Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants

Oshinbolu, Sheun; Shah, Rachana; Finka, Gary; Molloy, Mike J.; Uden, Mark; Bracewell, Daniel G.

doi:10.1002/jctb.5519

Cited by 39 publications

(45 citation statements)

References 27 publications

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“…A recent study with an early prototype Thioflavin T revealed that the dye exhibits a 6‐fold higher affinity towards BSA dimers than monomers . Conversely, there is also evidence to suggest that the dye can bind to specific parts of monomers, non‐ β ‐sheet cavities, and therefore fails to distinguish between different protein conformations …”

Section: Resultsmentioning

confidence: 99%

Chromatography process development aided by a dye‐based assay

Jasulaityte

Johansson²,

Bracewell

2019

J of Chemical Tech & Biotech

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BACKGROUND: The lifetime of chromatography resins typically averages between 10 and 300 cycles for the manufacture of a therapeutic protein. Developing and establishing the robustness of the method for each separation process represents a significant challenge and is subject to extensive regulatory oversight. This paper presents a novel fluorescence-based assay for residual aggregated proteins to aid the evaluation of the extent of resin regeneration. RESULTS: The versatility of this method was demonstrated by using strong anion and cation exchange agarose resins PraestoQ and SP in conjunction with bovine serum albumin and monoclonal antibody feed materials. The assay entails applying a molecular rotor dye to a sample of free resin and measuring the fluorescence intensity using a plate reader or visualizing under a confocal laser scanning microscope to gain a more detailed characterization. Following five consecutive chromatography cycles, both methods revealed a 10-fold increase in fluorescence intensity along with a proportional reduction in dynamic binding capacity. Furthermore, the use of the assay suggested that fouling was dependent on spatial bead position in the column, bead channel structure and cleaning conditions. CONCLUSION: This work presents a simple assay suitable for use in resin lifetime studies to enhance process understanding.

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Section: Resultsmentioning

confidence: 99%

Chromatography process development aided by a dye‐based assay

Jasulaityte

Johansson²,

Bracewell

2019

J of Chemical Tech & Biotech

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“…This would make it easier to assess the variations if the sample structure that may be induced during synthesis. Protein aggregation can be measured by evaluating the fluorescence spectra of the NBC [77]. Moreover, the mechanisms involved in photoactivated NBC cell death leading to free radical apoptosis of CSCs remain unknown, as cell death may be caused by distinct, but overlapping, signaling pathways that respond to different stimuli [78].…”

Section: Discussionmentioning

confidence: 99%

Effective Gold Nanoparticle-Antibody-Mediated Drug Delivery for Photodynamic Therapy of Lung Cancer Stem Cells

Crous

Abrahamse

2020

IJMS

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Cancer stem cells (CSCs) are a leading contributor to lung cancer mortality rates. CSCs are responsible for tumor growth and recurrence through inhibition of drug-induced cell death, decreasing the effect of traditional cancer therapy and photodynamic therapy (PDT). PDT can be improved to successfully treat lung cancer by using gold nanoparticles (AuNPs), due to their size and shape, which have been shown to facilitate drug delivery and retention, along with the targeted antibody (Ab) mediated selection of CSCs. In this study, a nanobioconjugate (NBC) was constructed, using a photosensitizer (PS) (AlPcS4Cl), AuNPs and Abs. The NBC was characterized, using spectroscopy techniques. Photodynamic effects of the NBC on lung CSCs was evaluated, using biochemical assays 24 h post-irradiation, in order to establish its anticancer effect. Results showed successful conjugation of the nanocomposite. Localization of the NBC was seen to be in integral organelles involved in cell homeostasis. Biochemical responses of lung CSCs treated using AlPcS4Cl-AuNP and AlPcS4Cl-AuNP-Ab showed significant cell toxicity and cell death, compared to free AlPcS4Cl. The PDT effects were enhanced when using the NBC, showing significant lung CSC destruction to the point of eradication.

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“…Two microliters of ProteoStat® Fluorescent Dye (Enzo Life Sciences, USA) was added to each test sample containing 200 μg/ml of IgG in a 96-well black, clear flat-bottomed plate (Greiner Bio-One, Austria). This dye is widely used to detect protein aggregates (27) and is benchmarked with IgG by the manufacturer (28). The microplate containing test samples was incubated in the dark for 15 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Low pH Exposure During Immunoglobulin G Purification Methods Results in Aggregates That Avidly Bind Fcγ Receptors: Implications for Measuring Fc Dependent Antibody Functions

et al. 2019

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Evaluating the biophysical and functional nature of IgG is key to defining correlates of protection in infectious disease, and autoimmunity research cohorts, as well as vaccine efficacy trials. These studies often require small quantities of IgG to be purified from plasma for downstream analysis with high throughput immunoaffinity formats which elute IgG at low-pH, such as Protein G and Protein A. Herein we sought to compare Protein G purification of IgG with an immunoaffinity method which elutes at physiological pH (Melon Gel). Critical factors impacting Fc functionality with the potential to significantly influence FcγR binding, such as IgG subclass distribution, N-glycosylation, aggregation, and IgG conformational changes were investigated and compared. We observed that transient exposure of IgG to the low-pH elution buffer, used during the Protein G purification process, artificially enhanced recognition of Fcγ Receptors (FcγRs) as demonstrated by Surface Plasmon Resonance (SPR), FcγR dimer ELISA, and a functional cell-based assay. Furthermore, low-pH exposed IgG caused conformational changes resulting in increased aggregation and hydrophobicity; factors likely to contribute to the observed enhanced interaction with FcγRs. These results highlight that methods employed to purify IgG can significantly alter FcγR-binding behavior and biological activity and suggest that the IgG purification approach selected may be a previously overlooked factor contributing to the poor reproducibility across current assays employed to evaluate Fc-mediated antibody effector functions.

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Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants

Cited by 39 publications

References 27 publications

Chromatography process development aided by a dye‐based assay

Chromatography process development aided by a dye‐based assay

Effective Gold Nanoparticle-Antibody-Mediated Drug Delivery for Photodynamic Therapy of Lung Cancer Stem Cells

Low pH Exposure During Immunoglobulin G Purification Methods Results in Aggregates That Avidly Bind Fcγ Receptors: Implications for Measuring Fc Dependent Antibody Functions

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