Analysis Methods for Peptide-Related Impurities in Peptide Drugs


Peptide drugs, as a type of pharmaceutical with molecular weight between small molecule chemical drugs and biopolymers, provide more options for medicinal chemists and have become a hot research topic in recent years. This article discusses the development of analysis methods for peptide-related impurities in peptide drugs.

Features of peptides

Peptide molecules are compounds formed by the connection of multiple amino acids through peptide bonds (amide bonds), which have the following characteristics:

(1) Peptide drugs have a larger molecular weight. For example, the molecular weight of the peptide star drug somatostatin (semaglutide) is 4114 Da, which is lower than that of biopolymer drugs but several times that of small molecule drugs. The increase in molecular weight leads to some important changes in chromatographic behavior, such as increased sensitivity to solvent strength, even slight changes in B% can cause rapid changes in retention time. Furthermore, diffusion in the mobile phase is slower, leading to a decrease in column efficiency and an increase in peak width.

(2)Peptide molecules have a tertiary structure. Peptide chains form secondary and tertiary structures through α helices and β folds, with their unique three-dimensional structure. Due to the molecular volume and three-dimensional structure, not all amino acids can simultaneously contact a fixed phase, only residues on the surface can interact with each other.

(3)Peptide molecules are connected through amide bonds, which have partial double-bond properties. The isomers of cis-trans exchange may cause peak distortion.

(4)Peptide molecules are amphipathic, with acidic carboxyl groups and basic amino groups, and have a specific isoelectric point.

(5)The mass spectrometry of peptide drugs is extremely complex. There are various types of process impurities and degradation impurities in synthetic peptide molecules, especially the structure and properties of racemic impurities are very similar to the main component, and their chromatographic behavior is also similar, which greatly increases the difficulty of separation and analysis.

Analysis methods for peptide-related impurities

Regarding the characteristics of peptide molecules, there are some special considerations in the development of relevant analytical methods. The commonly used method for substance analysis of peptide drugs is high-performance liquid chromatography, which can use reverse-phase chromatography, ion exchange chromatography, and size exclusion chromatography.

2.1 Reverse-phase chromatography (RP)

RP chromatographic column selection

When selecting a chromatographic column for peptide molecules with the characteristic of slow diffusion in the mobile phase, special consideration needs to be taken. 

Silica gel carriers have high mechanical strength, mature and stable technology, and high selectivity. Additionally, due to the slow diffusion of peptides, solute molecules only enter the silica gel layer on the surface of porous silica gel particles, resulting in a shorter path in the particles and reducing internal diffusion, which lead to smaller band broadening. Small particle size columns also help to solve band broadening and reduced column efficiency, although decreasing the particle size increases the column pressure significantly, the benefits of reducing the particle size for peptide molecules outweigh those for small molecule compounds. Similarly, selecting a narrow-bore column can also reduce band broadening when longitudinal diffusion occurs in the chromatographic column.

Common chromatographic columns with pore sizes of 10 nm and 12 nm can meet the analysis needs of most peptides, allowing peptide molecules to enter the interior pores of the particles.

Alkyl-bonded phases, particularly C18, are currently the most commonly used types of stationary phases and are also used for the analysis of peptide drugs. However, various types of phenyl columns can provide different selectivities, particularly for peptides containing aromatic residue structures such as tyrosine. Additionally, chromatographic columns with different surface modifications, end-capped/non-end-capped, and embedded functional groups can also offer different selectivities.

Currently, there are many manufacturers on the market offering different processing techniques and types of reverse-phase chromatography columns with a wide range of product categories. During the method exploration stage, it is necessary to try chromatographic columns with different selectivities, and some characterization methods can be used as a reference, such as the Snyder/Dolan method utilizing H, S, A, B, and C parameters or searching for chromatographic columns with significant selectivity differences through the USP or supplier websites. Furthermore, automated and intelligent software development methods can greatly save development time and are more suitable for peptide-related substance method development.

RP mobile phase selection

Trifluoroacetic acid is a commonly used buffer solution in peptide analysis, with good buffering capacity and the ability to form ion pairs with peptide amino acids, increasing retention. The commonly used concentration is 0.1% (V/V).

Phosphate is the most commonly used reverse-phase chromatography buffer salt, which can be configured to different pH values. High concentrations of phosphate, such as 50mmol/L, can provide higher ion strength and reduce peak broadening caused by the mutual exclusion of peptide molecules. Ion exchangers such as hexafluorophosphate and sodium perchlorate can not only increase ion strength and change selectivity but also have a higher solubility in acetonitrile water.

