze holograms of individual sub-visible particles. The analysis yields the size distribution of particles present and simultaneously determines the composition from the index of refraction of the particles. Particle symmetry provides additional information about the morphology of the particles. Spherical particles, such as those of emulsion droplets or air bubbles are distinguished from more irregularly shaped particles, by the symmetry of their holograms. We compare the size, refractive index and morphology of the control samples and the PS80 sample to identify the composition of the contaminants in the sample. Spherical particles with the refractive index of oleic acid are easily distinguishable from other contaminants. Additional experiments examine the impact of PS80 degradation on protein aggregation. Samples of IgG and PS80 are subjected to enzymatic degradation and compared to similar samples without IgG. This comparison shows that PS80 degradation in the presence of IgG results in dramatically increased formation of protein aggregates. Additional experiments eject a sample of PS80 that has undergone enzymatic degradation through a syringe. Ejection from the syringe adds trace amounts of silicone oil to the sample. Both silicone oil and oleic acid droplets are identical in size and shape but have different indexes of refraction and are therefore easily distinguished using THC.

Conclusion: These experiments demonstrate that degradation of PS80 can instigate the formation of protein aggregates. The presence of oleic acid can be used to identify PS80 degradation as the cause of protein aggregation. Oleic acid has the same size and shape as other contaminants such as silicone oil. In addition, protein aggregates themselves have a similar size distribution to the oleic acid emulsion droplets formed when PS80 degrades. These experiments demonstrate that THC can detect and distinguish oleic acid droplets from silicone oil droplets and protein aggregates to determine whether surfactant degradation is the cause of the presence of unexpected protein aggregates.

References: 1. Tiffany Markus, Juliana Lumer, Rivka Stasavage, David B. Ruffner, Laura A. Philips, Fook Chiong Cheong, Intern. J. Pharm., 652, 123843 (2024)
2. Winters A, Cheong FC, Odete MA, Lumer J, Ruffner DB, Mishra KI, Grier DG, Philips LA., J Pharm Sci. 109(8):2405-2412 (2020)
3. Priya N.O. Kasimbeg, Fook Chiong Cheong, David B. Ruffner, Jaroslaw M. Blusewicz, Laura A. Philips, J. Pharm. Sci., 108 (1):155-161 (2019)

Acknowledgements: Spheryx, Inc. manufactures and sells the instruments used in the experiments reported here.


Figure 1 Schematic of Total Holographic Characterization system


Figure 2 Scatter plot of refractive index vs diameter for (a) oleic acid, (b) silicone oil, (c) IgG aggregates, and (d) Mixture of emulsion and protein aggregates


Figure 3 Scattered plot refractive index vs diameter for (a) PS80 +IgG, (b) IgG + Lipase, (c) PS80 + Lipase (d) IgG + PS80 + Lipase; (e-h) the same data points as in (a-d) respectively, color-coded by particle morphology.