(M1330-09-57) Unveiling the Impact of Surface Chemistry on Adsorption of a Therapeutic Monoclonal Antibody Using a Quartz Crystal Microbalance

Purpose: Therapeutic proteins have attained significant prominence in current pharmaceutical products across the years. Among therapeutic proteins, monoclonal antibodies (mAbs) make up the biggest share of the market (1). These proteins, however, display inherent instability, and a variety of factors can lead to the degradation of proteins which would consequently impact the safety and efficacy of the products. The degraded products of monoclonal antibodies and in particular protein aggregates may cause acute or more long-term immune responses upon administration including immunogenicity. Interfacial stresses have been shown to be one of the leading causes of aggregation in biologics during the process of development, production, and clinical administration. Interface induced aggregation mechanisms are not well understood but adsorption of proteins across the many interfaces (liquid–liquid, solid–liquid, and vapor–liquid) and stressing of the adsorbed layer have been suggested to play a major role (2). Chemistry and nature of the surface in contact with the protein product play a critical role in the phenomenon of adsorption and may regulate the aggregate formation at these interfaces which needs to be established for therapeutic proteins.

So, this study focuses on exploring the adsorption of trastuzumab, a commercially available therapeutic protein, on glass and a few commonly used glass coatings using a quartz crystal microbalance with dissipation (QCM-D).

Methods: In order to investigate the interaction of mAbs with the solid surface in adsorption studies, the following four QCM-D sensors were prepared: A hydrophilic sensor was prepared by standard QCM-D cleaning of the borosilicate coated sensor as per the instrument supplier protocol (3); a water washed sensor was also prepared by a simple rinsing of the borosilicate sensor with ultrapure water; a hydrophobic sensor was prepared by treating the borosilicate coated sensor by piranha solution (mixture of H2SO4 and H2O2) and subsequent submerging in a solution of dimethyldichlorosilane in trichloroethylene (4); a PEGylated sensor was prepared by treating the borosilicate sensor by piranha solution and then coating with PEG-Silane as described previously (5). After preparing these surfaces, the adsorption of trastuzumab (10 mg/ml in histidine buffer 10 mM pH 6), was monitored by QCM-D. First, a histidine buffer run was performed to establish the baseline frequency. The protein solution run was conducted subsequently and trastuzumab adsorption was monitored through the changes in frequency. Finally, another buffer run was used to rinse away the unabsorbed protein and to allow for assessment of the number of adsorbed proteins by calculating the resonance frequency differences between the resonance frequency of baseline and the resonance frequency after adsorption and rinse. In order to obtain more information about the coatings and surfaces, the water contact angle on surfaces was measured by putting a 20 µl drop of the water on the sensor, taking a photograph and measuring the contact angle.

Results: The silanized sensor was the most hydrophobic surface and had a contact angle around 90°, while other surfaces were relatively hydrophilic (Fig. 1). The data obtained by QCM-D analysis provided valuable insights into the adsorption behavior of trastuzumab on different sensor surfaces. The results revealed a specific trend in the mass adsorbed on the sensor surfaces, with the lowest adsorption observed on the PEGylated surface, followed by the hydrophilic, water washed, and silanized surfaces. This trend was established based on the frequency change (Δf3) measurements, which correlated with the adsorbed mass. The corresponding frequency changes were recorded as -94, -111, -121, and -149 Hz, respectively. The higher adsorption observed on the hydrophobic surface can be attributed to the interaction forces that come into play. Hydrophobic surfaces tend to have stronger attractive forces towards proteins (hydrophobic interactions), resulting in increased adsorption. Conversely, the lower adsorption observed on the PEGylated surface can be attributed to the formation of a hydrated layer on the surface. PEG, being a hydrophilic polymer, has a high affinity for water molecules. When the sensor surface is modified with PEG, it forms a hydrated layer that acts as a steric barrier, reducing the adsorption of trastuzumab.

Conclusion: These results indicate that surface chemistry of glass, as exists in the primary packaging of mAbs, and its commonly used coatings greatly influences the adsorption of trastuzumab. Potential future studies could look into how trastuzumab aggregation will be influenced in borosilicate glass vials exposing these four surface chemistries.

References: 1- 4. Dimitrov DS. Therapeutic proteins. Method Mol Biol. 2012;899:1-26.
2- Li, J., Krause, M. E., Chen, X., Cheng, Y., Dai, W., Hill, J. J., Huang, M., Jordan, S., LaCasse, D., Narhi, L., Shalaev, E., Shieh, I. C., Thomas, J. C., Tu, R., Zheng, S., & Zhu, L. (2019). Interfacial Stress in the Development of Biologics: Fundamental Understanding, Current Practice, and Future Perspective. The AAPS Journal, 21(3), 44. https://doi.org/10.1208/s12248-019-0312-3
3- Clegg JR, Ludolph CM, Peppas NA. QCM-D assay for quantifying the swelling, biodegradation, and protein adsorption of intelligent nanogels. J Appl Polym Sci. 2020 Jul 5;137(25):48655. doi: 10.1002/app.48655. Epub 2019 Oct 31. PMID: 34732941; PMCID: PMC8562820.
4- Nejadnik, M. R., & Garcia, C. D. (2011). Staining proteins: A simple method to increase the sensitivity of ellipsometric measurements in adsorption studies. Colloids and Surfaces B: Biointerfaces, 82(1), 253-257. https://doi.org/https://doi.org/10.1016/j.colsurfb.2010.08.021
5- Gidi, Y., Bayram, S., Ablenas, C. J., Blum, A. S., & Cosa, G. (2018). Efficient One-Step PEG-Silane Passivation of Glass Surfaces for Single-Molecule Fluorescence Studies. ACS Applied Materials & Interfaces, 10(46), 39505-39511. https://doi.org/10.1021/acsami.8b15796

A drop of water on A: Silanized sensor, B: Water washed sensor, and C: PEGylated sensor.

Frequency changes over time for borosilicate glass and the coated glass surfaces described in this study.