Analytical Platform for Monitoring Aggregation of Monoclonal Antibody Therapeutics

Bansal, Rohit; Gupta, Sonia; Rathore, Anurag S.

doi:10.1007/s11095-019-2690-8

Cited by 41 publications

(35 citation statements)

References 44 publications

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“…[57][58][59][60] In this context, it is important to combine modeling activities with advances in analytical methods. The characterization of dimers and other aggregate end products involves a variety of orthogonal techniques [61][62][63] and can benefit from emerging approaches based on microfluidic technology. 64,65 We refer to recent reviews and reports for discussion about the strengths and shortcomings of these techniques.…”

Section: Aggregation Under Storage Conditions Correlates Poorly With mentioning

confidence: 99%

Accelerated Aggregation Studies of Monoclonal Antibodies: Considerations for Storage Stability

Wälchli

Vermeire

Massant

et al. 2020

Journal of Pharmaceutical Sciences

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Aggregation of mAbs is a crucial concern with respect to their safety and efficacy. Among the various properties of protein aggregates, it is emerging that their size can potentially impact their immunogenicity. Therefore, stability studies of antibody formulations should not only evaluate the rate of monomer loss but also determine the size distribution of the protein aggregates, which in turn depends on the aggregation mechanism. Here, we study the aggregation behavior of different formulations of 2 monoclonal immunoglobulins (IgGs) in the temperature range from 5 C to 50 C over 52 weeks of storage. We show that the aggregation kinetics of both antibodies follow non-Arrhenius behavior and that the aggregation mechanisms change between 40 C and 5 C, leading to different types of aggregates. Specifically, for a given monomer conversion, dimer formation dominates at low temperatures, while larger aggregates are formed at higher temperatures. We further show that the stability ranking of different molecules as well as of different formulations is drastically different at 40 C and 5 C while it correlates better between 30 C and 5 C. Our findings have implications for the level of information provided by accelerated aggregation studies with respect to protein stability under storage conditions.

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Section: Aggregation Under Storage Conditions Correlates Poorly With mentioning

confidence: 99%

Accelerated Aggregation Studies of Monoclonal Antibodies: Considerations for Storage Stability

Wälchli

Vermeire

Massant

et al. 2020

Journal of Pharmaceutical Sciences

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“…In case of flow imaging systems such as FlowCAM (Fluid Imaging Technologies, Maine), Sysmex Flow Particle Image Analyzer (FPIA) (Malvern Instruments, Germany), and Micro-Flow Imaging (MFI, Protein simple, Santa Clara, California), the dilution of sample can change the nature of aggregate. The requirement of a certain difference in refractive index between the solvent and the protein could result in inaccurate quantification for certain samples 4 , 5 , 12 . Analysis using fluorescence microscopy enables prevention of false detection of dust, air bubbles and non-proteinaceous particles that could plague the analysis 11 .…”

Section: Resultsmentioning

confidence: 99%

“…The aggregates of therapeutic IgG have altered properties as compared to the monomer 1 , 5 – 8 . When aggregates are introduced into the blood, various undesired outcomes such as blockage of blood capillaries, change in efficacy, clearance, and adverse immune reactions can occur 3 , 6 , 9 .…”

Section: Introductionmentioning

confidence: 99%

“…Serum has various proteins already present in it 3 , 8 . As a result, characterization of aggregates in serum is difficult, if not impossible, via the existing tools such as size exclusion chromatography (SEC), nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), asymmetrical field-flow fractionation (FFF), light obscuration (LO), particle counting, analytical ultracentrifugation (AUC) and flow-based imaging techniques 5 – 7 . As a result, the knowledge of how aggregates evolve once in blood or serum is quite limited 3 , 6 , 10 , 11 .…”

Section: Introductionmentioning

confidence: 99%

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Novel semi-automated fluorescence microscope imaging algorithm for monitoring IgG aggregates in serum

Sreenivasan

Sonawat

Mandal

et al. 2021

Sci Rep

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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.

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“…The cells were then subjected to H/R injury with or without melatonin. Lastly, the cell viability was measured at 450 nm using CCK-8 [65].…”

Section: Cell Counting Kit-8 (Cck-8) Assaymentioning

confidence: 99%

Melatonin improves mitochondrial biogenesis through the AMPK/PGC1α pathway to attenuate ischemia/reperfusion-induced myocardial damage

Wang

2020

Aging

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Cardiac ischemia/reperfusion injury is associated with reduced mitochondrial turnover and regeneration. There is currently no effective approach to stimulate mitochondrial biogenesis in the reperfused myocardium. In this study, we investigated whether melatonin could increase mitochondrial biogenesis and thus promote mitochondrial homeostasis in cardiomyocytes. Cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) injury with or without melatonin treatment, and various mitochondrial functions were measured. H/R injury repressed mitochondrial biogenesis in cardiomyocytes, whereas melatonin treatment restored mitochondrial biogenesis through the 5' adenosine monophosphate-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) pathway. Melatonin enhanced mitochondrial metabolism, inhibited mitochondrial oxidative stress, induced mitochondrial fusion and prevented mitochondrial apoptosis in cardiomyocytes subjected to H/R injury. The melatonin-induced improvement in mitochondrial biogenesis was associated with increased cardiomyocyte survival during H/R injury. On the other hand, silencing of PGC1α attenuated the protective effects of melatonin on cardiomyocyte viability, thereby impairing mitochondrial bioenergetics, disrupting the mitochondrial morphology, and activating mitochondrial apoptosis. Thus, H/R injury suppressed mitochondrial biogenesis, while melatonin activated the AMPK/PGC1α pathway and restored mitochondrial biogenesis, ultimately protecting the reperfused heart.

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Analytical Platform for Monitoring Aggregation of Monoclonal Antibody Therapeutics

Cited by 41 publications

References 44 publications

Accelerated Aggregation Studies of Monoclonal Antibodies: Considerations for Storage Stability

Accelerated Aggregation Studies of Monoclonal Antibodies: Considerations for Storage Stability

Novel semi-automated fluorescence microscope imaging algorithm for monitoring IgG aggregates in serum

Melatonin improves mitochondrial biogenesis through the AMPK/PGC1α pathway to attenuate ischemia/reperfusion-induced myocardial damage

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