Characterisation of Stress-Induced Aggregate Size Distributions and Morphological Changes of a Bi-Specific Antibody Using Orthogonal Techniques

Hamrang, Zahra; Hussain, Maryam; Tingey, Katie; Tracka, Malgorzata B.; Casas‐Finet, José R.; Uddin, Shahid; Walle, Christopher F. van der; Pluen, Alain

doi:10.1002/jps.24530

Cited by 14 publications

(15 citation statements)

References 28 publications

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“…7,8,49,51 Like NTA, DLS is sensitive to smaller particles than holographic characterization. As a sample-averaged measurement, however, DLS does not inherently account for heterogeneity in sample composition.…”

Section: Discussionmentioning

confidence: 99%

“…Established particle-characterization technologies such as dynamic light scattering (DLS) work well for in situ characterization of submicrometer-scale aggregates but are less effective for larger subvisible aggregates. 7,8 Other techniques, such as microflow imaging (MFI), 9,10 are better suited for aggregates larger than a micrometer or so 8,10 but do not provide information on composition. Comparatively few established techniques probe the size and composition of protein aggregates in the subvisible range from 100 nm to 10 μm.…”

Section: Introductionmentioning

confidence: 99%

“…Comparatively few established techniques probe the size and composition of protein aggregates in the subvisible range from 100 nm to 10 μm. 8 The need for enhanced characterization techniques is particularly acute in applications that require real-time monitoring of subvisible aggregates in their native environment.…”

Section: Introductionmentioning

confidence: 99%

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Holographic Characterization of Protein Aggregates

Wang

Zhong

Ruffner

et al. 2016

Journal of Pharmaceutical Sciences

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We demonstrate how holographic video microscopy can be used to detect, count, and characterize individual micrometer-scale protein aggregates as they flow down a microfluidic channel in their native buffer. Holographic characterization directly measures the radius and refractive index of subvisible protein aggregates and offers insights into their morphologies. The measurement proceeds fast enough to build up population averages for time-resolved studies and lends itself to tracking trends in protein aggregation arising from changing environmental factors. Information on individual particle’s refractive indexes can be used to differentiate protein aggregates from such contaminants as silicone droplets. These capabilities are demonstrated through measurements on samples of bovine pancreas insulin aggregated through centrifugation and of bovine serum albumin aggregated by complexation with a polyelectrolyte. Differentiation is demonstrated with samples that have been spiked with separately characterized silicone spheres. Holographic characterization measurements are compared with results obtained with microflow imaging and dynamic light scattering.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Holographic Characterization of Protein Aggregates

Wang

Zhong

Ruffner

et al. 2016

Journal of Pharmaceutical Sciences

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“…The number of runs was optimized for each sample prior to the initiation of measurements (a minimum of 10 runs was performed per measurement). The diffusion coefficient and the particle diameter by the cumulant method were calculated from the autocorrelation function using Dynamics software (Wyatt Technology) 50 .…”

Section: Methodsmentioning

confidence: 99%

AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG

Senga

Imamura

Miyafusa

et al. 2017

Sci Rep

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Therapeutic IgG becomes unstable under various stresses in the manufacturing process. The resulting non-native IgG molecules tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk factor for immunogenicity, rapid analysis of aggregation is required for quality control of therapeutic IgG. In this study, we developed a homogeneous assay using AlphaScreen and AF.2A1. AF.2A1 is a 25-residue artificial protein that binds specifically to non-native IgG generated under chemical and physical stresses. This assay is performed in a short period of time. Our results show that AF.2A1-AlphaScreen may be used to evaluate the various types of IgG, as AF.2A1 recognizes the non-native structure in the constant region (Fc region) of IgG. The assay was effective for detection of non-native IgG, with particle size up to ca. 500 nm, generated under acid, heat, and stirring conditions. In addition, this technique is suitable for analyzing non-native IgG in CHO cell culture supernatant and mixed with large amounts of native IgG. These results indicate the potential of AF.2A1-AlphaScreen to be used as a high-throughput evaluation method for process monitoring as well as quality testing in the manufacturing of therapeutic IgG.

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“…These developing in silico approaches complement established accelerated stress studies that are performed in vitro to predict the shelf‐life and stability of biologics (Jain et al, 2017; Yang et al, 2013). Various methods are employed to generate such data including heating (Cheng et al, 2012; Hamrang et al, 2015), stirring (Luo et al, 2011; Sediq, Van Duijvenvoorde, Jiskoot, & Nejadnik, 2016), shaking (Kiese, Papppenberger, Friess, & Mahler, 2008; Rudiuk, Cohen‐Tannoudji, Huille, & Tribet, 2012), and simulation of transportation (Fleischman, Chung, Paul, & Lewus, 2017). The extent of aggregation, however, can be heavily dependent on the type of accelerated stress employed (Fleischman et al, 2017; Joubert, Luo, Nashed‐Samuel, Wypych, & Narhi, 2011; Tamizi & Jouyban, 2016).…”

Section: Introductionmentioning

confidence: 99%

Using extensional flow to reveal diverse aggregation landscapes for three IgG1 molecules

Willis

Kumar

Dobson

et al. 2018

Biotech & Bioengineering

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Monoclonal antibodies (mAbs) currently dominate the biopharmaceutical sector due to their potency and efficacy against a range of disease targets. These proteinaceous therapeutics are, however, susceptible to unfolding, mis‐folding, and aggregation by environmental perturbations. Aggregation thus poses an enormous challenge to biopharmaceutical development, production, formulation, and storage. Hydrodynamic forces have also been linked to aggregation, but the ability of different flow fields (e.g., shear and extensional flow) to trigger aggregation has remained unclear. To address this question, we previously developed a device that allows the degree of extensional flow to be controlled. Using this device we demonstrated that mAbs are particularly sensitive to the force exerted as a result of this flow‐field. Here, to investigate the utility of this device to bio‐process/biopharmaceutical development, we quantify the effects of the flow field and protein concentration on the aggregation of three mAbs. We show that the response surface of mAbs is distinct from that of bovine serum albumin (BSA) and also that mAbs of similar sequence display diverse sensitivity to hydrodynamic flow. Finally, we show that flow‐induced aggregation of each mAb is ameliorated by different buffers, opening up the possibility of using the device as a formulation tool. Perturbation of the native state by extensional flow may thus allow identification of aggregation‐resistant mAb candidates, their bio‐process parameters and formulation to be optimized earlier in the drug‐discovery pipeline using sub‐milligram quantities of material.

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Characterisation of Stress-Induced Aggregate Size Distributions and Morphological Changes of a Bi-Specific Antibody Using Orthogonal Techniques

Cited by 14 publications

References 28 publications

Holographic Characterization of Protein Aggregates

Holographic Characterization of Protein Aggregates

AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG

Using extensional flow to reveal diverse aggregation landscapes for three IgG1 molecules

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