Analytical Challenges and Emerging Strategies for GLP-1 Analysis

Nitish Sharma of the Department of Pharmaceutical Analysis at the National Institute of Pharmaceutical Education and Research, Ahmedabad (NIPER-A), discusses the challenging aspects of detecting glucagon-like peptide-1 (GLP-1) impurities using high performance liquid chromatography (HPLC)/ultrahigh-performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (HRMS). Unlike small molecules, peptides have strong hydrophilic and hydrophobic interactions that complicate separation. HRMS struggles with isomers, ion suppression, and complex fragmentation. New approaches like two-dimensional liquid chromatography–HRMS (2D-LC–HRMS), electron transfer dissociation (ETD), and hydrogen-deuterium exchange mass spectrometry (HDX-MS) improve impurity detection and structure confirmation. Regulatory guidelines require low-level profiling and orthogonal methods to confirm impurity sameness. Size-exclusion chromatography (SEC) helps analyze aggregates, while optimized sample preparation and appropriate diluents reduce degradation and improve reliability.

What are the major technical and analytical challenges associated with the separation of glucagon-like peptide-1 (GLP-1) impurities using high performance liquid chromatography/ultrahigh-pressure liquid chromatography (HPLC/UHPLC)?

In reversed-phase liquid chromatography (RPLC), small molecules are retained primarily by a partitioning mechanism, where analytes distribute between the stationary and mobile phases. Adsorption to residual silanol groups on the silica surface is a minor factor.

In contrast, peptides are separated through an entirely different process. Due to their large size, only a portion of a peptide molecule interacts with the stationary phase. However, this hydrophobic interaction is so strong that the peptide remains adsorbed until a specific concentration of the organic phase is reached. At this point, the entire molecule desorbs, resulting in a single adsorption/desorption process that produces sharp peaks. This distinct mechanism sets peptide separation apart from that of small molecules. In this way, most of the peptide impurities elute either fronting or tailing the active peptide peak.

Regulatory bodies (for example, the Food and Drug Administration [USFDA]) require the identification and quantification of impurities at very low levels (0.5% for known impurities and 0.10% for unknown ones). Achieving this level of sensitivity and selectivity is quite difficult, especially when impurities coelute with the main peak or other matrix components.

Consistency and reproducibility are major challenges, as even minor variations in mobile phase preparation, column batches, and system cleanliness can impact retention times and peak areas. This complicates method validation and routine quality control.

What are the limitations of current high-resolution mass spectrometry (HRMS) methods for characterizing GLP-1 impurities, and what emerging strategies are being developed to overcome these challenges?

Current HRMS methods, while powerful, face several key challenges when analyzing GLP-1 impurities.

i.) Difficulty with isomeric and isobaric impurities: GLP-1 peptides are prone to forming isomeric compounds that have the same molecular formula and mass but different structures. HRMS is excellent at determining the exact mass of a molecule, but it can’t distinguish between these similar mass compounds on its own. For example, racemization (D-amino acid impurities) results in impurities with the same mass as the parent drug. These are quite difficult to differentiate with mass spectrometry alone.

ii.) Challenges with low-level impurities: Regulatory bodies require the detection and quantification of impurities at very low levels (for example, below 0.10%). While HRMS has high sensitivity, coeluting peaks from the main drug or other impurities can mask the signal of low-level impurities, making them difficult to characterize accurately if not properly separated.

iii.) Ion suppression: The mobile phase additives used for chromatographic separation for peptides, such as trifluoroacetic acid (TFA), can significantly suppress the ionization of peptide molecules in the mass spectrometer. This leads to reduced signal intensity and compromised sensitivity.

iv.) Complex fragmentation patterns: When analyzing large peptides with tandem mass spectrometry (MS/MS), the fragmentation patterns can be highly complex and quite difficult to interpret. Identifying the exact location of a minor change, such as a single amino acid substitution or a post-translational modification, requires sophisticated data analysis software and expertise.

v.) When using HRMS to analyze impurities in peptides (not actually related to GLP-1 peptides), the presence of disulfide bridges creates a unique set of challenges. One of the main issues is that these impurities often exist at very low concentrations. This makes it difficult to effectively reduce the disulfide bonds, which are crucial for getting useful fragmentation patterns during HRMS/MS analysis. Without proper fragmentation, it’s nearly impossible to determine the structure of the impurity, even with the high sensitivity of HRMS.

To address these limitations, a number of cutting-edge strategies are being developed and implemented.

Two-dimensional liquid chromatography high-resolution mass spectrometry (2D-LC–HRMS): This technique combines two different chromatographic separation mechanisms in a single analysis. The first dimension (for example, reversed-phase chromatography) separates the bulk of the sample, while the second dimension (hydrophilic interaction liquid chromatography, HILIC, or a different reversed-phase method) is used to re-separate a narrow section of the chromatogram containing the coeluting impurities. This provides superior resolving power, allowing for the separation of very similar impurities that would be impossible to resolve with a single column.

Advanced fragmentation techniques: In addition to standard collision-induced dissociation (CID), newer fragmentation methods like electron transfer dissociation (ETD) are being used. Conventional fragmentation techniques like CID or higher energy collisional dissociation (HCD) often yield incomplete backbone coverage leading to poor localization of subtle post-translational modifications. Advanced electron-based fragmentation methods such as ETD improve sequence coverage and facilitate precise localization of post-translational modifications. This is particularly useful for identifying complex isoforms and post-translational modifications.

