Image Analysis Algorithm-Based Platform for Determining Micron and Higher Aggregate Size Distribution of Therapeutic IgG Using Brightfield and Fluorescence Microscope Images

Sreenivasan, Shravan; Sonawat, Deepak; Rathore, Anurag S.

doi:10.1007/s11095-021-03108-7

Cited by 3 publications

(6 citation statements)

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“…DLS can estimate the size of aggregates accurately up to 5 μm. , The DLS size distribution plot of number (%) against size (Figure e,f) and Z -average (d. nm) (Table ) showed that sample (C) had the maximum average aggregate size. The aggregate size of sample (A) was 13.78 nm.…”

Section: Resultsmentioning

confidence: 98%

“…Fluorescein isothiocyanate (FITC) is one of the widely used dyes for fluorescence labeling of proteins. The dye reacts with the primary amine of protein. , To label mAb with FITC, the sample containing mAb was transferred to a carbonate buffer of pH 9. FITC was dissolved in dimethyl sulfoxide at a concentration of 1 mg/mL.…”

Section: Methodsmentioning

confidence: 99%

“…The images obtained by brightfield and fluorescence microscope were analyzed for the extraction of size distribution in diameter (μm). A detailed description of every step used in the processing of brightfield and fluorescence microscope images is mentioned in our previous publications. , The aggregate sizes obtained from multiple images of a sample were compiled together and arranged in the ascending order, and the aggregates having more than 5 μm size were plotted into a histogram with the y -axis showing the percentage number of aggregate particles and the x -axis showing aggregate size ranges with a bin center of 5 and a bin width of 10.…”

Section: Methodsmentioning

confidence: 99%

“…The final part of the second case study showed the aggregation of mAb in serum having the combined presence of Fe 2+ and H 2 O 2. The aggregation of mAb in extracellular saline and macromolecule free fraction of serum was analyzed by size exclusion chromatography (SEC), and aggregates of mAb were monitored in serum by using the previously published fluorescence microscope image processing algorithm. , …”

Section: Introductionmentioning

confidence: 99%

“…The aggregation of mAb in extracellular saline and macromolecule free fraction of serum was analyzed by size exclusion chromatography (SEC), and aggregates of mAb were monitored in serum by using the previously published fluorescence microscope image processing algorithm. 26,27…”

Section: Introductionmentioning

confidence: 99%

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Combined Presence of Ferrous Ions and Hydrogen Peroxide in Normal Saline and In Vitro Models Induces Enhanced Aggregation of Therapeutic IgG due to Hydroxyl Radicals

Sreenivasan

Rathore

2023

Mol. Pharmaceutics

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Therapeutic monoclonal antibodies (mAb) are known to form aggregates and fragments upon exposure to hydrogen peroxide (H 2 O 2 ) and ferrous ions (Fe 2+ ). H 2 O 2 and Fe 2+ react to form hydroxyl radicals that are detrimental to protein structures. In this study, aggregation of mAb in the combined presence of Fe 2+ and H 2 O 2 was investigated in saline and physiologically relevant in vitro models. In the first case study, forced degradation of mAb in saline (a fluid used for administration of mAb) was carried out at 55 °C in the combined presence of 0.2 mM Fe 2+ and 0.1% H 2 O 2 . The control and stressed samples were analyzed using an array of techniques including visual observation, size-exclusion chromatography (SEC), dynamic light scattering (DLS), microscopy, UV−vis, fluorescence, Fourier transform infrared spectroscopy, and cell-based toxicity assays. At the end of 1 h, samples having the combined presence of both Fe 2+ and H 2 O 2 exhibited more than 20% HMW (high molecular weight species), whereas samples having only Fe 2+ , H 2 O 2 , or neither resulted in less than 3% HMW. Aggregate-rich samples also exhibited altered protein structures and hydrophobicity. Aggregation increased upon increasing the time, temperature, and concentration of Fe 2+ and H 2 O 2 . Samples having both Fe 2+ and H 2 O 2 also showed higher cytotoxicity in red blood cells. Samples of mAb with chlorides of copper and cobalt with H 2 O 2 also resulted in multifold degradation. The first case study showed enhanced aggregation of mAb in the combined presence of Fe 2+ and H 2 O 2 in saline. In the second case study, aggregation of mAb was investigated in artificially prepared extracellular saline and in vitro models such as macromolecule free fraction of serum and serum. In the presence of both Fe 2+ and H 2 O 2 , %HMW was higher in extracellular saline compared to macromolecule free fraction of serum. Further, in vitro models having the combined presence of Fe 2+ and H 2 O 2 resulted in enhanced aggregation of mAb compared to models that had neither.

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Section: Resultsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%