Accelerate screening during drug discovery with high-throughput peptide purification

The absence of impurities in peptides, synthesized via SPPS, is vital for reliable assay results. However, especially in drug discovery screenings (number of peptides > 24), potentially low-purity crude peptides are often used because parallel purification with chromatography is not possible. We overcome this bottleneck by parallelization of the orthogonal PurePep® EasyClean (PEC) purification technology to a 96 well-plate format.

Introduction

In peptide drug discovery, testing hundreds of different peptides for validation or structure activity relation (SAR) is essential. Modern parallel solid-phase peptide synthesis (SPPS) supplies the synthetic peptide samples through rapid and automated assembly of multiple peptides, using the Fmoc strategy. However, during synthesis, by-products such as truncations or deletion sequences typically form due to incomplete coupling. These impurities can be removed by preparative reversed-phase high-pressure liquid chromatography (RP-HPLC). However, RP-HPLC is limited in throughput as only one peptide can be purified per column. As a result, current screening is mostly based on crude peptides, which increases the risk of false-positive or false-negative results, hindering the further development of peptide drugs.¹ In this case study, we present a well-plate format together with a vacuum manifold for PEC processing that allows the rapid purification of 96 peptides in a 10 μmol scale simultaneously for highly reliably development work.

Methods

Synthesis

Two sets of 96 peptides were synthesized in parallel in a 10 μmol scale on Ramage-Amide-DP-Resin in 96-well plates (1 x 45 min with a 5-fold excess of aa), followed by routine capping (2 M Ac2O, 2 M pyridine in DMF 1x5 min). This capping enabled selective coupling of the reductively cleavable linker RC+ (4 eq.) to the full-length peptide as the last building block in DMF using DIPEA (6 eq.) and Oxyma (6 eq.) for 2h. Sequences were kindly provided by Steven Cobb, Department of Chemistry, Durham University, UK. TFA cleavage was performed for 2h using TFA/ H2O/EDT/PhSMe/TIS (83:5:5:5:2, 0.5 mL cleavage cocktail per 10 μmol peptide). The crude peptides were collected, precipitated, centrifuged and dried in a 96-well plate. Figure 1 shows the weighted distribution of the amino acids in the 96 sequences.
Figure 1. Weblogo of the used residues for the peptide library. X is used as placeholder for peptides with a length of 8, 9 or 10 AAs.

PEC purification

For parallel purification of the library, the PEC procedure was adapted to the 96 well plate format. Hence, after dissolution of the crude linker-modified peptides, all further steps were performed in a fritted 96-well filter plate with the help of a vacuum manifold, which allows for a seamless workflow from immobilization to final release. The PEC-grade peptides were collected in a deep well plate and directly lyophilized. Only the peptides of the first synthesis plate were purified using the PEC technology and lyophilized. The peptides of the second plate were cleaved, precipitated, dissolved in H2O/ MeCN (7:3) + 0.1% TFA, analyzed, and lyophilized without further purification to determine crude purities and yields.

Analysis

UPLC-UV and mass spectra were recorded with an analytical Acquity H-Class UPLC-ESI-MS system from Waters on a C-18 column (1.7 μm, 2.1 x 500 mm). As the mobile phase, mixtures of water (A) and MeCN (B) with 0.1% TFA were used. Recoveries were calculated using an internal standard with a known extinction coefficient at 210 nm (0.1 mg/mL Fmoc-LGlutamic acid (OtBu) monohydrate solution). The addition of extinction coefficients (γabs) of the amino acids at 214 nm allowed us to calculate the ψabs of the target sample.²
Figure 2. UPLC-UV/VIS-Chromatograms of crude and PEC-grade SCP12 and SCP31 at 210 nm

Results and discussion

The mean purity from 96 peptides could be increased by 28% using PEC technology (Table 1).

Table 1. Mean results for crude and PEC-grade peptides

No truncated sequences could be detected. One peptide showed a purity below 70% (Figure 3). This is a major improvement compared to the crude plate, where only 18 peptides showed purities above 80% (Figure 3). The overall recovery of PEC-grade peptides was 48%.

Figure 2 shows the crude and PEC-purified chromatograms for a more detailed view of two representative peptides.

The example of SCP31 (Figure 2, left) highlights the strength of PEC: the efficient removal of truncated sequences. This feature is essential in unoptimized syntheses, such as in the prepara- tion of peptide libraries. Impurities caused by aspartimide formation were dominant in this set as 76 of 96 peptides have an Asp-Gly-segment in their sequences. An example is shown in Figure 2 (right). On average, impurities caused by aspartimide formation account for 7%.

Even though PEC could not remove these impurities, these results highlight the impressive potential: Right measures, such as using Asp(Dmb/Hmb)Gly building block, would enable a mean purity well above the 90% level for the parallel purification of 96 peptides in a filter plate format.

Figure 3. Crude and PEC-grade purity analysis of 96 peptides

Results at a glance

• access massive parallel purification of peptides with PEC in a 96-well filter plate format
• the one-method-fits-all procedure creates PEC-grade peptides with an average purity of 86%
• improve your assay reliability through absence of peptide-related impurities
• can be automated using Biotage® PeptiPEC-96 High-Throughput Kit, which is based on the PurePep® EasyClean (PEC™) technology from Gyros Protein Technologies

References

1. Currier, J. R.; et al., Clin. Vaccine Immunol. 2008, 15, 267-276.
2. Kuipers, B.J.H.; Gruppen, H., Agr. Food Chem. 2007, 55, 5445-5451.

Acknowledgements

Application note reproduced by kind permission of Gyros Protein Technologies.

Literature Number: AN1006