Particle Analysis of Biotherapeutics in Human Serum Using Machine Learning

Schuster, Joachim; Koulov, Atanas V.; Mahler, Hanns‐Christian; Joerg, Susanne; Huwyler, Jörg; Schleicher, Kai D.; Detampel, Pascal; Mathaes, Roman

doi:10.1016/j.xphs.2020.02.015

Cited by 10 publications

(10 citation statements)

References 19 publications

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“…8,9 Other methods previously employed to track the physical stability of biotherapeutics in serum included asymmetrical flow field-flow fractionation 7 and widefield fluorescence microscopy. 3 Limitations of such methods may include small analysis volumes (e.g., fluorescence microscopy) or lack of particle images (e.g., flow cytometry).…”

Section: Discussionmentioning

confidence: 99%

“…Labeling of mAbs with AF was done as previously described by Schuster et al 3 Briefly, all mAbs were covalently labeled at a fourfold molar excess of AF dye at 25 C for 2 h. Excessive (unbound) dye was removed with Princeton pro spin columns hydrated with a 20 mM histidine-HCl buffer (pH 6.0).…”

Section: Labeling Of Mabsmentioning

confidence: 99%

“…Conventional pharmaceutical methods for SbVP quantitation are unable to discriminate particles derived from therapeutic protein from other particles that may form in the biological fluid over incubation time. 2,3 Previous reports have indicated red blood cells undergo lysis and endogenous proteins can undergo aggregation and precipitation, i.e., forming proteinaceous particles, once isolated from animals/humans. 2e6 Fluorescence methods have been applied to enable the detection of therapeutic proteins in biological fluids 3,7e10 ; however, analytical challenges remain due to the autofluorescence of biological fluid components.…”

Section: Introductionmentioning

confidence: 99%

“…2e6 Fluorescence methods have been applied to enable the detection of therapeutic proteins in biological fluids 3,7e10 ; however, analytical challenges remain due to the autofluorescence of biological fluid components. 3,11 In this study, we investigated the stability of seven different monoclonal antibodies (mAbs) under simulated physiologic conditions (after potential intravenous administration) by mixing mAbs with human serum or phosphate-buffered saline (PBS) and tracking particle formation over a period of five days. To enable detection of the therapeutic protein within the serum matrix, mAbs were fluorescent-labeled by conjugation of Alexa Fluor 488 (AF) as described previously.…”

Section: Introductionmentioning

confidence: 99%

“…To enable detection of the therapeutic protein within the serum matrix, mAbs were fluorescent-labeled by conjugation of Alexa Fluor 488 (AF) as described previously. 3 Particle quantitation was performed using LO and FI, analytical methods commonly applied for assessment of SbVPs. Because LO and FI lack fluorescence measurements, particles were additionally measured using imaging flow cytometry (IFC), which simultaneously collects brightfield (BF), side-scatter, and fluorescence images of particles in flow, thereby enabling differentiation of fluorescent and non-fluorescent particles.…”

Section: Introductionmentioning

confidence: 99%

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Assessing Particle Formation of Biotherapeutics in Biological Fluids

Schuster

Probst

Mahler

et al. 2021

Journal of Pharmaceutical Sciences

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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.

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