(W1130-09-59) Low Molecular Weight Hydroxypropyl Cellulose as a Novel Protein Stabilizer for Parenteral Pharmaceuticals

Purpose: The protein drugs are known with their high-order conformational molecular structure and various functional moieties that makes them highly sensitive to chemical degradation and aggregation in aqueous solutions¹. Surfactants, such as polysorbates, are often used to stabilize the protein molecule in the final dosage formulations¹. Some authors report that several factors, such as enzymatic hydrolysis by residual proteases and oxidation due to light exposure, can lead the degradation of polysorbate and the generation of free fatty acid in the protein formulation, resulting in the formation of fatty acid particle^2,3. The need for alternative stabilizers in pharmaceutical protein formulations forces scientists to look for different molecules. Among the novel excipient candidates are some cellulose ethers, like hydroxypropyl cellulose (HPC), already known as effective solubilizers. In this study, we report the potential use of low molecular weight HPC as an alternative protein stabilizer for parenteral pharmaceutical formulations.

Methods: Hydroxypropyl cellulose (NISSO HPC-SSL and UL) from Nippon Soda, Japan and abatacept (ORENCIA FOR I.V. INFUSION 250 mg; Bristol Myers Squibb Company, USA) were used. The abatacept solution (10 mM phosphate, pH 7.0) was sterilized by filtration through a 0.22 μm filter (SLGVR33RS, Merck, USA) under aseptic conditions and then mixed to HPC solution with different concentrations. Each sample was subjected to static storage test at 25 °C and 40 °C for 4 weeks or 12 weeks. Shaking test was performed using an in vitro shaker (Mix-VR, TAITEC, Japan) at room temperature with a speed of 500 r/min for 3 hours (Table 1). Subsequently, soluble protein components were analyzed using size exclusion chromatography (SEC, Alliance HPLC system, Waters Corporation, USA). The relative proportions of monomer and high molecular weight (H.M.W.) species were calculated from their respective peak areas.

Results: After centrifugation (15,000×g, 4 °C, 30 min) of the collected samples, 20 μL of the supernatant was injected into a TSKgel G3000SW_XL column (0008541, Tosoh, Japan). From the area values of monomer and H.M.W. species on the chromatogram (280 nm), their relative ratios (%) were calculated (Table 1). In the static storage test, no significant differences were observed between formulations with and without HPC addition after storage at 25 °C for 4 and 12 weeks (Figure 1). When using a storage condition of 40 °C for 4 weeks, the sample without HPC addition (No. 5) demonstrated significant changes. The percentage of H.M.W. species increased, while the percentage of monomers decreased under the same conditions (Figure 1). These changes demonstrated that HPC addition improved the stability of abatacept. In the shaking test (Figure 2), No. 2 and No. 4, which contained HPC concentrations below their respective critical micelle concentrations (CMC), demonstrated significant increases in H.M.W. species and significant decreases in monomer content to a similar extent as the additive-free control (No. 6). When HPC was added at concentrations above the CMC, the stability of abatacept was improved.

Conclusion: The 40°C static storage test showed that HPC addition successfully suppressed protein aggregation. The shaking test comparison confirmed the aggregation suppression efficiency of HPC, however, only at concentrations above the HPC CMC.
These results indicate the HPC efficiency as a surfactant/stabilizer for pharmaceutical protein formulations.

References: [1] V. Gervasi et al., Parenteral protein formulations: An overview of approved products within the European Union, Eur. J. Pharm. Biopharm. 131 (2018) 8–24.
[2] B. Kerwin et al., Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways, J Pharm Sci. 97 (2008) 2924-2935
[3] N. Glücklich et al., An in-depth examination of fatty acid solubility limits in biotherapeutic protein formulations containing polysorbate 20 and polysorbate 80, Int. J. Pharm. 591 (2020) 119934

Table 1. Composition of abatacept solutions. (a) Static storage test samples, (b) Shaking test samples.

Figure 1. Percentage of (a) H.M.W. species and (b) Monomer detected by SEC analysis. *: 0.01 < p < 0.05, **: p <0.01, Initial: start of storage, w: storage period, Error bar represents standard deviation (n =3).

Figure 2. Percentage of (a) H.M.W. species and (b) Monomer detected by SEC analysis. *: 0.01 < p < 0.05, **: p <0.01, Initial: before shaking, Shake: after 3 hours shaking, Error bar represents standard deviation (n =3).