(T1230-01-05) Impact of Excipient Composition on the Stability of Trastuzumab Biosimilars

Purpose: Commercial biosimilar formulations can vary significantly in both the types of excipients used and their concentrations. This study specifically investigates the role of excipients in preventing protein destabilization during stresses induced by freeze-drying and storage at high temperature. Trastuzumab (Herceptin®) serves as the model protein for this research. Several trastuzumab biosimilars have been approved by the FDA. While all these formulations use histidine as a buffering agent, the selection and concentration of other excipients differ. Stabilizers like trehalose, sucrose, and sorbitol are commonly employed. Notably, one product contains more than double the trehalose content compared to others using the same sugar. Additionally, a formulation combining sorbitol with PEG 3350 has also been introduced. These differences highlight several important considerations, including a) the comparability of the stabilizing effects of sucrose and trehalose, b) the potential for higher trehalose concentrations to improve protein stability against processing stresses, and c) the relative performance of commonly used disaccharides (sucrose and trehalose) compared to the combination of PEG and sorbitol as stabilizers.

Methods: Trastuzumab (Tmab) obtained from a single market product was reformulated to mimic four distinct formulation compositions, designated as F1, F2, F3, and F4 (Figure 1). All formulations utilized histidine as the buffering agent, with polysorbate included in three of the four formulations (excluded in F4). The stabilizers varied across formulations: sucrose in F3, sorbitol in F4, and trehalose in F1 and F2 (Figure 1). The pre-lyophilization solutions were subjected to freeze-drying and the freeze-dried forms were further subjected to heat stress at 40 C° and 50 C° (Figure 1). A comprehensive array of analytical techniques—UV–Vis spectroscopy, size-exclusion chromatography, dynamic light scattering, fluorescence spectroscopy, circular dichroism spectroscopy, and flow imaging microscopy was employed to assess monoclonal antibody stability (Figure 1).

Results: Overall, the aggregation was influenced by the excipient composition. Specifically, aggregation of sucrose and trehalose-containing formulations were slightly affected by freeze-drying and heat, respectively. The formation of higher molecular weight (HMW) species was evaluated by size-exclusion chromatography (Figure 2). After freeze-drying the formulation, The HMW species concentration was the highest in F3 (~ 3.3%) where sucrose is used as a lyoprotectant. For the other formulations, it was < 2% (Figure 2). DLS showed similar particle diameters for all formulations. The average diameter (in nm) for F1, F2, F3 and F4 were 10.1 (±0.2), 10.0 (±0.1), 10.2 (±0.1) and 10.3 (±0.2) respectively. These differences are not statistically significant (p > 0.05). The corresponding polydispersity index (PDI) values for F1, F2, F3 and F4 were 0.20 (±0.22), 0.05 (±0.01), 0.29 (±0.09) and 0.11. In the accelerated stability study, F3 showed more stability during the storage at 50 C° for 4 and 8 weeks. On the other hand, the other formulations with trehalose and sorbitol had a significant increase in their HMW species concentration (figure 3).

Conclusion: This study demonstrated that excipient composition plays a critical role in the stability of trastuzumab formulations under freeze-drying and heat stress. Formulations containing trehalose exhibited greater stability during freeze-drying, whereas sucrose-based formulations showed improved stability during accelerated storage at elevated temperatures.

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Acknowledgements: This project is supported by the Food and Drug Administration (FDA) of the U.S. Department of Health and Human Services (HHS) as part of a financial assistance award U01FD007758 to the National Institute for Pharmaceutical Technology and education (NIPTE) totaling $1,976,692 with 100 percent funded by FDA/HHS. The contents are those of the author(s) and do not necessarily represent the official views of, nor an endorsement, by FDA/HHS, or the U.S. Government

Figure 1. Schematic representation of isolation of Tmab from one commercial formulation and reformulation into four different formulation compositions. The reformulated Tmab was exposed to stress conditions and was monitored using different analytical techniques.

Figure 2. HMW species concentration of four different formulation exposed to freeze drying stress

Figure 3. HMW species concentration of four different formulations exposed to heat stress.