Using Microwave-Assisted Amino Acid Hydrlysis of Liraglutide

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Introduction

Peptide therapeutics are increasingly being used for the treatment of diseases. New generations are being discovered at a rapid pace. These drugs often mimic the function of native proteins and thus are generally regarded as safe and well tolerated because they can be broken down by the human body.1 Peptides for this purpose can be synthesized by bacterial, insect, or mammalian cells and then purified. Alternatively, they can be prepared chemically via solid-phase peptide synthesis (SPPS). Ideally, the synthesis should be accomplished in a rapid and controlled manner at a multikilogram scale. CEM’s Liberty PRO™ automated peptide synthesizer allows for the high-throughput microwave-assisted SPPS of functional peptides at the production scale.

Unsurprisingly, peptide therapeutics are held to high regulatory standards. Whether biologically or synthetically produced, the amino acid composition and content of these peptides can serve as an important quality control check. Amino acid analysis can be used to verify a successful synthesis, identify isolated peptides, detect atypical amino acid residues, and support structural analysis. Typically, amino acid analysis of these macromolecules is accomplished via acidic or alkaline hydrolysis in a sealed vessel using conventional heating for up to 24 hrs. Microwave power can be used to speed up this reaction, often requiring only 15 minutes to complete the hydrolysis.2

In this work, the CEM Discover PrepTM microwave system was used to confirm the amino acid content of Liraglutide, a peptide synthesized using the Liberty PRO. In order to hydrolyze amide bonds and liberate the individual amino acid residues, acidic and alkaline hydrolysis of the peptide was performed using the Discover Prep. Alkaline hydrolysis is generally applied for the quantification of tryptophan, as its indole side chain is destroyed under acidic conditions. Following Discover Prep amino acid hydrolysis, the Waters AccQ-Tag™ Ultra Derivatization kit was used in conjunction with a Waters Acquity H-Class UPLC system and a PDA detector to measure the amino acid content of the hydrolysates.3 The Discover Prep successfully prepared the peptide for LC-PDA analysis for amino acid quantification. Upon comparison to the theoretical yield of each amino acid residue present in the synthesized peptide, the microwaveassisted hydrolysis resulted in comparable recoveries in a very rapid and efficient manner.