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  • Review Article
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Protein drug stability: a formulation challenge

Key Points

  • Recombinantly expressed proteins are increasingly important in drug therapy. This makes it crucial to assess how their properties as proteins affect drug efficacy, targeting and side effects, as well as the ability to survive long-term storage.

  • Amino-acid substitutions have led to therapeutically improved variants of, for example, insulin and interleukin-2, but modifications such as acylation and PEGylation can be just as effective, by causing a decrease in the clearing rate and reducing immunogenicity.

  • Aggregation and misfolding is a fundamental issue in the long-term storage of protein therapeutics before administration. Although the mechanisms of aggregation are complex and can differ between even closely related proteins, methods have been developed to predict how amino-acid substitutions can affect this process.

  • An easier approach might be to modify drug formulations. Simple additives, such as detergents, amino-acid pairs or cyclodextrins, can markedly reduce aggregation. Furthermore, judicious use of lyophilization can also provide a very reliable way to extend shelf-life.

Abstract

The increasing use of recombinantly expressed therapeutic proteins in the pharmaceutical industry has highlighted issues such as their stability during long-term storage and means of efficacious delivery that avoid adverse immunogenic side effects. Controlled chemical modifications, such as substitutions, acylation and PEGylation, have fulfilled some but not all of their promises, while hydrogels and lipid-based formulations could well be developed into generic delivery systems. Strategies to curb the aggregation and misfolding of proteins during storage are likely to benefit from the recent surge of interest in protein fibrillation. This might in turn lead to generally accepted guidelines and tests to avoid unforeseen adverse effects in drug delivery.

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Figure 1: Barriers to biomacromolecular drug transport.
Figure 2: Transport of insulin across biological membranes.
Figure 3: Schematic illustration of protein release from a polymer sensitive to external stimuli, such as change in pH, ionic strength and temperature.
Figure 4: Protein structure and immunogenicity.
Figure 5: The many conformational choices for a polypeptide chain.
Figure 6: The double funnel of folding and aggregation.

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Acknowledgements

D.O. is supported by the Technical Science Research Foundation and the Villum Kann Rasmussen Foundation.

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Correspondence to Daniel E. Otzen.

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DATABASES

Entrez Gene

GLP1

IL-2

OMIM

Alzheimer's disease

Huntington's disease

Parkinson's disease

FURTHER INFORMATION

FoldX — a force field for energy calculations in proteins:

Tango — computer algorithm for prediction of aggregating regions in unfolded polypeptide chains:

Glossary

PARENTERAL

A substance that is introduced into the body anyway except by mouth.

PHARMACOKINETIC

The study of the absorption, distribution, metabolism, excretion and interactions of a drug.

PEGylation

The covalent binding of polyethylene glycol (PEG) to a protein.

EPITOPE

An alternative term for an antigenic determinant. These are particular chemical groups on a molecule that elicit a specific immune response.

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Frokjaer, S., Otzen, D. Protein drug stability: a formulation challenge. Nat Rev Drug Discov 4, 298–306 (2005). https://doi.org/10.1038/nrd1695

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