Multi-Step Preparative LC–MS Workflow for Peptide Purification

The manufacturing of “true” biopharmaceuticals such as antibody drugs can be challenging, and so-called middle-molecule drugs, including peptide therapeutics, have gained significant attention. Peptide therapeutics offer advantages such as low manufacturing costs, easy cellular uptake because of their small molecular weight, and protection from degradation through specific three-dimensional (3D) structures upon entry into the human body (1). This study addresses the critical need for efficient and high-purity peptide purification and leverages an analytical/preparative switching liquid chromatography–mass spectrometry (LC–MS) system to integrate optimized separation conditions, scaling‑up, fractionation, and purity confirmation within a single setup. The workflow significantly improves the efficiency and accuracy of peptide purification while reducing labor and costs. By combining UV and MS triggers for fractionation, the exclusion of impurities is ensured, resulting in highly pure peptide products. This not only meets the rigorous demands of drug development but also has broader implications for biochemical research and production.

Experimental

The LC–MS system can be optimized for both analytical and preparative workflows, as shown in the schematic in Figure 1. In the analytical mode (Figure 1[a]), the system fine-tunes separation conditions, assesses column loadability, and ensures the purity and recovery of components. The preparative mode (Figure 1[b]) is dedicated to the large-scale separation of target compounds. The liquid handler module integrates both paths to enable direct injection from fraction tubes into the analytical flowpath. This integration enables an effective preparative purification workflow (2). The system also incorporates a single quadrupole MS detector, providing mass information of target compounds during optimization, and utilizes a combined UV and MS signal trigger during fractionation (3). This ensures the recovery of compounds with high purity. The preparative flow path is designed to split, directing a portion of the mobile phase, combined with a make‑up solvent, into the MS system, thereby facilitating both fractionation and MS detection.

Figure 1: Schematic of analytical (a) and preparative (b) flow path in a dual-functionality setup. Colored flow path is in operation. Click the figure to enlarge.

Analytical Conditions: System: Nexera Prep + LCMS-2050 (Shimadzu); sample: 2 mg/mL parathormone (PTH) in N-methylpyrrolidone; mobile phase A: 0.1% TFA in water; mobile phase B: 0.1% TFA in acetonitrile; column: 150 mm × 4.6 mm, 5-µm Shim-pack Scepter C18-120 (Shimadzu); injection volume: 10 µL.

HPLC Conditions: Gradient: X%B → Y%B (10 min) → 90%B (10.01–15 min) → X%B (15.01–20 min), X: 10, 15, 20, 25, 30, Y: 30, 35, 40, 45, 50; temperature: ambient; flow rate: 1 mL/min; sample loop size: 500 µL; syringe size: 500 µL; detection: PDA 220 nm (conventional cell).

LC–MS Conditions: Ionization: ESI/APCI (DUIS), positive mode SCAN (m/z 500–2000); nebulizing gas: 2.0 L/min; drying gas: 5.0 L/min; heating gas: 7.0 L/min; DL temp.: 200 °C; desolvation temp.: 250 °C; interface voltage: 0.5 kV.

Preparative Analysis Conditions: System: Nexera Prep + LCMS-2050 (Shimadzu); sample: 10 mg/mL PTH in N-methylpyrrolidone; mobile phase A: 0.1% TFA in water; mobile phase B: 0.1% TFA in acetonitrile; column: 150 mm × 20 mm, 5-µm Shim-pack Scepter C18-120 (Shimadzu); injection volume: 1000 µL.

Preparative HPLC Conditions: Gradient: 25%B → 35%B (10 min) → 90%B (10.01–15 min) → 25%B (15.01–20 min); temperature: ambient; flow rate: 20 mL/min; make-up flow rate: 1.5 mL/min; make-up solvent: 0.1% propionic acid in 90:10 (v/v) water–methanol; sample loop size: 2 mL; syringe size: 5 mL; detection: UV 220 nm (preparative cell).

Discussion

Separation conditions for a crude synthetic sample containing the target synthetic peptide (parathormone [1-34]: PTH) were investigated in analytical scale. Initially, the UV chromatogram (see Figure 2[a]) showed poor separation between PTH and impurities under standard conditions. Increased sample loading exacerbated this issue, making it imperative to refine the separation process for high-purity PTH recovery. To achieve this, a commercial method development software, offering an examination of high performance liquid chromatography (HPLC) separation parameters with 25 gradient profiles and five combinations of initial and final organic solvent concentrations, was used. The optimal separation result for PTH from impurities (indicated by the arrow) in the crude sample was successfully obtained with an initial solvent concentration of 25% and a final concentration of 35% (Figure 2[c]).

Figure 2: Example of chromatograms obtained in the process of method optimization in analytical scale. Click the figure to enlarge.

After optimizing the conditions at the analytical scale (Figure 2[c]), the loading capacity was evaluated with injection volumes of 5, 10, 20, and 50 µL using a synthetic sample with a concentration of 10 mg/mL. Figure 3 shows that the separation efficiency of PTH remained consistent regardless of the increased injection volume, indicating that the separation quality did not diminish. It was therefore decided to scale up and proceed with the preparative analysis with an injection volume of 50 µL.

Figure 3: Chromatograms obtained during the evaluation of loading capacity. Click the figure to enlarge.

PTH was fractionated using both UV and MS triggers. The preparative conditions resulted in the chromatogram illustrated in Figure 4, where the blue area indicates the fractionated interval. The flow rate was scaled up to 20 mL/min, maintaining the same linear velocity as the analytical scale, based on the cross‑sectional area ratio (approximately 20-fold) between the preparative column (20 mm internal diameter [i.d.]) and the analytical column (4.6 mm i.d.). The injection volume was correspondingly increased to 1 mL. This scaling maintained the separation pattern and purity observed in the analytical scale, ensuring effective separation from impurities. The combined use of MS and UV triggers resulted in highly selective fractionation of PTH.

Figure 4: Chromatogram of the preparative analysis of PTH, with blue mark indicating the fractionation band. Click the figure to enlarge.

To confirm the purity of the collected fraction, the fractionated PTH was reinjected into the analytical path, and its chromatogram was compared to that of a synthetic sample prepared at the same theoretical concentration. As can be seen in Figure 5, the chromatogram of the fractionated PTH matches the expected profile, demonstrating that the target PTH was successfully purified.

Conclusion

The preparative purification workflow was effectively and efficiently executed using an analytical/preparative switching LC–MS method. By
using modern method development software, an analytical screening
batch table was automatically generated to assist with the optimization of
HPLC separation conditions. Additionally, highly selective fractionation of
target compounds was achieved using not only UV but also MS data as a trigger for collection. Both the analytical and preparative flow paths ensured an efficient workflow for the preparative purification of peptides, including confirmation of purity.

References

1. Fujisaki, S.; Masuda, Y. Seamless Purification Workflow from Analytical to Preparative in Single LC-MS System, Shimadzu Application Note 01-00650-EN, November 2023.
2. Fujisaki, S.; Masuda, Y. High Purity Preparative Purification Enabled by UV/MS Trigger on LC-MS System, Shimadzu Application Note 01-00651-EN, November 2023.
3. Suzuki, Y.; Masuda, Y. Efficient Preparative Purification Workflow of Synthetic Peptide Using Analytical/Preparative Switching LC-MS System, Shimadzu Application Note 01-00758-EN, June 2024.