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Thermodynamic Unfolding and Aggregation Fingerprints of Monoclonal Antibodies Using Thermal Profiling

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Abstract

Purpose

Predicting thermal protein stability is of major interest in the development of protein-based biopharmaceuticals. Therefore, this study provides a predictive tool for determining transition enthalpies, which can be used for ranking different proteins according to their thermal stability.

Methods

Unfolding and aggregation profiles of eight different therapeutic monoclonal antibodies (mAbs) of type G, isotype 1 were investigated. The unfolding profiles were determined by intrinsic fluorescence (IF) spectroscopy and differential scanning calorimetry (DSC). A three-state unfolding fitting model was used to determine thermodynamic parameters for macromolecular multi-domain mAbs in IF experiments, like the van’t Hoff enthalpy change (∆Hvh) and the entropy change (∆S) of the unfolding event. The derived values were compared to thermodynamic parameters obtained directly by calorimetry. Moreover, differences in the Fab enthalpies were used to predict aggregation behavior and protein thermal stabilities. To do so, the liquid-formulated mAbs were investigated exemplarily by size exclusion chromatography (SEC) after accelerated thermal-induced stress conditions.

Results

Comparing the thermodynamic parameters derived from IF spectroscopy and DSC resulted in similar values. Data generated by thermal-induced stress at 40°C show similar stability ranking as postulated through the Fab enthalpies for mAbs in two different formulations, while at 25°C a meaningful ranking is not possible, because distinct differences in the thermal stability cannot be observed. The additional consideration of Fab enthalpies to predict the 40 °C SEC ranking seems to be more reliable compared to the use of exclusively the melting temperatures or aggregation onset temperatures and times.

Conclusion

We show that thermodynamic profiling can help predicting unfolding and aggregation properties of therapeutic mAbs at 40°C. Therefore, analyzing thermodynamic unfolding parameters is a useful and supportive tool discriminating thermal stability profiles of mAbs for further pharmaceutical development and clinical studies.

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Abbreviations

∆cp :

heat capacity change

∆G:

Gibbs free energy

∆Hcal :

calorimetric enthalpy

∆Hvh :

van’t Hoff enthalpy

∆S:

entropy

CH-domain:

constant antibody domain of the heavy chain

CHO:

Chinese Hamster Ovary

cp,Tm :

heat capacity of the melting temperature

c.u.:

cooperative unit

DSC:

differential scanning calorimetry

F:

folded state

f:

state fraction

Fab:

fragment antigen binding

Fc:

fragment crystallizable

FE330 :

fluorescence emission at 330 nm

FE350 :

fluorescence emission at 350 nm

HP/UP-SEC:

high performance / ultra performance size exclusion chromatography

I:

intermediate state

IF:

intrinsic fluorescence spectroscopy

IgG1:

immunoglobulin type G isotype 1

K:

equilibrium constant

LENP:

Lumry-Eyring nucleated polymerization

mAb:

monoclonal antibody

mAU:

milli arbitrary units

MWCO:

molecular weight cut off

n:

reaction order

R:

gas constant

S:

slope

T:

temperature

Tagg :

aggregation onset temperature

tagg :

aggregation onset time

Tm :

melting temperature

U:

unfolded state

y:

signals of the IF thermograms

Y:

single state signals

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Authors and Affiliations

  1. Institute of Chemistry, Martin-Luther-Universität Halle-Wittenberg, von-Danckelmann-Platz 4, DE-06120, Halle, Saale, Germany

    Richard Melien & Dariush Hinderberger

  2. Pharmaceutical Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorferstrasse 65, DE-88397, Biberach, Germany

    Richard Melien, Patrick Garidel & Michaela Blech

Authors
  1. Richard Melien
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  2. Patrick Garidel
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  3. Dariush Hinderberger
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  4. Michaela Blech
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Contributions

Richard Melien designed and Michaela Blech designed and supervised the study. Experimental research, data analysis, and evaluation was performed by Richard Melien. All authors contributed to the data analysis. The first draft was written by Richard Melien and was reviewed by Patrick Garidel, Dariush Hinderberger, and Michaela Blech.

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Correspondence to Michaela Blech.

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Guest Editors: Ahmed Besheer and Hanns-Christian Mahler

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Melien, R., Garidel, P., Hinderberger, D. et al. Thermodynamic Unfolding and Aggregation Fingerprints of Monoclonal Antibodies Using Thermal Profiling. Pharm Res 37, 78 (2020). https://doi.org/10.1007/s11095-020-02792-1

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