Hydrogen-deuterium exchange (HDX) mass spectrometry: Thisis a powerful biophysical technique that provides information on the higher-order structure of a molecule, not just its mass. It works by monitoring the exchange of labile hydrogen atoms on the peptide backbone with deuterium atoms from a deuterated solvent. The rate and extent of this exchange are dependent on the molecule’s solvent accessibility and hydrogen-bonding patterns.

How can one effectively prove the similarity between an active pharmaceutical ingredient (API) and drug product impurity profiling, especially if the API is outsourced?

A comprehensive impurity profile for the outsourced API has to be established using validated analytical methods, such as HPLC coupled with MS, to identify and quantify all impurities above the specified threshold. This is followed by a similar impurity analysis for the final drug product as well, including samples from forced degradation and long-term stability studies. The impurity profile for these two has to be compared. Through the use of advanced chromatographic and mass spectrometric techniques, manufacturers must ensure that the impurities in the final drug product are either already present in the API at acceptable levels or are well characterized.

Evaluating the potential for impurities to be introduced or to increase during the formulation process should be recommended. This includes looking at interactions between the API and excipients, as well as potential contamination from the manufacturing environment. Any new impurities identified as degradation products could potentially be due to the interaction with the excipients. If a manufacturer changes the analytical method for a drug product analysis, it is recommended to re-establish the drug substance’s impurity profile using the newly developed drug product method or else establish method equivalency between the drug substance or drug product analytical method. This ensures a strong correlation between the analytical methods for both the drug substance and the drug product’s related substances.

What is the best strategy for meeting the FDA’s requirement to prove impurity sameness using at least two orthogonal analytical methods?

The best strategy for meeting the regulatory requirement to prove impurity sameness using at least two orthogonal analytical methods is to leverage techniques that rely on fundamentally different principles. This approach provides a robust and comprehensive impurity profile, as the weaknesses of one method can be compensated for by the strengths of another. A one-size-fits-all approach is rarely sufficient, as the best combination of methods depends on the specific drug substance and its known or potential impurities.A primary method for routine impurity analysis for peptide products can be developed using HPLC–UV/diode array detection (DAD). Reverse-phase chromatography stationary phases, such as C4, C8, and C18, may be the primary choice for impurity separation. For an orthogonal approach, HILIC chromatography or anion exchange/cation exchange stationary phases can be an alternate choice to separate impurities. By coupling LC–HRMS, we can generate a powerful data set where an impurity’s identity is confirmed by both its chromatographic behavior and its precise mass.

What are the key factors to consider in developing a robust size-exclusion chromatography (SEC) method for the analysis and quantification of aggregates in GLP-1 analysis?

The parameters affecting resolution in the SEC column are particle size, column length, internal diameter, pore size, and the volume of sample injected. Ideally, the resolution is considered good if the technique can measure a molecular weight difference of twofold. To attain that, the long column with a smaller internal diameter, moderate flow rate, small particle size, and lower volume of sample should be used. Also, the viscosity of the sample should be the same as that of the mobile phase. A small change in pH, ionic strength, and composition of the sample buffer will not significantly affect resolution as long as these parameters do not alter the size or stability of the peptide oligomer. Also, the sample is exchanged into the running buffer during the separation, which is an added benefit of SEC.

The resolution of the peaks in SEC is governed by pore size and particle size of the gel matrix; that is, the smaller the particle size, the better the resolution. Pore size controls the exclusion volume of the molecules entering the column. Extremes in pH and ionic strength, as well as the presence of denaturing agents or detergents, can cause conformational changes. Buffer conditions that are compatible with peptide stability should be chosen. The product of interest should be collected in a suitable buffer. It is preferable to use a buffer concentration that maintains buffering capacity and a constant pH. Sodium chloride with optimized concentration is used to avoid nonspecific ionic interactions with the matrix, which can be seen as a delay in the peak elution.

How should sample preparation be optimized for GLP-1 peptides to avoid loss, adsorption, or artefactual modifications before HPLC/LC–MS analysis?

Diluent selection for peptide molecules during a chromatographic run often proves challenging since these molecules can adsorb to the glass or plastic consumables used during the analysis. Lower recovery is often observed at the limit of quantitation (LOQ) level if an improper diluent is selected. The parameters that should be considered during diluent selection include the isoelectric point (pI) of the peptide and the HPLC index. During diluent selection for peptides, the pH of the buffer needs to be optimized in such a way that the ionic interaction in a peptide molecule can be minimized. At pH lower than pI, the molecule contains a net positive charge, while as the pH becomes greater than pI, there is an overall negative charge on the molecule. It is advisable to compare solution stability at laboratory temperature and at refrigerated storage in relevant containers such as HPLC vials, centrifuge tubes, or volumetric flasks. The HPLC index provides the degree of hydrophobicity of a peptide. The higher the value of the HPLC index, the more hydrophobic the compound. For peptide molecules with a high HPLC index, the organic ratio should be increased in the diluent and mobile phases. In other words, the organic ratio can be optimized to reduce the hydrophobic interaction between the stationary phase and the molecule. Formulation buffer with optimized organic ratio can be an ideal choice for diluent selection.

Nitish Sharma currently serves as assistant professor in the Department of Pharmaceutical Analysis at the National Institute of Pharmaceutical Education and Research, Ahmedabad (NIPER-A), a position he has held since April 2021. He has almost 14 years of research experience in pharmaceutical analysis. He has published ~44 research/review papers in international journals.