Formulation Development of Therapeutic Monoclonal Antibodies Using High-Throughput Fluorescence and Static Light Scattering Techniques: Role of Conformational and Colloidal Stability

Goldberg, Deborah S.; Bishop, Steven M.; Shah, Ambarish; Sathish, Hasige A.

doi:10.1002/jps.22371

Cited by 135 publications

(121 citation statements)

References 32 publications

(33 reference statements)

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“…The long term loss of monomer is generally predicted through quantitative monitoring of aggregation under accelerated and stress conditions over weeks to months. Recent progress has been made in: (i) the development of detailed kinetic models [2,4,5,34,42,49,60,96]; (ii) correlating aggregation kinetics with protein structure and folding [11,15,16,32,35,52,53,54,67,88]; (iii) and with native-state protein-protein interaction measurements [36,46,57,74,75,79,82].…”

Section: Introductionmentioning

confidence: 99%

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Austerberry

Dajani

Panova

et al. 2017

European Journal of Pharmaceutics and Biopharmaceutics

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a b s t r a c tThe aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations.

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Section: Introductionmentioning

confidence: 99%

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Austerberry

Dajani

Panova

et al. 2017

European Journal of Pharmaceutics and Biopharmaceutics

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“…In this section, thermostability was considered indicative of aggregation propensity where a high thermostability value and hence high‐protein conformational stability was taken as an indicator of low aggregation propensity (Goldberg et al, 2011). …”

Section: Manufacturability Index Resultsmentioning

confidence: 99%

Multi‐criteria manufacturability indices for ranking high‐concentration monoclonal antibody formulations

Yang

Velayudhan

Thornhill

et al. 2017

Biotech & Bioengineering

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The need for high‐concentration formulations for subcutaneous delivery of therapeutic monoclonal antibodies (mAbs) can present manufacturability challenges for the final ultrafiltration/diafiltration (UF/DF) step. Viscosity levels and the propensity to aggregate are key considerations for high‐concentration formulations. This work presents novel frameworks for deriving a set of manufacturability indices related to viscosity and thermostability to rank high‐concentration mAb formulation conditions in terms of their ease of manufacture. This is illustrated by analyzing published high‐throughput biophysical screening data that explores the influence of different formulation conditions (pH, ions, and excipients) on the solution viscosity and product thermostability. A decision tree classification method, CART (Classification and Regression Tree) is used to identify the critical formulation conditions that influence the viscosity and thermostability. In this work, three different multi‐criteria data analysis frameworks were investigated to derive manufacturability indices from analysis of the stress maps and the process conditions experienced in the final UF/DF step. Polynomial regression techniques were used to transform the experimental data into a set of stress maps that show viscosity and thermostability as functions of the formulation conditions. A mathematical filtrate flux model was used to capture the time profiles of protein concentration and flux decay behavior during UF/DF. Multi‐criteria decision‐making analysis was used to identify the optimal formulation conditions that minimize the potential for both viscosity and aggregation issues during UF/DF. Biotechnol. Bioeng. 2017;114: 2043–2056. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Perodicals, Inc.

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“…Recently, differential static light scattering was developed to follow protein aggregation in a highthroughput format in combination with differential scanning flurimetry for screening of protein formulation conditions (6). However, this approach requires use of a different set of equipments.…”

Section: Resultsmentioning

confidence: 99%

“…Currently, there are a variety of high-throughput methods including intrinsic and extrinsic fluorescence spectroscopy to study protein unfolding and detect soluble aggregates (5). A few of the most commonly used fluorescent dyes to probe protein unfolding include SYPRO Orange and 1-anilino-8-naphthalene sulfonate (6)(7)(8). Similarly, aggregate formation has been studied using thioflavin T (ThT), Nile Red, and Congo Red which have been known to bind specifically to amyloid-type fibrils (9)(10)(11)(12)(13).…”

Section: Introductionmentioning

confidence: 99%

Orthogonal High-Throughput Thermal Scanning Method for Rank Ordering Protein Formulations

et al. 2013

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Abstract. A high-throughput thermal-scanning method to rank-order formulation conditions for therapeutic proteins is described. Apparent transition temperatures for unfolding and aggregation of four different proteins are determined using the dyes SYPRO Orange and thioflavin T (ThT) under a variety of buffer conditions. The results indicate that the ThT-based thermal scanning method offers several advantages over the previously described SYPRO Orange-based thermal scanning and allows rapid rank ordering of solution conditions relevant toward long-term storage of therapeutic molecules. The method is also amenable to high protein concentration and does not require sample dilution or extensive preparation. Additionally, this parallel use of SYPRO Orange and ThT can be readily applied to the screening of mutants for their unfolding and aggregation propensities.

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Formulation Development of Therapeutic Monoclonal Antibodies Using High-Throughput Fluorescence and Static Light Scattering Techniques: Role of Conformational and Colloidal Stability

Cited by 135 publications

References 32 publications

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Multi‐criteria manufacturability indices for ranking high‐concentration monoclonal antibody formulations

Orthogonal High-Throughput Thermal Scanning Method for Rank Ordering Protein Formulations

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