Most therapeutic proteins are rapidly cleared from circulation, typically by renal filtration. Increasing the <i>in vivo </i>residence times of protein therapeutics could decrease their dosing frequencies. PEGylation of proteins is one of the most common strategies. However, PEGylation can be expensive, and non-specific. PEGylated proteins have been observed to cause renal tubular vacuolation. Renally cleared PEGylated proteins and their metabolites may accumulate in the kidney, causing the formation of PEG hydrates that interfere with normal glomerular filtration. In addition, animals and humans can be induced to make antibodies to PEG. A new class of fusion tags are being developed, which are composed of unstructured long amino acid sequences. These tags provide a more homogeneous end-product than PEGylation, and have been shown to increase the plasma half-life of therapeutic proteins by at least five-fold. In the present study, we examined how this 40-amino acid long fusion tag affects the structure and stability of a pharmaceutical protein using various techniques that include heteronuclear 2D NMR, chemical denaturant melts, and time-resolved fluorescence anisotropy. The tag is predominantly random coil with no stable structure, and did not affect the secondary or tertiary structures of the pharmaceutical protein. In addition, there is no effect of tag on the conformational stability of the protein. However, the tag prevented the oligomerization of the protein thus promoting its functional monomer. Taken together, these results indicate that long amino acid fusion tags can be advantageous in significantly extending the plasma half-life of pharmaceutical proteins without affecting their structure, stability, and function.

Effect of a long genetic fusion tag on the structure and stability of a pharmaceutical protein

2019 Spring National Meeting

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