Sweet Corn Phytoglycogen as a Protein Stabilizing Excipient

Introduction:: Introduction: Preservation of protein biotherapeutics is a major manufacturing burden. Cold-chain storage of wet formulations can maintain biotherapeutic function but is expensive and resource-intensive, while lyophilization into a dry powder can damage protein structure. Sugar-based compounds that can protect proteins against physical and chemical degradation are receiving considerable attention as excipients that can be added to biopharmaceutical formulations. But, many of these compounds are acquired through complex synthesis and purification processes. Here, we demonstrate that the glucose dendrimer phytoglycogen (“PG”) produced by sweet corn can be used as an excipient to stabilize proteins during lyophilization. The benefits of PG include its low cost, abundant natural source, “green” extraction process, and biocompatibility.


Materials and Methods:: Materials and methods: We extracted PG from different maize sources (i.e., “PG1”-“PG16”). Solubility of each PG was tested by adding 1x PBS, a common biotherapeutic vehicle, to a final expected PG concentration of 20 mg/mL concentration. Solutions without precipitate were considered soluble PG, whereas precipitated solutions were excluded. PG cytotoxicity was tested using an established NIH 3T3 fibroblast cell assay [1]. Protein preservation was measured by subjecting lysozyme (2.5, 1, 0.5, 0.25 mg/mL), green fluorescent protein (GFP) (15, 8, 4 µM), β-galactosidase (β-gal) (300, 200, 100, 50 µg/mL) or horse radish peroxidase (HRP) (300, 200, 100, 50 µg/mL) to repeated cycles of freezing, lyophilization, and resuspension in the presence or absence of PG (1-20 mg/mL) (Figure 1). 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, according to manufacturer’s instructions. 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 H₂O₂ stop solution. The protein structure was analyzed by FTIR spectroscopy. A1650 cm^-1 represents amide1 band, and A1550 cm^-1 corresponds to amide2 band. The ratio of the area under the amide1/amide2 curve was used to assess changes in protein structure.


Results, Conclusions, and Discussions:: Results and Discussion: PG1, 2, 5, 8, 9, 11, 13, 15, and 16 were soluble up to 20 mg/mL in PBS. PG1, 2, 9, and 13 were not cytotoxic to NIH3T3 fibroblasts at all concentrations in vitro (Figure 2). Insoluble and toxic PG variants were excluded from lyophilization studies. GFP stocks retained more than 80% fluorescence over 10 lyophilization cycles when 20 mg/mL PG1, 2, 9, and 13 was added as an excipient, compared to less than 10% fluorescence when no excipient was added. For example, GFP with PG13 maintained at least 88.1% fluorescence, whereas in the absence of PG13, GFP fluorescence dropped to 5% after 10 cycles of lyophilization (Figure 3). As the concentration of PG decreased, the GFP fluorescence after 10 cycles of lyophilization also decreased, demonstrating that the excipient effect of PG is dose-dependent. Lysozyme maintained more than 88.7% activity over 10 lyophilization cycles when 20 mg/mL PG13 was included in the formulation, whereas a significant decrease in activity was observed, dropping to 58.3%, when PG was absent. According to the series of experiments, PG13 was selected as the most promising candidate. β-gal activity was evaluated over 4 cycles of lyophilization. With 20 mg/mL PG13 excipient, 93.3% of β-gal activity was preserved, while only 6.7% of activity was maintained without PG13. Finally, HRP maintained at least 96.6% activity over 3 lyophilization cycles with 20 mg/mL PG13 excipient, while only 74.3% of HRP activity was maintained in the absence of PG. FTIR measurements indicated that a PG excipient preserved 81% of lysozyme structural integrity whereas only 57% of lysozyme structure was maintained in the absence of PG. Collectively, these data demonstrate that PG excipients can stabilize proteins during lyophilization by maintaining protein structure. Future work will seek to identify the stabilizing mechanism of PG and PG as an excipient in lyophilized biopharmaceutical formulations.

Conclusions: In conclusion, our data demonstrate that corn-derived PG can provide a simple, biocompatible, abundant, and economically-favorable excipient to stabilize proteins during lyophilization, thereby reducing the need for expensive and complicated cold-chain storage.


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


References (Optional): : References: [1] Liu, R., Molecules, 2020, 25(3), 637. [2] U.S. Patent App 63/311,524 Apr 2022.