As a zwitterionic substance, the change in the flow phase pH has a profound impact on the separation of peptides, so pH screening is very important. It should be noted that peptide molecules have the lowest solubility near the isoelectric point, and the pH of the isoelectric point should be avoided by at least ±1.0 units.

Acetonitrile, as an organic phase (B%), has obvious advantages such as low UV absorption and low viscosity, and its peak shape is better than that of alcohol solvents. Alcohol solvents such as methanol and isopropanol can be added in small amounts to provide different selectivity.

As mentioned earlier, peptides are sensitive to the organic phase B%, and slight changes can significantly affect retention time. Therefore, when setting a gradient program, B% should be slowly increased to achieve optimal separation.

Other RP chromatographic parameters

High column temperature reduces the viscosity of the mobile phase, increases molecular diffusion rate, improves peak shape, changes selectivity, and increases separation efficiency. It can also solve the problem of peak shape differences of cis/trans isomers, which is a characteristic of peptide analysis. It should be noted that if a high column temperature is used, the mobile phase before the column needs to be preheated. However, high column temperature also has some disadvantages, such as sample degradation and shortened column life.

Peptides lack conjugated structures. Generally, the absorption wavelength range of 210nm~220nm at the C-terminus is selected, that is, the absorption of the amide bond. Although some peptides contain aromatic amino acids such as phenylalanine, the absorption intensity is significantly lower than that of the C-terminus.

Ensure that the pH and solvent strength of the dilution solution is the same or similar to the initial mobile phase to avoid solvent effects.

Ultra-high-performance liquid chromatography is the preferred method for analyzing peptide drugs, and some high-performance liquid chromatography methods may also meet performance requirements.

2.2 Ion exchange chromatography (IEC)

Due to the complex impurity profile of peptide drugs, reverse-phase chromatography alone often cannot meet the requirements for impurity analysis and research. Ion exchange chromatography provides different separation selectivity and can be an effective supplement to reverse-phase chromatography.

IEC mobile phase selection

Buffer is used to control the pH of the mobile phase, which can change the ionization degree of the sample and affect retention and selectivity. For peptide drugs, the mobile phase pH should generally be controlled within ±1 pH unit of the isoelectric point. For cation exchange, the mobile phase pH should be at least 1 pH unit lower than the isoelectric point; for anion exchange, the mobile phase pH should be at least 1 pH unit higher than the isoelectric point. Common types of buffers used in reverse-phase chromatography can be used, but phosphate is still preferred.

Counter ions play an important role in IEC, and their types and concentrations are used to regulate retention and selectivity. Commonly used counter ions include Cl and other halide anions, ClO4, and SO42- as anions, and Na+ and other alkali metal ions as cations. If the software for mobile phase selection is available, time and effort can be saved.

Acetonitrile is still the preferred choice as the organic solvent, which affects peak shape and selectivity.

IEC chromatographic column selection

The carrier can be silica gel, but pH tolerance should be ensured. Many column manufacturers also provide polymer-carriers. The specifications can refer to the recommendations for reverse-phase chromatography.

IEC stationary phase generally has four types: strong anion exchange -N(CH3)3+; strong cation exchange -SO3; weak anion exchange -NH2; weak cation exchange -COOH.

Other chromatography parameters can refer to reverse-phase chromatography.

2.3 Size exclusion chromatography (SEC)

This method is commonly used for the separation of polymeric impurities in peptide drugs based on the difference in volume between polymer molecules and drug molecules.

SEC chromatographic column selection

Silica gel is used as the filling material with silane alcohol (diol-based column) modification to reduce interaction between silica gel and peptide molecules. Generally, 12nm pore-size SEC columns offered by manufacturers can meet the separation needs of peptide polymers. Chromatography column specifications are not as diverse as reverse-phase columns and smaller particle sizes can be chosen.

SEC mobile phase selection

Buffer solution: commonly used trifluoroacetic acid or phosphate buffer, pH and ion strength can be regulated to reduce ionization and higher concentrations can be used as needed;

Organic solvent: acetonitrile or methanol to reduce hydrophobicity;

Flow rate: commonly used at 1ml/min, but lower flow rates can be used to increase column efficiency due to the low diffusion coefficient of peptide molecules.

Summary of the development of analytical methods for peptide drugs

The development of analytical methods for peptide drugs is difficult and time-consuming. As stated in the guiding principles, various separation modes and parameters need to be explored, such as chromatography columns with different stationary phases and different flow pHs. As the impurity research progresses, each parameter also needs to be continuously optimized. Based on these characteristics, the development of analytical methods for peptide drugs is a systematic and technically challenging task, requiring method developers to continuously learn and deepen their understanding of molecular properties and separation principles through practice. Finally, they must maintain sufficient patience and confidence.