Comparison of High-Throughput Biophysical Methods to Identify Stabilizing Excipients for a Model IgG2 Monoclonal Antibody: Conformational Stability and Kinetic Aggregation Measurements

Cheng, Weiqiang; Joshi, Sangeeta B.; He, Feng; Brems, David N.; He, Bing; Kerwin, Bruce A.; Volkin, David B.; Middaugh, C. Russell

doi:10.1002/jps.23076

Cited by 59 publications

(73 citation statements)

References 71 publications

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“…The high-throughput format and the low sample volume make DSF an ideal tool for analyzing various formulation conditions simultaneously. In earlier reports DSF with SYPRO Orange was shown to identify formulation conditions that enhance thermal stability of proteins (He et al, 2010;Cheng et al, 2012;Goldberg et al, 2011). The use of DSF with SYPRO Orange is however limited to formulations without surfactants, since interaction with the highly hydrophobic part of surfactants limits the use of SYPRO Orange-based DSF in protein formulation studies (Samra and He, 2012;He et al, 2011).…”

Section: Introductionmentioning

confidence: 96%

“…DSF is an excellent method to identify solution conditions and excipients that stabilize proteins. It has been previously applied to investigate protein stability under different formulation conditions (He et al, 2010;Cheng et al, 2012;Goldberg et al, 2011). In DSF, the protein is heated by a multiwell RT PCR instrument, in the presence of a fluorescence dye, to monitor thermally induced structural changes of the protein conformation.…”

Section: Introductionmentioning

confidence: 99%

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Differential scanning fluorescence approach using a fluorescent molecular rotor to detect thermostability of proteins in surfactant-containing formulations

Ablinger

Leitgeb

Zimmer

2013

International Journal of Pharmaceutics

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Section: Introductionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Differential scanning fluorescence approach using a fluorescent molecular rotor to detect thermostability of proteins in surfactant-containing formulations

Ablinger

Leitgeb

Zimmer

2013

International Journal of Pharmaceutics

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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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“…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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Comparison of High-Throughput Biophysical Methods to Identify Stabilizing Excipients for a Model IgG2 Monoclonal Antibody: Conformational Stability and Kinetic Aggregation Measurements

Cited by 59 publications

References 71 publications

Differential scanning fluorescence approach using a fluorescent molecular rotor to detect thermostability of proteins in surfactant-containing formulations

Differential scanning fluorescence approach using a fluorescent molecular rotor to detect thermostability of proteins in surfactant-containing formulations

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

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

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