Local Dynamics and Their Alteration by Excipients Modulate the Global Conformational Stability of an lgG1 Monoclonal Antibody

doi:10.1002/jps.23332

Journal of Pharmaceutical Sciences

Volume 101, Issue 12, December 2012, Pages 4444-4457

https://doi.org/10.1002/jps.23332 Get rights and content

ABSTRACT

A molecular understanding of excipient effects on the interrelationship(s) between dynamics and conformational stability of proteins, such as monoclonal antibodies (mAbs), can be important for their pharmaceutical development. The current study examines stabilizing and destabilizing effects of excipients on the conformational stability and local dynamics of distinct solvent‐exposed regions within an IgG1 monoclonal antibody (mAb‐B). The principles of site‐selective photoselection upon red‐edge excitation, accompanied by acrylamide quenching of tryptophan fluorescence were employed in this study. The initiation of mAb‐B thermal unfolding occurs by structural alterations in the more solvent‐exposed regions of the antibody, which subsequently leads to a cascade of structural alterations in its relatively more solvent‐shielded regions. In addition, an increase in internal dynamics of solvent‐shielded regions made mAb‐B more susceptible to thermally induced structural perturbations resulting in its global destabilization. Sucrose and arginine exert their stabilizing and destabilizing effects by predominantly influencing the conformational stability of solvent‐exposed regions in mAb‐B. The complex molecular effects of sucrose and arginine on local dynamics of different regions in mAb‐B and their correlation with the protein's conformational stability are described within the pretransition range, at the onset temperature (T_onset) and at the thermal melting temperature (T_M).

Section snippets

INTRODUCTION

The three‐dimensional structure of native, functionally active proteins is considered to be a large ensemble of dynamic intraconvertible microstates that are stable in solution.¹,² Proteins typically exhibit a wide variety of molecular motions ranging on the timescale from 10⁻¹⁵ to 10⁴ s. These fluctuations are composed of local motions (10⁻¹⁵–10⁻¹ s; 0.01–5 Å) such as atomic and side chain fluctuations, rigid body motions (10⁻⁹–1 s; 1–10 Å) such as helix or hinge bending deflections, or

Materials

The IgG1 monoclonal antibody (mAb‐B) was supplied by MedImmune (Gaithersburg, Maryland) and stored in its formulation buffer at 2°C–8°C. The protein was dialyzed into 20 mM citrate–phosphate buffer at pH 4.5, and the final ionic strength was maintained at 0.1 using NaCl. All chemicals, including the buffer components, were purchased either from Sigma (St. Louis, Missouri) or Fisher Scientific (Pittsburgh, Pennsylvania). Sucrose from Ferro Pfanstiehl Laboratories (Mayfield Heights, Ohio) was used

Intrinsic Trp Fluorescence, Thermal Melting Temperature, and Excipient Effects as a Function of Fluorescence Excitation Wavelength

Changes in mAb‐B higher order structure and conformational stability with temperature and excitation wavelength are illustrated in Figure 1, in which the Trp emission maximum (peak position) is plotted as a function of temperature at different excitation wavelengths. At 10°C and throughout the pretransition temperature range (10°C–35°C), the Trp emission maxima for mAb‐B have shifted to a higher wavelength upon REE from 292 to 308 nm (Figs. 1a and 1d). This observation suggests that the sampled

DISCUSSION

Excipients such as sugars and amino acids are frequently used to increase the conformational stability of therapeutic proteins' native structure and prevent aggregation both in solution and the dried state.⁴⁶,⁵⁴ These excipients are known to influence the forces and interactions involved in maintaining the stability of protein(s)⁴⁶ in part by altering the organization of hydration water.⁵⁵ Our previous studies show that sucrose and arginine had distinct effects on the stability and global

ACKNOWLEDGEMENTS

The authors thank MedImmune for providing IgG1 monoclonal antibody(mAb‐B) and financial support for these studies.

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BiotechnologyLocal Dynamics and Their Alteration by Excipients Modulate the Global Conformational Stability of an lgG1 Monoclonal Antibody

ABSTRACT

Section snippets

INTRODUCTION

Materials

Intrinsic Trp Fluorescence, Thermal Melting Temperature, and Excipient Effects as a Function of Fluorescence Excitation Wavelength

DISCUSSION

ACKNOWLEDGEMENTS

Curr Opin Struct Biol

J Pharm Sci

Biophys J

J Pharm Sci

J Pharm Sci

J Pharm Sci

Adv Biophys

Methods Enzymol

Biophys J

Biophys J

Biophys Chem

Trends Biochem Sci

J Pharm Sci

Biophys J

Adv Drug Deliv Rev

J Pharm Sci

J Biol Chem

Adv Drug Deliv Rev

Biophys Chem

Chem Phys Lett

Chem Phys Lett

Int J Pharm

Dynamic personalities of proteins

Nature

Protein folding funnels: A kinetic approach to the sequence‐structure relationship

Proc Natl Acad Sci U S A

Chemical physics of protein folding

Proc Natl Acad Sci U S A

Binding induced conformational changes of proteins correlate with their intrinsic fluctuations: A case study of antibodies

BMC Struct Biol

Millisecond time scale conformational flexibility in a hyperthermophile protein at ambient temperature

Proc Natl Acad Sci U S A

Do ultrastable proteins from hyperthermophiles have high or low conformational rigidity

Proc Natl Acad Sci U S A

Immunoglobulin dynamics, conformational fluctuations, and nonlinear elasticity and their effects on stability

J Phys Chem B

A relationship between protein stability and protein function

Proc Natl Acad Sci U S A

Native protein fluctuations: The conformational‐motion temperature and the inverse correlation of protein flexibility with protein stability

J Biomol Struct Dyn

Biotechnology
Local Dynamics and Their Alteration by Excipients Modulate the Global Conformational Stability of an lgG1 Monoclonal Antibody