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Expanding the toolbox for predictive parameters describing antibody stability considering thermodynamic and kinetic determinants

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Abstract

Purpose

Introduction of the activation energy (Ea) as a kinetic parameter to describe and discriminate monoclonal antibody (mAb) stability.

Methods

Ea is derived from intrinsic fluorescence (IF) unfolding thermograms. An apparent irreversible three-state fit model based on the Arrhenius integral is developed to determine Ea of respective unfolding transitions. These activation energies are compared to the thermodynamic parameter of van´t Hoff enthalpies (∆Hvh). Using a set of 34 mAbs formulated in four different formulations, both the apparent thermodynamic and kinetic parameters together with apparent melting temperatures are correlated collectively with each other to storage stabilities to evaluate its predictive power with respect to long-term effects potentially reflected in shelf-life.

Results

Ea allows for the discrimination of (i) different parent mAbs, (ii) different variants that originate from parent mAbs, and (iii) different formulations. Interestingly, we observed that the Ea of the CH2 unfolding transition shows strongest correlations with monomer and aggregate content after storage at accelerated and stress conditions when collectively compared to ∆Hvh and Tm of the CH2 transition. Moreover, the predictive parameters determined for the CH2 domain show generally stronger correlations with monomer and aggregate content than those derived for the Fab. Qualitative assessment by ranking Ea of the Fab domain showed good agreement with monomer content in storage stabilities of individual mAb sub-sets.

Conclusion

Ea from IF unfolding transitions can be used in addition to other commonly used thermodynamic predictive parameters to discriminate and characterize thermal stability of different mAbs in different formulations. Hence, it shows great potential for antibody engineering and formulation scientists.

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Abbreviations

α:

degree of conversion

∆Hvh :

change of van´t Hoff enthalpy

A:

frequency factor

ANOVA:

analysis of variance

Ar :

solution of the Arrhenius integral

B:

heating rate

c:

intercept of the irreversible three-state model

CH domain:

constant domain of the antibody heavy chain

CHO:

Chinese hamster ovary

D1 :

first denatured state

D2 :

second denatured state

DSC:

differential scanning calorimetry

Ea :

activation energy

F:

folded state

f:

state fraction

Fab:

fragment antigen binding

FE330 :

fluorescence emission at 330 nm

FE350 :

fluorescence emission at 350 nm

I:

intermediate state

IF:

intrinsic fluorescence

IgG1:

immunoglobulin type G isotype 1

K:

equilibrium constant

m:

linear slope of the irreversible three-state model

mAb:

monoclonal antibody

MWCO:

molecular weight cut off

NES:

novel experimental setup

R:

gas constant

S:

slope of the reversible three-state model

SEC:

size exclusion chromatography

T:

temperature

Tm :

melting temperature

U:

unfolded state

HP/UP-SEC:

high- performance /ultra- performance size exclusion chromatography

y:

data of the IF thermograms

Y:

single state data

Y0 :

intercept of the reversible three-state model

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ACKNOWLEDGMENTS

The authors thank Philipp Baaske and David Ng from NanoTemper Technologies GmbH for ongoing support and the “German Federal Ministry of Education and Research, funding program KMU-innovativ Photonics Research Germany, contract number 13N14245“ for funding. We also thank Felicitas Wahl, Marina Rechsteiner, Sebastian Kube, and Gabriele Richter from Boehringer Ingelheim Pharma GmbH & Co. KG for technical assistance and Anna Schulze from NanoTemper Technologies GmbH for review.

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Authors and Affiliations

  1. Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, Pharmaceutical Development Biologicals, 88397, Biberach an der Riss, Germany

    Michaela Blech, Richard Melien & Patrick Garidel

  2. Institute of Chemistry, Martin-Luther-Universität Halle-Wittenberg, von-Danckelmann-Platz 4, 06120, Halle (Saale), Germany

    Richard Melien & Dariush Hinderberger

  3. Nanotemper Technologies GmbH, Flößergasse 4, 81369, München, Germany

    Nuska Tschammer

  4. Analytical Development Biologicals, CMC Statistics, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Str. 65, 88397, Biberach an der Riss, Germany

    Beate Presser

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  1. Michaela Blech
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Blech, M., Melien, R., Tschammer, N. et al. Expanding the toolbox for predictive parameters describing antibody stability considering thermodynamic and kinetic determinants. Pharm Res 38, 2065–2089 (2021). https://doi.org/10.1007/s11095-021-03120-x

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