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December 26, 2016

Optimizing Peptide Synthesis in Demanding Peptide Drug Development

  • There has been a recent revival in interest in peptide therapeutic development, with five of the top-selling drugs being peptides or proteins (Enbrel, Remicade, Humira, Avastin, MabThera) and many more currently in clinical trials [1]. Among their many advantages, peptide therapies provide high levels of receptor recognition that reduce toxicity profiles and drug-drug interaction potential [1]. However, in some cases these therapies have disadvantages, such as low membrane permeability, easy degradation by proteases, and unspecific proteolysis that limit duration of action and oral bioavailability. Researchers are working on a variety of solutions to overcome these roadblocks, including structural modifications such as D-amino acids substitutions, modifying peptide terminals, covalent attachment of fatty acids or PEG, and peptide cyclization.

  • Case Study 1: Heat-Assisted Synthesis of Linear and Cyclic Peptides on Prelude X

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    Figure 1

    The renewed interest in peptide therapeutics in industry and academia has increased the demand for rapid peptide development, which has been met by automated solid phase peptide synthesis (SPPS). The synthesis of complex peptides can be challenging due to steric and conformational factors, and this has driven the development of synthesizers with enabling technologies such as rapid heating methods. Prelude® X automated peptide synthesizer, with six reaction vessels and induction heating on each vessel enables independent, simultaneous and rapid heating of multiple reactions with increased efficiency. In addition, Intellisynth® real-time UV monitoring enables the amino acid deprotection solution to be monitored while it is mixing, making this system suitable for method optimization.

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    Table 1

    To demonstrate the efficacy of the novel induction heating technology available on Prelude X, the difficult peptide sequences (Figure 1) Jung-Redemann (JR) 10-mer [2]and Aib-Enkephalin (Leu-enkephalin with Aib replacing both glycines) were synthesized with heating during every cycle. The effects of heating on peptide cyclization were also assessed by synthesizing the potent melanocortin receptor agonist Melanotan II (MT-II) [3]. Heat-assisted synthesis has been shown to speed up production of high-purity linear peptides and also cyclic peptides. Therefore, multiple temperature profiles were tested in parallel for the optimization of the cyclization reaction (Table 1).

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    Figure 2


    Difficult peptide sequences, JR10-mer and Aib-enkephalin, along with a cyclic peptide, MT-II, were successfully synthesized with short synthesis times using Prelude X. Increasing the temperature up to 90°C improved the crude purities of the linear peptides (Figure 2) from 15% to 66%. By using induction heating, at 85°C for 5 min, the cyclization time of MT-II was significantly reduced and resulted in an increased purity of 73%. In conclusion, multivariable conditions were tested in parallel for the optimization of JR10-mer, Aib-enkephalin and MT-II syntheses and increased temperatures provided the best results.

  • Case Study 2: Simultaneous Screening of Multiple Synthesis Methods on Symphony X

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    Figure 3

    The ability to rapidly synthesize peptide libraries for biological evaluation is another important aspect of peptide drug development and one for which the Symphony® X peptide synthesizer is well suited. In addition, this instrument also offers independent method optimization and the greatest flexibility of any peptide synthesizer on the market, making it the industry standard for high-throughput peptide process development.

    To assist in process development and optimization, the capabilities of Symphony X were used for simultaneous synthetic method screens. Optimization of the synthesis of the linear 25-mer ziconotide (Prialt®) (Figure 3) was used as a model and the following parameters were investigated to determine the conditions that would result in the best purity: variations in resin, coupling reagents, and reaction times. This ω-conotoxin contains 25 amino acid residues, including six cysteines that, in ziconotide’s native conformation, form three specific disulfide bonds. [5]

  • Table 2

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    Ziconotide is a synthetically challenging sequence due in part to the presence of multiple cysteines. The methods described in Table 2 were used to successfully synthesize this peptide on Symphony X using independent methods for the screening of 12 different conditions simultaneously with crude purities ranging from 28.5% to 44%. The crude purity of ziconotide increased with increasing coupling time, with 

  • Conclusions

    Automated solid phase peptide synthesis provides a wide range of options for method optimization as well as synthesis of peptide libraries for peptide drug development. Prelude X provides the researcher with a range of capabilities, including induction heating and real-time UV monitoring for the rapid synthesis of both simple and complex peptides with high crude purities. Symphony X, with 24 reaction vessels, is the ideal optimizer for the simultaneous screening of different methods.

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