Here we explore the essential methods and processes for peptide purification, covering:

• Removal of cytotoxic reagents
• Purification with RP-HPLC
• Alternatives and challenges in purification
• Scalability and lyophilization
• Multicolumn countercurrent solvent gradient purification (MCSGP)

Peptide properties are determined by their amino acid composition and sequence. After solid-phase peptide synthesis (SPPS), the cleavage process produces a crude product that contains the desired peptide alongside various impurities, including deletion peptides, truncated peptides, incompletely deprotected peptides, modified peptides, residual reagents, and by-products from cleaved protecting groups. These contaminants must be removed to isolate the final product.

How are peptides purified?

Removal of cytotoxic reagents

During synthesis, cytotoxic reagents (except trifluoroacetic acid, TFA) are removed in the washing steps before final cleavage or during the purification process. This ensures that harmful reagents do not remain in the final peptide.

Purification with RP-HPLC

The standard method for peptide purification is reversed-phase high-performance liquid chromatography (RP-HPLC), using C18-modified silica as the stationary phase and UV detection. In RP-HPLC, the retention of peptides and impurities depends on their hydrophobicity. The purification process begins with polar contaminants being eluted first with aqueous 0.1% TFA. The polarity of the eluent is then gradually reduced by increasing the proportion of acetonitrile (also containing 0.1% TFA). This separates the target peptide from other impurities, which are monitored at 210–220 nm. Fractions containing sufficiently pure target peptide, as determined by analytical HPLC, are pooled and freeze-dried or subjected to additional purification if necessary.

Alternatives and challenges in purification

If standard RP-HPLC methods do not yield sufficient purity, alternate RP-HPLC methods with different solvents or buffers may be used. In some cases, alternative column packing materials might be necessary, especially for less polar peptides. For peptides synthesized in solution, countercurrent distribution (CCD)is often used for purification (see figure 12).

Scalability and lyophilization

Peptide purification can become a bottleneck in large-scale production, especially if the raw peptide quality is poor. To expedite the process, methods like CCD may be more suitable for large-scale synthesis. Once purified, the peptide is obtained as a solution, which is then lyophilized to remove the solvent and produce a fine white powder. While it is not possible to completely remove all water, this does not impact the final product.

Peptides with basic groups

Peptides containing basic groups (such as N-terminus, Arg, Lys, or His) bind to TFA (in Fmoc-SPPS peptides) or acetic acid to form salts. These salts cannot be removed during purification but do not affect the quality of the final peptide.

Multicolumn countercurrent solvent gradient purification (MCSGP)

The Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) technology marks a significant advancement in downstream processing for peptide and oligonucleotide manufacturing. Compared to traditional single-column batch purification, MCSGP reduces solvent consumption by over 30%, promoting greater sustainability. This process also offers higher capacity, achieving target product purity with yields that are typically 10% higher.

The automated system operates 24/7, enabling further reductions in cycle time. In 2021, Bachem acquired two process-scale MCSGP systems for High-Performance Liquid Chromatography (HPLC) and Anion Exchange Chromatography (AEX) purification, featuring 20 cm and 30 cm diameter columns. These systems are fully GMP-compliant, and large-scale purifications have been successfully conducted.

Figure 13: Process scheme of MCSGP (P = pure product; W = weakly adsorbing impurities; S = strongly adsorbing impurities; W/P = weakly adsorbing impurities overlapping with the product; P/S = product is overlapping with strongly adsorbing impurities). The numbers indicate theoretical steps in the process. The right part of the figure represents the UV profile of the feed injected into MCSGP.

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