Comparative Signature Diagrams to Evaluate Biophysical Data for Differences in Protein Structure Across Various Formulations

Iyer, Vidyashankara; Maddux, Nathan; Hu, Lei; Cheng, Weiqiang; Youssef, Adel F.; Winter, Gerhard; Joshi, Sangeeta B.; Volkin, David B.; Middaugh, C. Russell

doi:10.1002/jps.23367

Cited by 15 publications

(20 citation statements)

References 22 publications

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“…In addition, this method is difficult for people with vision color deficiencies. To overcome these disadvantages, several new data visualization approaches (Kim et al, 2012; Iyer et al, 2013) have been recently introduced.…”

Section: High Throughput Biophysical Methods and Data Visualization Tmentioning

confidence: 99%

“…Finally, comparative signature diagrams (CSDs) (Iyer et al, 2013) are designed to visualize statistically significant differences in biophysical stability data sets between two samples. The mean and standard deviation of multiple datasets from the same biophysical techniques for the two target samples to be compared are calculated first for each variable such as wavelength and temperature.…”

Section: High Throughput Biophysical Methods and Data Visualization Tmentioning

confidence: 99%

“…In these case studies, the structural integrity of several model proteins such as acidic fibroblast growth factor-1 (FGF-1), an IgG1 monoclonal antibody, and granulocyte colony stimulating factor (GCSF) were characterized using various biophysical methods as a function of temperature and solution pH. The large physical stability data sets obtained were combined into various types of diagrams for data visualization (Maddux et al, 2011; Kim et al, 2012; Iyer et al, 2013). Examples include EPDs, radar charts and CSDs as described above.…”

Section: Case Studies Using Data Visualization Techniques To Compare mentioning

confidence: 99%

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Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles

et al. 2014

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In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs), radar charts, and comparative signature diagrams (CSDs) for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1) mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF). Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress). Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.

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Section: High Throughput Biophysical Methods and Data Visualization Tmentioning

confidence: 99%

Section: High Throughput Biophysical Methods and Data Visualization Tmentioning

confidence: 99%

Section: Case Studies Using Data Visualization Techniques To Compare mentioning

confidence: 99%

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Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles

et al. 2014

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“…Prior work [25,26] has shown that a mass balance on all protein species allows one to determine from and m via Equation [8]. can be significantly different depending on which conditions were used to create aggregates.…”

Section: Aggregate Morphologymentioning

confidence: 99%

“…Although the relationship between protein structure and morphology has been developed for low concentrations, it has not been established for intermediate to high concentrations where strong nonideal protein-protein interactions are expected [7,8]. The behavior of protein solutions at intermediate to high concentrations is difficult to predict based on protein properties or measurements at lower concentrations [9].…”

Section: Introductionmentioning

confidence: 98%

Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations

Barnett

Wei

Amin

et al. 2015

Biophysical Chemistry

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Non-native aggregation is a common issue in a number of degenerative diseases and during manufacturing of protein-based therapeutics. There is a growing interest to monitor protein stability at intermediate to high protein concentrations, which are required for therapeutic dosing of subcutaneous injections. An understanding of the impact of protein structural changes and interactions on the protein aggregation mechanisms and resulting aggregate size and morphology may lead to improved strategies to reduce aggregation and solution viscosity. This report investigates non-native aggregation of a model protein, α-chymotrypsinogen, under accelerated conditions at elevated protein concentrations. Far-UV circular dichroism and Raman scattering show structural changes during aggregation. Size exclusion chromatography and laser light scattering are used to monitor the progression of aggregate growth and monomer loss. Monomer loss is concomitant with increased β-sheet structures as monomers are added to aggregates, which illustrate a transition from a native monomeric state to an aggregate state. Aggregates grow predominantly through monomer-addition, resulting in a semi-flexible polymer morphology. Analysis of aggregation growth kinetics shows that pH strongly affects the characteristic timescales for nucleation (τn) and growth (τg), while the initial protein concentration has only minor effects on τn or τg. Low-shear viscosity measurements follow a common scaling relationship between average aggregate molecular weight (Mw(agg)) and concentration (σ), which is consistent with semi-dilute polymer-solution theory. The results establish a link between aggregate growth mechanisms, which couple Mw(agg) and σ, to increases in solution viscosity even at these intermediate protein concentrations (less than 3w/v %).

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High-Throughput Biophysical Analysis and Data Visualization of Conformational Stability of an IgG1 Monoclonal Antibody After Deglycosylation

Alsenaidy

Kim

Majumdar

et al. 2013

Journal of Pharmaceutical Sciences

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The structural integrity and conformational stability of an IgG1 monoclonal antibody (mAb), after partial and complete enzymatic removal of the N-linked Fc glycan, was compared to the untreated mAb over a wide range of temperature (10° to 90°C) and solution pH (3 to 8) using circular dichroism, fluorescence spectroscopy, and static light scattering combined with data visualization employing empirical phase diagrams (EPDs). Subtle to larger stability differences between the different glycoforms were observed. Improved detection of physical stability differences was then demonstrated over narrower pH range (4.0-6.0) using smaller temperature increments, especially when combined with an alternative data visualization method (radar plots). Differential scanning calorimetry and differential scanning fluorimetry were then utilized and also showed an improved ability to detect differences in mAb glycoform physical stability. Based on these results, a two-step methodology was used in which mAb glycoform conformational stability is first screened with a wide variety of instruments and environmental stresses, followed by a second evaluation with optimally sensitive experimental conditions, analytical techniques and data visualization methods. With this approach, high-throughput biophysical analysis to assess relatively subtle conformational stability differences in protein glycoforms is demonstrated.

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Comparative Signature Diagrams to Evaluate Biophysical Data for Differences in Protein Structure Across Various Formulations

Cited by 15 publications

References 22 publications

Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles

Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles

Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations

High-Throughput Biophysical Analysis and Data Visualization of Conformational Stability of an IgG1 Monoclonal Antibody After Deglycosylation

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