Optimizing a therapeutic humanized follicle-stimulating hormone-blocking antibody formulation by protein thermal shift assay

Authors

Damini Sant, Center for Translational Medicine and Pharmacology and Departments of Medicine and of Pharmacology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Satish Rojekar, Center for Translational Medicine and Pharmacology and Departments of Medicine and of Pharmacology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Sakshi Gera, Center for Translational Medicine and Pharmacology and Departments of Medicine and of Pharmacology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Anusha R. Pallapati, Center for Translational Medicine and Pharmacology and Departments of Medicine and of Pharmacology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Judit Gimenez-Roig, Center for Translational Medicine and Pharmacology and Departments of Medicine and of Pharmacology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Tan-Chun Kuo, Center for Translational Medicine and Pharmacology and Departments of Medicine and of Pharmacology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Clifford J. Rosen, MaineHealth Institute for Research, Scarborough, ME.Follow

Document Type

Article

Publication Date

1-11-2023

Institution/Department

MaineHealth Institute for Research

Journal Title

Annals of the New York Academy of Sciences

Abstract

Biopharmaceutical products are formulated using several Food and Drug Administration (FDA) approved excipients within the inactive ingredient limit to maintain their storage stability and shelf life. Here, we have screened and optimized different sets of excipient combinations to yield a thermally stable formulation for the humanized follicle-stimulating hormone (FSH)-blocking antibody, MS-Hu6. We used a protein thermal shift assay in which rising temperatures resulted in the maximal unfolding of the protein at the melting temperature (T ). To determine the buffer and pH for a stable solution, four different buffers with a pH range from 3 to 8 were screened. This resulted in maximal T s at pH 5.62 for Fab in phosphate buffer and at pH 6.85 for Fc in histidine buffer. Upon testing a range of salt concentrations, MS-Hu6 was found to be more stable at lower concentrations, likely due to reduced hydrophobic effects. Molecular dynamics simulations revealed a higher root-mean-square deviation with 1 mM than with 100 mM salt, indicating enhanced stability, as noted experimentally. Among the stabilizers tested, Tween 20 was found to yield the highest T and reversed the salt effect. Among several polyols/sugars, trehalose and sucrose were found to produce higher thermal stabilities. Finally, binding of recombinant human FSH to MS-Hu6 in a final formulation (20 mM phosphate buffer, 1 mM NaCl, 0.001% w/v Tween 20, and 260 mM trehalose) resulted in a thermal shift (increase in T ) for the Fab, but expectedly not in the Fc domain. Given that we used a low dose of MS-Hu6 (1 μM), the next challenge would be to determine whether 100-fold higher, industry-standard concentrations are equally stable.

Recommended Citation

Sant D, Rojekar S, Gera S, et al. Optimizing a therapeutic humanized follicle-stimulating hormone-blocking antibody formulation by protein thermal shift assay [published online ahead of print, 2023 Jan 11]. Ann N Y Acad Sci. 2023;10.1111/nyas.14952. doi:10.1111/nyas.14952