Sweet corn phytoglycogen dendrimers as a lyoprotectant for dry‐state protein storage

Abstract

Protein biotherapeutics typically require expensive cold‐chain storage to maintain their fold and function. Packaging proteins in the dry state via lyophilization can reduce these cold‐chain requirements. However, formulating proteins for lyophilization often requires extensive optimization of excipients that both maintain the protein folded state during freezing and drying (i.e., “cryoprotection” and “lyoprotection”), and form a cake to carry the dehydrated protein. Here we show that sweet corn phytoglycogens, which are glucose dendrimers, can act as both a protein lyoprotectant and a cake‐forming agent. Phytoglycogen (PG) dendrimers from 16 different maize sources (PG1‐16) were extracted via ethanol precipitation. PG size was generally consistent at ~70–100 nm for all variants, whereas the colloidal stability in water, protein contaminant level, and maximum density of cytocompatibility varied for PG1‐16. 10 mg/mL PG1, 2, 9, 13, 15, and 16 maintained the activity of various proteins, including green fluorescent protein, lysozyme, β‐galactosidase, and horseradish peroxidase, over a broad range of concentrations, through multiple rounds of lyophilization. PG13 was identified as the lead excipient candidate as it demonstrated narrow dispersity, colloidal stability in phosphate‐buffered saline, low protein contaminants, and cytocompatibility up to 10 mg/mL in NIH3T3 cell cultures. All dry protein‐PG13 mixtures had a cake‐like appearance and all frozen protein‐PG13 mixtures had a Tg' of ~ −26°C. The lyoprotection and cake‐forming properties of PG13 were density‐dependent, requiring a minimum density of 5 mg/mL for maximum activity. Collectively these data establish PG dendrimers as a new class of excipient to formulate proteins in the dry state.