(W1030-02-07) Comparative Analyses of Thermal Stability Amplifying Peptide Dendrons and Polysorbates

Purpose: Monoclonal antibodies (mAbs) are widely used therapeutics that are prone to degradation. Degradation can be reduced by low-temperature storage (-80 to -10 ^oC), yet the maintenance of such temperatures can prove challenging and costly. The development of formulations that impart greater stability at or above room temperature ( >25 ^oC) is therefore of great interest. Previous reports of a peptide dendron additive demonstrated a greater degree of protein stability at 55 ^oC when comparing protein aggregation and fragmentation, as well as structure and activity against samples treated with polysorbate (PS), a common surfactant in mAb formulations. To explore the potential of L6 and similar molecules as an alternate surfactant, several stress factors were considered in a comparative study design.

Methods: Protein carbonylation was measured by a previously established ELISA assay after incubating protein with CuSO₄ and ascorbic acid at 37 ^oC for 24 hours. Particle formation was measured using a micro-flow imaging system. Degradation of L6 by oxidative stress was conducted by stressing L6 at 37 ^oC for one week with iron, copper and 2,2'-Azobis(2-amidinopropane) dihydrochloride and analyzing the oxidized products by MALDI-TOF mass spectrometry. Immunogenicity potential assessment and cell viability was measured by THP1 cells including an NF-kB reporter. Critical micelle concentration (CMC) was measured using coumarin as a fluorescent indicator over a range of surfactant concentrations.

Results: Trace metal impurities can oxidatively damage proteins and excipients, therefore, protein carbonylation and resultant particle formation was measured, showing similar levels when treated with L6 or PS. Additional characterization of L6 by MALDI-TOF-MS showed only trace degradation, supporting its resistance to oxidative stress. Previous results demonstrated the biocompatibility of L6 as it did not affect receptor binding or cause hemolysis, and our findings expand this compatibility. L6 showed minimal inflammatory signal and cytotoxicity at low concentrations using an NfκB reporter assay. The measurement of the critical micelle concentration, often used to compare surfactants, showed similar values between L6 and PS80.

Conclusion: These results, coupled with the added benefit of another agent to support stability at elevated temperatures, demonstrate similar and non-inferior performance of L6 compared to PS under additional stressors and characterizations, highlighting the potential of such novel materials for further formulation development.

References: Bansal, R., et al. (2017). "Peptide Dendrons as Thermal-Stability Amplifiers for Immunoglobulin G1 Monoclonal Antibody Biotherapeutics." Bioconjug Chem 28(10): 2549-2559.
Uehara, H. and V. A. Rao (2015). "Metal-mediated protein oxidation: applications of a modified ELISA-based carbonyl detection assay for complex proteins." Pharm Res 32(2): 691-701.
Fluksman, A. and O. Benny (2019). "A robust method for critical micelle concentration determination using coumarin-6 as a fluorescent probe." Analytical Methods 11(30): 3810-3818.

Acknowledgements: This project was supported in part by an appointment to the Research Participation Program at the Office of Biotechnology Products in the Center for Drug Evaluation and Research at the U.S. Food and Drug Administration (FDA), administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and FDA. The funding source had no part in the study design, data collection/analysis, decision to publish, or preparation of the manuscript, the authors have no conflicts of interests. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the United States Food and Drug Administration and the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government.