Peptide drugs have a molecular size between small molecule drugs and large molecule proteins, thus possessing advantages that small molecule drugs and large molecule proteins cannot match, such as higher activity and selectivity than small molecule drugs, lower immunogenicity and production accessibility than large molecule proteins. With these unique pharmacological advantages and the increasingly mature Peptide synthesis technology, peptide drugs have become a hot topic in Drug Development and have broad development prospects.

Like small molecules in gift based drugs, impurities (related substances) in peptide drugs are also important factors affecting drug safety. How to characterize and control these impurities is one of the important tasks in the development of peptide drugs. Among numerous impurity characterization tools, LC-MS is widely recognized as the most effective method for molecular weight analysis and has significant advantages in impurity research of peptide drugs.

However, based on the special properties of peptide drugs, in order to achieve ideal separation effects, most of the additives used in peptide related substance detection are incompatible with MS. Among them, trifluoroacetic acid is the most commonly used additive because it is a strong hydrophobic acid and also a very effective ion pairing agent, which can minimize secondary chromatographic interactions, obtain excellent peak shapes and separations, while trifluoroacetic acid has a significant ion inhibitory effect, The incompatibility of gift mass spectrometry increases the difficulty of impurity research on peptides. So, how to conduct impurity research on peptide drugs, and what are the methods to solve the difficulties?

1. Direct substitution method

The most direct and simple method is to replace trifluoroacetic acid with a mass spectrometry compatible acid or salt. The replaceable acids (salts) include formic acid (ammonium formate), acetic acid (ammonium acetate), difluoroacetic acid, trifluoroacetic acid, etc.

1) Formic acid, acetic acid, and their volatile buffer salts

The most common method is to replace trifluoroacetic acid with formic acid.

For example, in the impurity spectrum study of a recombinant GLP-1 analog, formic acid was chosen instead of trifluoroacetic acid due to its damage to the instrument, resulting in a better mass spectrometry response (see Figure 1). Combined with MALDI high-resolution mass spectrometry and amino acid sequencing results, the impurities were analyzed and the possible amino acid sequence was confirmed (Figure 2,4-dihydroxy-6-methylnicotinic acid ethyl ester).

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In addition to acids, corresponding volatile salts can also be used.

For example, in the study of the degradation mechanism of antimicrobial peptide (cbf-14), ammonium formate buffer (pH 3.0) was used as the mobile phase to determine the molecular weight information of the degraded impurities. Based on the molecular weight, their structure was preliminarily inferred (see Figure 3), and the sources and formation mechanisms of these impurities were elucidated, providing reference for subsequent quality research of cbf-14 and other peptide products.

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2,4-dihydroxy-6-methylnicotinic acid ethyl ester) difluoroacetic acid

Replacing trifluoroacetic acid with formic acid, acetic acid, and their corresponding volatile salts is the most common and effective method. However, in practical applications, it can be found that many peptides have poor peak shapes in mobile phases such as formic acid or ammonium formate, and their separation is severely compromised. Moreover, the retention time and peak sequence of components that are sensitive to pH may undergo unpredictable changes, affecting impurity identification. At this point, it can be considered to replace trifluoroacetic acid with difluoroacetic acid (see Figure 4).

Image.png difluoroacetic acid has stronger acid strength than formic acid, which can ensure that the peak sequence does not change to the maximum extent, and at the same time, it can obtain better chromatographic peak type and resolution. In addition, because of its low hydrophobicity, it is conducive to influencing the surface tension of electric spray droplets, which has no obvious inhibitory effect on the mass spectrum signal, and can obtain a strong mass spectrum response while ensuring the peak capacity as far as possible. Scholars have used eight different peptide components, with 0.1% formic acid, 0.1% difluoroacetic acid, and 0.1% trifluoroacetic acid as mobile phases, to compare their mass spectrometry responses. It has been confirmed that the mass spectrometry response of difluoroacetic acid is significantly improved compared to trifluoroacetic acid (see Figure 5). image.png

Unfortunately, there are currently no chromatographic or mass spectrometry grade products available for this type of reagent. The purchased products typically contain high concentrations of sodium and potassium, which may compromise the interpretability of the mass spectrometry. Therefore, it is necessary to carefully evaluate their purity before use and, if necessary, distill them before use.

