Poster O8 - Sweet Corn Phytoglycogen Dendrimers as a Lyoprotectant for Dry-State Protein Storage

Introduction: Preserving protein biotherapeutics is a significant manufacturing challenge. Cold-chain storage of liquid formulations maintains biotherapeutic function but is expensive and resource-intensive, while lyophilization into dry powder can damage protein structure. Sugar-based compounds, like sucrose and trehalose, can protect proteins from lyophilization stresses. However, these “lyoprotectants” typically require additional excipients such as bulking agents, complicating formulation processes. Here, we report that the glucose dendrimer phytoglycogen (“PG”) from sweet corn can stabilize proteins and act as a bulking agent during lyophilization. The benefits of PG include its low cost, abundance, simple extraction, and biocompatibility.

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. Solutions that demonstrated a constant optical density over 500 h were considered colloidally stable, whereas PG extracts that precipitated were excluded from subsequent studies. PG cytotoxicity was tested using an established NIH 3T3 fibroblast cell assay (Figure 1) [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 horseradish 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 was measured after 10 cycles of lyophilization via fluorimetry (ex/em 485/510 nm) using a SpectraMax M3 plate reader. Lysozyme activity was measured after 10 cycles of lyophilization with the EnzChek™ Lysozyme Assay Kit. β-gal activity was evaluated after 4 cycles of lyophilization by adding O-nitrophenyl-β-D-galactoside (ONPG), and the absorbance of samples was read at 405 nm for 30 mins. HRP activity was measured after 10 cycles of lyophilization via Tetramethylbenzidine (TMB, 4 mg/mL) and 50 μL of H2SO4 stop solution. Protein structure was analyzed by FTIR spectroscopy. A1650 cm-1 represents amideⅠ band, and A1550 cm-1 corresponds to amideⅡ band. The ratio of the area under the amide Ⅰ/amide Ⅱ curve was used to assess changes in protein structure.

Results, Conclusions, and Discussions: PG1, 2, 8, 9, 12, 13, 15, and 16 were colloidally stable up to 10mg/mL in PBS. PG1, 2, 9, and 13 were not cytotoxic to NIH3T3 fibroblasts at the highest concentrations (Figure 1). Unstable and cytotoxic PG variants were excluded from further studies. Due to its low sweet corn protein contaminant level, PG13 was selected as the most promising candidate. GFP stocks retained more than 80% fluorescence over 10 lyophilization cycles when 10mg/mL PG13 was added as an excipient, compared to less than 10% fluorescence without PG13 (Figure 2a). Lysozyme maintained over 75% activity after 10 lyophilization cycles with 10 mg/mL PG13, whereas a significant decrease in activity was observed, dropping to 60% without PG13 (Figure 2b). β-gal activity was evaluated over 4 cycles of lyophilization. With 10mg/mL PG13 excipient, at least 90% of β-gal activity was preserved, while only 6% of activity was maintained without PG13 (Figure 2c). Finally, HRP maintained at least 50% activity over 3 lyophilization cycles with 10mg/mL PG13 excipient, while only 25% of HRP activity was maintained in the absence of PG (Figure 2d). All PG+protein formulations formed a smooth and stable lyocake, demonstrating that PG can also act as a bulking agent during sublimation.
FTIR measurements indicated that PG13 maintained a lysozyme secondary structure that was comparable to that of lysozyme before lyophilization, whereas lysozyme structure was compromised by lyophilization in the absence of PG13 (Figure 3a, 3b). PG has a soft and flexible hairy surface. We hypothesized that this amorphous structure, coupled with its favorable freeze-concentrate glass transition temperature (Tg′, -26 C), underlies the formation of a glassy matrix during lyophilization, which stabilizes protein structure by restricting protein mobility. Collectively, these data demonstrate that PG excipients can stabilize proteins during lyophilization by forming a lyocake that maintains protein structure. Future work will seek to identify the stabilizing mechanism of PG and characterize PG as an excipient in lyophilized biopharmaceutical formulations.
These data demonstrate corn-derived PG as a simple, biocompatible, abundant, and economically-favorable excipient that can stabilize proteins during lyophilization, thereby reducing the need for expensive and complicated cold-chain storage.

Acknowledgements (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.