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High Throughput Differential Scanning Fluorimetry (DSF) Formulation Screening with Complementary Dyes to Assess Protein Unfolding and Aggregation in Presence of Surfactants

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Abstract

Purpose

The purpose was to evaluate DSF for high throughput screening of protein thermal stability (unfolding/ aggregation) across a wide range of formulations. Particular focus was exploring PROTEOSTAT® – a commercially available fluorescent rotor dye – for detection of aggregation in surfactant containing formulations. Commonly used hydrophobic dyes (e.g. SYPRO™ Orange) interact with surfactants, complicating DSF measurements.

Methods

CRM197 formulations were prepared and analyzed in standard 96-well plate rT-PCR system, using SYPRO™ Orange and PROTEOSTAT® dyes. Orthogonal techniques (DLS and IPF) are employed to confirm unfolding/aggregation in selected formulations. Selected formulations are subjected to non-thermal stresses (stirring and shaking) in plate based format to characterize aggregation with PROTEOSTAT®.

Results

Agreement is observed between SYPRO™ Orange (unfolding) and PROTEOSTAT® (aggregation) DSF melt temperatures across wide range of non-surfactant formulations. PROTEOSTAT® can clearly detect temperature induced aggregation in low concentration (0.2 mg/mL) CRM197 formulations containing surfactant. PROTEOSTAT® can be used to explore aggregation due to non-thermal stresses in plate based format amenable to high throughput screening.

Conclusions

DSF measurements with complementary extrinsic dyes (PROTEOSTAT®, SYPRO™ Orange) are suitable for high throughput screening of antigen thermal stability, across a wide range of relevant formulation conditions – including surfactants –with standard, plate based rT-PCR instrumentation.

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Abbreviations

λem :

Fluorescence emission wavelength

λex :

Fluorescence excitation wavelength

λweighted :

Weighted average (tryptophan) emission wavelength

ANS:

8-Anilinonaphthalene-1-sulfonic acid fluorescent dye

CCVJ:

9-(2-carboxy-2-cyanovinyl)julolidine fluorescent rotor dye

CRM197:

Modified diphtheria toxin (CRM197)

DCVJ:

9-Julolidinylmethylenemalononitrile fluorescent rotor dye

dH,min :

Hydrodynamic radius

DLS:

Dynamic light scattering

DSF:

Differential scanning fluorimetry

HT:

High throughput

IPF:

Intrinsic protein fluorescence

mAb:

Monoclonal antibody

MRL:

Merck research laboratories

rT-PCR:

Real time polymerase chain reaction

Tagg :

Aggregation transition temperature of CRM197

Tm :

Melting (unfolding) transition temperature of CRM197

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors gratefully acknowledge Henryk Mach for guidance with intrinsic protein fluorescence measurements and useful discussions. We gratefully acknowledge Brian K. Meyer and Christopher L. Daniels for reviewing the manuscript. We also thank MRL Vaccine Bioprocess for supplying CRM197 for this study. S. M. McClure, P. L. Ahl, and J.T. Blue are employees of Merck Sharp & Dohme Corp.

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Authors and Affiliations

  1. Center for Materials Science and Engineering, Merck Sharp & Dohme Corp, WP75B-210, 770 Sumneytown Pike, West Point, PA, 19486, USA

    Sean M. McClure

  2. Vaccine Drug Product Development, Merck Sharp & Dohme Corp, West Point, PA, 19486, USA

    Patrick L. Ahl & Jeffrey T. Blue

Authors
  1. Sean M. McClure
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Corresponding author

Correspondence to Sean M. McClure.

Electronic supplementary material

Figure S1

Additional results from Fig. 1 in text. (a). Tm vs. pH, by NaCl [mM] from 96 well pH-NaCl screen of CRM197 using SYPRO™ Orange dye (unfolding)). (b). Tagg vs. pH, by NaCl [mM] from 96 well pH-NaCl screen of CRM197 using PROTEOSTAT® dye (aggregation)). (c). Tagg vs. Tm pH-NaCl screen data, where linear least square fit line is Tagg = 0.97Tm + 3.46°C (R2 = 0.93). Note: Tm and Tagg values for pH = 4.5 (no transitions observed) and control (no pH buffer) are not included. (GIF 179 kb)

High resolution image (TIFF 105 kb)

Figure S2

SYPRO™ Orange Tm results from CRM197 excipient screen (excipient, by concentration). Bars colored by excipient type. Error bars represent standard deviation of duplicate measurements. Red line in figure is Tm for the base formulation (30 mM HEPES, pH = 7.5, 0 mM NaCl, no excipient) for reference. (GIF 284 kb)

High resolution image (TIFF 321 kb)

Figure S3

PROTEOSTAT® Tagg results from CRM197 excipient screen (excipient, by concentration). Bars colored by excipient type. Error bars represent standard deviation of duplicate measurements. Red line in figure is Tagg for the base formulation (30 mM HEPES, pH = 7.5, 0 mM NaCl) for reference. (GIF 314 kb)

High resolution image (TIFF 370 kb)

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McClure, S.M., Ahl, P.L. & Blue, J.T. High Throughput Differential Scanning Fluorimetry (DSF) Formulation Screening with Complementary Dyes to Assess Protein Unfolding and Aggregation in Presence of Surfactants. Pharm Res 35, 81 (2018). https://doi.org/10.1007/s11095-018-2361-1

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  • DOI: https://doi.org/10.1007/s11095-018-2361-1

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