Finding the Needle in the Haystack: High-Resolution Techniques for Characterization of Mixed Protein Particles Containing Shed Silicone Rubber Particles Generated During Pumping

Abstract

During the manufacturing process of biopharmaceuticals, peristaltic pumps are employed at different stages for transferring and dosing of the final product. Commonly used silicone tubings are known for particle shedding from the inner tubing surface due to friction in the pump head. These nanometer sized silicone rubber particles could interfere with proteins. Until now, only mixed protein particles containing micrometer-sized contaminations such as silicone oil have been characterized, detected, and quantified. To overcome the detection limits in particle sizes of contaminants, this study aimed for the definite identification of protein particles containing nanometer sized silicone particles in qualitative and quantitative manner. The mixed particles consisted of silicone rubber particles either coated with a protein monolayer or embedded into protein aggregates. Confocal Raman microscopy allows label free chemical identification of components and 3D particle imaging. Labeling the tubing enables highresolution imaging via confocal laser scanning microscopy and counting of mixed particles via Imaging Flow Cytometry. Overall, these methods allow the detection and identification of particles of unknown origin and composition and could be a forensic tool for solving problems with contaminations during processing of biopharmaceuticals.

Proteins on the Rack: Mechanistic Studies on Protein Particle Formation During Peristaltic Pumping

“…22 We could find mixed particles of protein containing nanometer sized silicone contaminants, but no evidence for the induction of protein aggregation by shed silicone particles. 23 Additionally, spiking silicone particles did not induce aggregation in two different mAb formulations up to 6 months storage at 2−8, 25, and 40°C. 24 In contrast, silicone oil droplets have been shown to trigger protein aggregation.…”

Machine learning approaches to root cause analysis, characterization, and monitoring of subvisible particles in monoclonal antibody formulations

“…Subvisible particles generated by the peristaltic pumping of mAb solutions using silicone tubing can be comprised of both protein and silicone in addition to homogenous, proteinaceous particles (Deiringer et al, 2021). Subvisible, heterogenous particles may form due to shedding of larger micron-sized silicone particles from the tubing's wetted surface and subsequent coating with protein, or due to the agglomeration of nanometer-sized, shed silicone tubing particles with protein aggregate films adsorbed (Deiringer et al, 2021). Saller et al did not observe increased levels of subvisible particles after spiking a mAb solution with tubing-derived silicone particles and incubating for 6 months at 40°C (Saller et al, 2018).…”

Catching Speedy Gonzales: Driving forces for Protein Film Formation on Silicone Rubber Tubing During Pumping

“…37 AFM revealed a rough surface of the tubing with height differences of approximately 1.2 µm, which is in good agreement with 3D laser scanning microscopy results from a previous study. 38 After incubation with the mAb, a rather flat film with superficial protein clusters of around 40 nm in height was obtained. The dimensions of the clusters, around 100 nm, indicate that these clusters are formed by multiple antibody entities.…”