(W0930-12-77) An Insight into the Interrelationship between Colloidal Stability of Protein Therapeutics in Formulation and Their Adsorption to a Solid Surface

Purpose: The exponential surge in the development and manufacturing of biologic drug products has created an immense need for understanding and control of product quality in which protein aggregation presents a formidable obstacle, as it can compromise both efficacy and safety (Roberts, 2014). Understanding the molecular mechanism of protein aggregation is essential for developing successful mitigation strategies. Stability of protein therapeutics relies not only on the intrinsic properties of protein molecules but also on their surrounding milieu including the concentration at which the protein is formulated and the buffer composition. For an effective and optimal formulation, stability must be screened as a function of specific ion types, their concentration, excipient profile, and pH. Several biophysical methods have been explored to understand the mechanisms of aggregation and to assess protein aggregates in the particle size range ≤10 µm, the size range that may elicit immunogenicity, as emphasized by U.S. Food and Drug Administration (FDA, 2014). Colloidal stability of protein solutions has been shown to be a major driver of stability while surface-induced protein aggregation has been challenging to predict and comprehend (Roberts, 2014) (Pham and Meng, 2020). Studying adsorption of proteins to a solid surface may shed light on both the colloidal stability of the protein formulations and surface-induced aggregation. However, studies aiming at the correlation of the aggregation in the subvisible/visible range with protein adsorption to a solid surface are scarce (Defante et al., 2020). The present work investigates potential correlations between protein aggregation studied with flow imaging microscopy and surface adsorption studied with quartz crystal microbalance using BSA as a model protein.

Methods: The stability of BSA in different buffers namely acetate, histidine, and phosphate with pH 4.5, 6.0, and 7.5, respectively, was studied in stability tests with the storage condition of 5°C for 5 days. The number of aggregates generated in the stability study was monitored by assessing the number of particles in the micrometer-size range using a flow imaging microscopy method (FlowCam). Quartz crystal microbalance, with dissipation monitoring, was employed to evaluate the adsorbed protein layer on a hydrophilic gold surface. The adsorption of BSA on the surface in the above-mentioned formulations was monitored in terms of frequency shift (ΔF) of the third overtone for all formulations.

Results: The irreversibly adsorbed layer of BSA, was detected by QCM-D in all three formulations and it was found that ΔF of the adsorbed layer in the case of acetate buffer was highest while the lowest ΔF was associated with the phosphate buffer (Fig 1 represents the QCM-D profile for adsorption from 2 mg/ml BSA in all three formulations). The formation of protein aggregates in different buffers in the stability studies was monitored employing flow imaging. It was found that the highest number of aggregates were formed in the case of acetate buffer, while the lowest was in the case of phosphate buffer. Table 1 depicts the interrelationship between the amount of protein adsorbed on the gold surface using QCM-D and the number of aggregates generated in the respective formulation for 2 mg/ml BSA concentration and indicates a strong correlation between these values. The finding may be attributed to the fact that the pH of acetate is 4.5 which is near the isoelectric point of BSA, reducing the electrostatic repulsive forces between the protein molecules, thereby, increasing their propensity to aggregate. While in the case of phosphate buffer, the protein is highly charged rendering the formulation to be stable.

Conclusion: The present study indicated that the irreversibly adsorbed layer of protein from QCM-D could predict the stability of protein formulation with different pHs. Additional research using representative therapeutic proteins such as monoclonal antibodies and other formulation conditions (ionic strength and presence of other excipients) will have to be performed to further assess the potentials of adsorption studies in the screening of therapeutic formulations.

References: (a) Roberts, C.J., 2014. Protein aggregation and its impact on product quality. Curr. Opin.
Biotechnol. 30, 211–217
(b) FDA, Guidance for industry: immunogenicity assessment for therapeutic protein products.
2014
(c) Pham, N. B., Meng, W. S. 2020. Protein aggregation and immunogenicity of biotherapeutics. International journal of pharmaceutics, 585, 119523
(d) Defante, A. P., Kalonia, C. K., Keegan, E., Bishop, S. M., Satish, H. A., Hudson, S. D., Santacroce, P. V. (2020). The impact of the metal interface on the stability and quality of a therapeutic fusion protein. Molecular Pharmaceutics, 17, 569-578.

Acknowledgements: Research reported in this publication was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R21AI178218. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Fig.1. Frequency plots showing adsorption of BSA (2 mg/ml) onto a gold surface from acetate, histidine, and phosphate buffer with pH 4.5, 6.0, and 7.5, respectively.

Table.1. Interrelationship between frequency shifts due to adsorption and the number of aggregates following stability study in the size >2 µm for BSA for all three formulations.