Light Obscuration Measurements of Highly Viscous Solutions: Sample Pressurization Overcomes Underestimation of Subvisible Particle Counts

Abstract

Light obscuration (LO) is the current standard technique for subvisible particle analysis in the quality control of parenterally administered drugs, including therapeutic proteins. Some of those, however, exhibit high viscosities due to high protein concentrations, which can lead to false results by LO measurements. In this study, we show that elevated sample viscosities, from about 9 cP, lead to an underestimation of subvisible particle concentrations, which is easily overlooked when considering reported data alone. We evaluated a solution to this problem, which is the application of sample pressurization during analysis. The results show that this is an elegant way to restore the reliability of LO analysis of highly viscous products without the necessity of additional sample preparation.

Factors Governing the Accuracy of Subvisible Particle Counting Methods

“…One possible cause of this observation is the high frailty of the particles of this model (a consequence of the relative low stress level applied for its generation) that could not endure the mechanical stress of the pumping through the glass tube orifice. Such mechanical stress due to particle transit inside the instrument has been suggested for other techniques 28 and is probably also present during particle dilution. These observations are of the highest importance given that CC is a technique that often requires particle dilution to increase sample conductivity.…”

“…In an attempt to evaluate the individual methods' performance a number of studies have examined different analytical aspects of the corresponding techniques, 12,13,[17][18][19][22][23][24][25][27][28][29][30][31][32][33] but to date, no systematic analysis which includes a comparison between all the different methods using a number of different standard systems, has been carried out. Recently, we reported on the factors governing the precision of subvisible particle measurement methods 26 in a detailed analysis of such an extensive analytical toolbox for subvisible particle characterization.…”

Effects of Tubing Type, Formulation, and Postpumping Agitation on Nanoparticle and Microparticle Formation in Intravenous Immunoglobulin Solutions Processed With a Peristaltic Filling Pump

“…It is well known in the field that LO is not an appropriately sensitive method for quantifying protein particle levels. [15][16][17][18] Often the levels measured in a given sample by LO are several-fold lower than those quantified by flow imaging. Perhaps the most important problem with LO measurements are that they are unable to detect important increases in protein particles: during processing steps and manufacturing scale-up; because of changes in formulation, container, and closure systems; or due to handling and mishandling by end users.…”

“…Although several published studies have documented that LO is relatively insensitive to detecting and quantifying subvisible protein particles [15][16][17] compared to flow imaging methods, in many instances in industry, LO is the only method used for assessment of particle formation during bioprocessing steps. A comparison of the LO and flow imaging is particularly important for filling pumpinduced particle formation in protein products because it is critical that the best method(s) be selected.…”

“…2) show relatively transparent particles in pumped and agitated IVIG in PBS; a property that often renders LO ineffective at detecting and quantifying protein particles. [15][16][17]…”