Detection of IgG Aggregation by a High Throughput Method Based on Extrinsic Fluorescence

He, Feng; Phan, Duke H.; Hogan, Sabine; Bailey, Robert W.; Becker, Gerald W.; Narhi, Linda O.; Razinkov, Vladimir I.

doi:10.1002/jps.22036

Cited by 58 publications

(52 citation statements)

References 36 publications

(59 reference statements)

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“…SYPRO Orange dye, which features high selectivity and sensitivity, can interact with hydrophobic regions of proteins and yields a strong fluorescence signal that is readily measured. 31,32 Figure 8A-C showed that the extrinsic fluorescence intensity was increased after deglycosylation for all three antibodies, which indicated an increase in hydrophobic regions after removal of oligosaccharides. Above all, both CDRs in the Fab region and the CH 2 domain in the Fc region are involved in the aggregate formation, and the less stable CH 2 domain after deglycosylation contributes substantially to the aggregation rate increase at elevated temperatures.…”

Section: Discussionmentioning

confidence: 97%

The impact of glycosylation on monoclonal antibody conformation and stability

Zheng

Bantog

Bayer

2011

mAbs

280

210

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Section: Discussionmentioning

confidence: 97%

The impact of glycosylation on monoclonal antibody conformation and stability

Zheng

Bantog

Bayer

2011

mAbs

280

210

View full text Add to dashboard Cite

“…An increase in surface hydrophobicity of the stressed samples was demonstrated by ANS and SYPRO Orange binding. ANS and SYPRO Orange act as general probes of the hydrophobic surface of the protein that might be exposed to changes in conformation and unfolding (37,57), by fluorescing when bound to hydrophobic surfaces. An increase in surface hydrophobicity was found in all of the mAb1 particle pellets; however, the protein that remained in solution also displayed hydrophobic exposure in extreme cases, such as heat treatment.…”

Section: Discussionmentioning

confidence: 99%

“…The pellet contains very large sized aggregates, which may result from association of the hydrophobic patches of individual molecules or smaller aggregates, resulting in protection of these regions within the pellet and thus blocking dye binding, a process that may become accelerated upon centrifugation. All samples were also tested for their ability to bind a second hydrophobic dye, SYPRO Orange (37). Trends in SYPRO Orange binding were similar to ANS binding with the highest binding seen for the total metal, stir-20h, stir-3d, agitate-so ϩ , 65C/pH 8.5, and 90C samples, in increasing order (data not shown).…”

mentioning

confidence: 91%

Classification and Characterization of Therapeutic Antibody Aggregates

Joubert

Luo

Nashed-Samuel

et al. 2011

Journal of Biological Chemistry

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283

269

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A host of diverse stress techniques was applied to a monoclonal antibody (IgG 2 ) to yield protein particles with varying attributes and morphologies. Aggregated solutions were evaluated for percent aggregation, particle counts, size distribution, morphology, changes in secondary and tertiary structure, surface hydrophobicity, metal content, and reversibility. Chemical modifications were also identified in a separate report (Luo, Q., Joubert, M. K., Stevenson, R., Narhi, L. O., and Wypych, J. (2011) J. Biol. Chem. 286, 25134 -25144). Aggregates were categorized into seven discrete classes, based on the traits described. Several additional molecules (from the IgG 1 and IgG 2 subtypes as well as intravenous IgG) were stressed and found to be defined with the same classification system. The mechanism of protein aggregation and the type of aggregate formed depends on the nature of the stress applied. Different IgG molecules appear to aggregate by a similar mechanism under the same applied stress. Aggregates created by harsh mechanical stress showed the largest number of subvisible particles, and the class generated by thermal stress displayed the largest number of visible particles. Most classes showed a disruption of the higher order structure, with the degree of disorder depending on the stress process. Particles in all classes (except thermal stress) were at least partially reversible upon dilution in pH 5 buffer. High copper content was detected in isolated metal-catalyzed aggregates, a stress previously shown to produce immunogenic aggregates. In conclusion, protein aggregates can be a very heterogeneous population, whose qualities are the result of the type of stress that was experienced.

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“…Highly hydrophobic regions can be exposed in the constructs, which are different from the typical IgG-like molecules. The increase of fluorescence in the presence of unfolded protein has been used for the direct measurement of mAb aggregation in the study by He et al 17 In this study, the aggregation caused by high temperature has been detected at a level as low as 3% and was confirmed by size exclusion chromatography. Turbidity measurement is an older, established method for the measurement of protein aggregation.…”

Section: Screening For Conformational Stabilitymentioning

confidence: 71%

Accelerated Formulation Development of Monoclonal Antibodies (mAbs) and mAb-Based Modalities: Review of Methods and Tools

2015

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More therapeutic monoclonal antibodies and antibody-based modalities are in development today than ever before, and a faster and more accurate drug discovery process will ensure that the number of candidates coming to the biopharmaceutical pipeline will increase in the future. The process of drug product development and, specifically, formulation development is a critical bottleneck on the way from candidate selection to fully commercialized medicines. This article reviews the latest advances in methods of formulation screening, which allow not only the high-throughput selection of the most suitable formulation but also the prediction of stability properties under manufacturing and long-term storage conditions. We describe how the combination of automation technologies and high-throughput assays creates the opportunity to streamline the formulation development process starting from early preformulation screening through to commercial formulation development. The application of quality by design (QbD) concepts and modern statistical tools are also shown here to be very effective in accelerated formulation development of both typical antibodies and complex modalities derived from them.

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Detection of IgG Aggregation by a High Throughput Method Based on Extrinsic Fluorescence

Cited by 58 publications

References 36 publications

The impact of glycosylation on monoclonal antibody conformation and stability

The impact of glycosylation on monoclonal antibody conformation and stability

Classification and Characterization of Therapeutic Antibody Aggregates

Accelerated Formulation Development of Monoclonal Antibodies (mAbs) and mAb-Based Modalities: Review of Methods and Tools

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