Abstract
Analysis of therapeutic IgG aggregates in serum is a potential area of investigation as it can give deeper insights about the function, immunogenic issues and protein interaction associated with the aggregates. To overcome various complexities associated with the existing analytical techniques for analyzing aggregates in serum, a novel florescence microscopy-based image processing approach was developed. The monoclonal antibody (mAb) was tagged with a fluorescent dye, fluorescein isothiocyanate (FITC). Aggregates, generated by stirring, were spiked into serum and images were captured at various time points. After denoising, thresholding by weighted median, 1D Otsu, and 2D Otsu was attempted and a modified 2D Otsu, a new mode of thresholding, was developed. This thresholding method was found to be highly effective in removing noises and retaining analyte sizes. Out of 0–255, the optimized threshold value obtained for the images discussed in modified 2D Otsu was 9 while 2D Otsu’s overestimated values were 38 and 48. Other morphological operations were applied after thresholding and the area, perimeter, circularity, and radii of the aggregates in these images were calculated. The proposed algorithm offers an approach for analysis of aggregates in serum that is simpler to implement and is complementary to existing approaches.
Highlights
Abbreviations monoclonal antibody (mAb) Monoclonal antibodies kDa Kilodalton size exclusion chromatography (SEC) Size exclusion chromatography nanoparticle tracking analysis (NTA) Nanoparticle tracking analysis dynamic light scattering (DLS) Dynamic light scattering FFF Asymmetrical field-flow fractionation light obscuration (LO) Light obscuration analytical ultracentrifugation (AUC) Analytical ultracentrifugation MFI Micro flow imaging PBS Phosphate buffered saline high performance liquid chromatography (HPLC) High performance liquid chromatography RGB Red green blue confocal laser scanning microscopy (CLSM) Confocal laser scanning microscopy fluorescein isothiocyanate (FITC) Fluorescein isothiocyanate 3D 3-Dimensional 1D 1-Dimensional 2D 2-Dimensional dimethyl sulfoxide (DMSO) Dimethyl sulfoxide NaCl Sodium chloride pI Isoelectric point SEM Scanning electron microscopy
The knowledge of how aggregates evolve once in blood or serum is quite limited[3,6,10,11]. Specialized techniques such as fluorescence single particle tracking, confocal laser scanning microscopy (CLSM), AUC with fluorescence detection system (FDS), optical microscopy, and flow cytometry (FCM) have been reported[3,6,9,11], they suffer from drawbacks such as limited size range, complex optimization of settings, sample dilution in instrument fluid and application of forces such as centrifugation[3,4,6,9,11,12]
The SEC chromatogram of the supernatant did not show the presence of any protein indicating that majority of the tagged mAb was degraded (Supplementary Fig. 2b)
Summary
Abbreviations mAb Monoclonal antibodies kDa Kilodalton SEC Size exclusion chromatography NTA Nanoparticle tracking analysis DLS Dynamic light scattering FFF Asymmetrical field-flow fractionation LO Light obscuration AUC Analytical ultracentrifugation MFI Micro flow imaging PBS Phosphate buffered saline HPLC High performance liquid chromatography RGB Red green blue CLSM Confocal laser scanning microscopy FITC Fluorescein isothiocyanate 3D 3-Dimensional 1D 1-Dimensional 2D 2-Dimensional DMSO Dimethyl sulfoxide NaCl Sodium chloride pI Isoelectric point SEM Scanning electron microscopy. Therapeutic monoclonal antibodies (mAbs) form aggregates during various stages of production, transportation, and storage These aggregates can result in altered biological activity and adverse immune r esponses[1,2]. The knowledge of how aggregates evolve once in blood or serum is quite limited[3,6,10,11] Specialized techniques such as fluorescence single particle tracking (fSPT), confocal laser scanning microscopy (CLSM), AUC with fluorescence detection system (FDS), optical microscopy, and flow cytometry (FCM) have been reported[3,6,9,11], they suffer from drawbacks such as limited size range, complex optimization of settings, sample dilution in instrument fluid and application of forces such as centrifugation[3,4,6,9,11,12]. Images in a fluorescence microscope can be acquired with reduced exposure times, which can further enable recording of fast-moving particles[4,11,12]
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