Ever wonder what the inside of the venomous Gila Monster’s mouth was like? Yeah, we did too.

Exenatide, a synthetic version of excendin-4, is a hormone found in the saliva of the Gila Monster. A glucagon-like peptide-1 agonist, Exenatide was approved in April 2005 for the treatment of diabetes mellitus type 2. Exenatide is a 39 amino acid peptide, its chemical structure is represented in Figure 1.

For a purification development experiment, a crude Exenatide was used as the starting material. The purification of complex crude synthetic peptide such as Exenatide often employs a multi-step chromatographic purification process.

Since Exenatide is relatively non-polar, the Luna C8 column was an appropriate stationary phase choice. Acetonitrile was initially chosen for the strong solvent eluent component because the final product was a lyophilized solid. And based on Table 1, the pH could be an effective tool for altering the chromatographic selectivity between Exenatide and its related impurities.

Table 1: Amino Acids Distribution in Exenatide

The initial screening of possible PREP conditions evaluated 4 different aqueous eluent components covering a pH range of 2-8. The isolated material from the optimized PREP screening experiments were evaluated with analytical HPLC methodology on Kinetex EVO.

The first assessment of Exenatide was to determine if a single step purification process was feasible, which showed that it was not in fact achievable, as it couldn’t meet the purity requirement of 98.5% with a suitable yield, meaning that a multi-step purification process was required. Since the desired final product was an acetate salt, the use of acetate in the final step had significant processing advantages.

The initial crude material had an estimated purity of 66% using Kinetex EVO as the analytical column (Figure 2). First, a simple loading step was conducted and it was determined that the crude material could be loaded at 1% of the column bed mass (Figure 3). The main component for the first step was collected as a series of fractions. These fractions were evaluated by analytical HPLC and the appropriate fractions were pooled together. The obtained material had an estimated purity of 96.5%.

Figure 2. Initial Crude Purity on Kinetex EVO

Figure 3a. Multi-Step Purification Process of Crude Exenatide

Next, the pooled material was processed with the second step acetate methodology. Again, the main component was collected as a series of fractions. These fractions were evaluated by analytical HPLC and the appropriate fractions were pooled together. The second step pooled material was dried by lyophilization and evaluated by analytical HPLC. The purity of the final material was 99.3%

Figure 3b. Multi-Step Purification Process of Crude Exenatide

The column used for large scale chromatography can be a significant part of the cost for a large scale purification process. This cost is not solely based on the actual stationary phase inside the column, but needs to include the time and hardware needed to pack, unpack, and switch between columns. A considerable amount of time and expense can be saved by using the same column packed with a single stationary phase for each step in a multi-step purification.

This work used Exenatide to demonstrate the development of a 2-step purification methodology for a synthetic peptide. To sum it up, the initial crude mixture was upgraded to 96.5% purity after the first step. The second step polished the final material to a purity of 99.3%. Luna 10 µm-PREP C8(3) was used as the single stationary phase for the purification of Exenatide. This phase as introduced by Phenomenex in 2013 and is available in large quantity for packing in dynamic axial compression columns (DAC).

This heavy, slow-moving lizard can go from deadly to life saving with the help of this simplified peptide purification. What can’t chromatography do!

If Gila Monsters saliva can help treat diabetes, we can only wonder what a nerdbird’s could do…

Look for the full length article on the purification of Exenatide in our Preparative Newsletter- Purify