Assessing Particle Formation of Biotherapeutics in Biological Fluids

Abstract

The stability of therapeutic proteins can be impacted in vivo after administration, which may affect patient safety or treatment efficacy, or both. Stability testing of therapeutic proteins using models representing physiologic conditions may guide preclinical development strategy; however, to date only a few studies assessing the physical stability are available in the public domain. In this manuscript, the stability of seven fluorescently labeled monoclonal antibodies (mAbs) was evaluated in human serum and phosphate-buffered saline, two models often discussed to be representative of the situation in humans after intravenous administration. Subvisible particles were analyzed using light obscuration, flow imaging, and imaging flow cytometry. All methods showed that serum itself formed particles under in vitro conditions. Imaging flow cytometry demonstrated that mean particle size and counts of mAbs increased substantially in serum over five days; however, particle formation in phosphate-buffered saline was comparably low. Stability differences were observed across the mAbs evaluated, and imaging flow cytometry data indicated that fluorescently labeled mAbs primarily interacted with serum components. The results indicate that serum may be more suitable as in vitro model to simulate physiologic intravenous conditions in patients closely and evaluate the in vivo stability of therapeutic proteins. Fluorescence labeling and detection methods may be applied to differentiate particles containing therapeutic protein from high amounts of serum particles that form over time.

Imaging Flow Cytometry for Sizing and Counting of Subvisible Particles in Biotherapeutics

“…16,17 Another application of IFC FL imaging described in the literature is the evaluation of particles formed by fluorescently labelled antibodies within complex biological fluids like serum. 18 Associated with the additional imaging options, analysis of IFC data is rather complex, with the definition of masks representing a crucial step. 19 Depending on the underlying algorithm masks determine the area occupied by an object in the image and from this masked area, output parameters (so-called features) such as particle diameter are calculated.…”

Assessment of Antibody Stability in a Novel Protein-Free Serum Model

“…Different in vitro models and analytical approaches have been developed to evaluate the in vivo stability of therapeutic proteins [ 4 , 5 , 6 , 7 ]. However, simulating physiological conditions and detecting the protein of interest in biological fluids is accompanied by challenges [ 8 , 9 ]. Purification techniques and/or fluorescence labeling have been successfully used to detect the target protein [ 10 , 11 ].…”