Two dimensional chromatography of 2,4-dihydroxy-6-methylnicotinic acid ethyl ester

In addition to the direct substitution method mentioned above, two-dimensional chromatography can also be attempted. Two dimensional chromatography can preserve the relevant substances of peptide drugs in their original liquid phase conditions, while avoiding the problem of poor mass spectrometry compatibility.

Two dimensional chromatography was originally used to solve the problem of difficult separation of multi-component samples in one-dimensional chromatography. In recent years, it has gradually been used to solve the problem of impurity research that is difficult to be compatible with mass spectrometry, and impurity research of peptide drugs is one of them. Usually, first dimensional chromatography uses relevant substance chromatography conditions, and then selects the impurity components to be studied and cuts them into two-dimensional chromatography. Two dimensional chromatography is a chromatographic condition compatible with gift mass spectrometry. Two dimensional chromatography usually uses simple universal large gradients or isometrics, and sends the tested components to the mass spectrometer detector through two-dimensional chromatography to determine their molecular weight.

For example, for a certain peptide based original preparation CBTC, according to its import registration standards, the mobile phase for the relevant substance method is phosphate buffer solution. Due to the fact that phosphate is a non-volatile salt, gift mass spectrometry is difficult to be compatible. In its impurity research, the R&D personnel used two-dimensional liquid chromatography technology to achieve rapid desalination treatment of the primary separation components, and then performed mass spectrometry analysis. Finally, the impurities mentioned in the standard were qualitatively analyzed and their structures were inferred. The results are shown in the table below (see Table 1).

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3 and a half preparations

If suitable alternative additives cannot be found and two-dimensional chromatography is not available, a slightly more complex method can be attempted, which is to extract the test component from the liquid phase, enrich it, and then determine its molecular weight through LC-MS. This method is suitable for determining impurities with relatively high content and for separating adjacent impurity baselines. In a certain project, we need to determine the molecular weight of a peptide acid degradation impurity. The additive used in the material method is trifluoroacetic acid. At that time, replacing trifluoroacetic acid with formic acid resulted in a significant decrease in resolution and a change in the order of impurity peaks, making it impossible to distinguish which impurity was an acid degradation impurity. There was also no two-dimensional chromatography available at that time. Therefore, we first used high concentration analytical liquid phase injection, and then received the acid degradation impurity fraction, which was injected into the mass spectrometer in large volume to obtain the molecular weight of the impurity.

Summary

The study of impurities in peptide drugs is an important component of their quality control. When the developed substance detection methods are incompatible with mass spectrometry, we may try the above methods. No matter which peptide it is, we believe there will always be a method that suits you.

reference

[1] Liang Tao, Study on Impurities of GLP-1 Analogues (Research Paper)

[2,4-dihydroxy-6-methylnicotinic acid ethyl ester] Yitong Huo，Characterization of structurally related peptide impurities using HPLC‑QTOF‑MS/MS: application to Cbf‑14, a novel antimicrobial peptide，Analytical and Bioanalytical Chemistry (2,4-dihydroxy-6-methylnicotinic acid ethyl ester02,4-dihydroxy-6-methylnicotinic acid ethyl ester2,4-dihydroxy-6-methylnicotinic acid ethyl ester) 414:6485–6495.

[3] Jennifer M. Nguyen，High sensitivity LC-MS profiling of antibody-drug conjugates with difluoroacetic acid ion pairing，Taylor &Francis ,2,4-dihydroxy-6-methylnicotinic acid ethyl ester019, VOL. 11, NO. 8, 1358–1366.

[4]Jennifer Nguyen，Improving LC-MS Separations of Peptide with Difluoroacetic Acid Ion Pairing, Waters Corporation ,Milford MA USA.

[5] Wang Shushan, Application of Two Dimensional Liquid Chromatography Mass Spectrometry in the Study of Peptide Drug Related Substances (Research Paper)