Sweet Corn Phytoglycogen as a Lyoprotectant for Dry-State Protein Storage

Introduction: : Preserving protein biotherapeutics is a significant manufacturing challenge. Cold-chain storage of liquid formulations mantains biotherapeutic function but is expensive and resource-intensive, while lyophilization into dry powder can damage protein structure. Sugar-based compounds are widely used to protect proteins during lyophilization stresses. However, these lyoprotectants typically require additional excipients such as bulking agents, complicating optimization processes. Here, we demonstrate that the hyperbranched glucose polymer phytoglycogen (“PG”) from sweet corn can act as a lyoprotectant and a bulking agent. The benefits of PG include its low cost, abundant natural source, easy extraction process, and cytocompatibility.

Materials and

Methods: : We extracted PG from different maize sources (i.e., “PG1”-“PG16”). Colloidal stability of each PG was tested by adding 1x PBS, to a final expected PG concentration of 20 mg/mL concentration. Solutions maintained optical density over 500 hours were considered stable PG, whereas precipitated were excluded (Figure 1a). PG cytotoxicity was tested using an established NIH 3T3 fibroblast cell assay (Figure 1b) [1]. Protein preservation was measured by subjecting lysozyme (1.25, 0.5, 0.25, 0.125 mg/mL), green fluorescent protein (GFP) (7.5, 4, 2 µM), β-galactosidase (β-gal) (150, 100, 50, 25 µg/mL) or horse radish peroxidase (HRP) (150, 100, 50, 25 µg/mL) to repeated cycles of freezing, lyophilization, and resuspension in the presence or absence of PG (1-20 mg/mL) (Figure 2). Solutions were frozen at -80 °C and lyophilized. GFP fluorescence, Lysozyme, β-gal, and HRP activity were measured after multiple cycles of lyophilization using fluorimetry (ex/em 485/510 nm), EnzChek™ Lysozyme Assay Kit, o-nitrophenyl-β-D-galactoside (ONPG), Tetramethylbenzidine (TMB, 4 mg/mL), respectively. Antibody stabilization was tested by immunostaining. α-Smooth muscle actin (α-SMA) of NIH 3T3 fibroblasts was treated with lyophilized primary antibody, with or without PG, followed by treatment with Alexa-594 conjugated secondary antibody for observation. IgG1 monoclonal anti-GFP antibody was mixed with either 20 mg/mL PG, 140 mg/mL sucrose, 120 mg/mL trehalose, or PBS, then lyophilized and stored at 4 °C and room temperature. The antibody activity was measured and compared using an enzyme-linked immunosorbent assay (ELISA).

Results, Conclusions, and Discussions:: PG1, 2, 8, 9, 12, 13, 15, and 16 were colloidally stable up to 10 mg/mL in PBS. PG1, 2, 9, and 13 were not cytotoxic to NIH3T3 fibroblasts at the highest concentrations (Figure 1). Precipitated and cytotoxic PG variants were excluded from further studies. Based on screening experiments, PG13 was selected as the most promising candidate. GFP stocks retained more than 80% fluorescence over 10 lyophilization cycles when 10 mg/mL PG13 was added as an excipient, compared to less than 10 % fluorescence without PG13(Figure 2a). Lysozyme, β-gal, and HRP maintained enzyme activity after multiple cycles of lyophilization, whereas a significant decrease in activity was observed without PG13 (Figure 2b, c, d). An anti-α-SMA primary antibody bound to the cell structure in NIH 3T3 fibroblasts after lyophilization with PG13. In contrast, without PG13, aggregated antibody was observed after staining, which hindered the observation of cell structures (Figure 3). Finally, long-term antibody stabilization was tested with PG13, sucrose, trehalose, or PBS. Immediately after lyophilization, the antibody activity was 98% with PG13, 88% with sucrose, 86% with trehalose, but 4% without excipient. The antibody activity with sucrose and trehalose dropped rapidly during both room temperature and 4 °C storage conditions. The antibody activity with PG13 was approximately 84% for 2 months at room temperature, and 98% for 2 months at 4°C. These data demonstrate that PG excipients can stabilize various model proteins during lyophilization. According to the DLS, TEM results, and other research (data not shown), PG is known for its soft and flexible hairy surface. We hypothesized that the unique structure and high glass transition temperature (Tg′) of PG provide a glassy matrix during lyophilization that stabilizes protein structure by restricting protein mobility. These data demonstrate that PG excipients can protect proteins during lyophilization by maintaining protein structure. Future work will seek to identify the stabilizing mechanism of PG and establish PG as an excipient in lyophilized biopharmaceutical formulations.
In conclusion, our data demonstrate that corn-derived PG is a simple, biocompatible, abundant, and inexpensive excipient to stabilize proteins during lyophilization, thereby reducing the need for expensive and complicated cold-chain storage.

Acknowledgements and/or References (Optional):: Acknowledgements: USDA 2020-67014-30900 - Phytoglycogen: A Sweet Corn Product with Potential Use as Nanoparticle Ingredient

References: [1] Liu, R., Molecules, 2020, 25(3), 637. [2] U.S. Patent App 63/311,524 Apr 2022.
[3] Junha Park et al., Journal of Biomedical Materials Research Part A (2024).