Characterization of Particles in Protein Solutions: Reaching the Limits of Current Technologies

Abstract

Recent publications have emphasized the lack of characterization methods available for protein particles in a size range comprised between 0.1 and 10 μm and the potential risk of immunogenicity associated with such particles. In the present paper, we have investigated the performance of light obscuration, flow microscopy, and Coulter counter instruments for particle counting and sizing in protein formulations. We focused on particles 2–10 μm in diameter and studied the effect of silicon oil droplets originating from the barrel of pre-filled syringes, as well as the effect of high protein concentrations (up to 150 mg/ml) on the accuracy of particle characterization. Silicon oil was demonstrated to contribute significantly to the particle counts observed in pre-filled syringes. Inconsistent results were observed between different protein concentrations in the range 7.5–150 mg/ml for particles <10 μm studied by optical techniques (light obscuration and flow microscopy). However, the Coulter counter measurements were consistent across the same studied concentration range but required sufficient solution conductivity from the formulation buffer or excipients. Our results show that currently available technologies, while allowing comparisons between samples of a given protein at a fixed concentration, may be unable to measure particle numbers accurately in a variety of protein formulations, e.g., at high concentration in sugar-based formulations.

Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments

“…For this approach, flow imaging microscopy has been successfully applied in several studies (22)(23)(24). In general, flow imaging microscopy tends to be more sensitive than LO for small transparent protein particles and therefore usually detects higher particle numbers (13,15,25). An increased RI of the formulation, leading to a decreased RI difference between particles and formulation, can impede a correct detection of protein particles by light-based techniques.…”

“…An increased RI of the formulation, leading to a decreased RI difference between particles and formulation, can impede a correct detection of protein particles by light-based techniques. Compared to LO, Micro-Flow Imaging (MFI) was shown to be slightly more robust against such a decreased RI difference (13,26).…”

“…It is currently being discussed whether this chapter should include particle analysis starting already from 2 μm as well as the use of additional techniques such as flow imaging microscopy. Flow imaging microscopy has already been used extensively in research and development (13)(14)(15)(16)(17)(18)(19) and more recently also for quality control/ routine testing (own experiences). However, it needs to be considered that the calculation of particle size depends on the underlying measurement principle and may differ between LO and flow imaging microscopy.…”

Factors Governing the Accuracy of Subvisible Particle Counting Methods

“…1,2 This quality attribute has received particular attention and presented specific challenges in the development of biotechnological products. [3][4][5] Specifically, concerns had been raised about the possible relevance of protein particles in the subvisible range. [6][7][8] In this context, it has become necessary to develop and adopt analytical tools capable of the assessing and characterizing particles smaller than the current 10-mm limit established in the United States Pharmacopeia <788> and other harmonized compendial methods.…”

Particulate Matter in Injectable Drugs: Evaluation of Risks to Patients

“…The reconstitution of L-Asparaginase in siliconised syringes induces protein aggregation [39]. Various technologies may be used to measure particle numbers in protein formulation but they are inadequate to characterise particles in a variety of protein solutions [40]. The siliconisation process may have an impact on the generation of particles in protein formulations in pre-filled syringes [41].…”