Managing Impurities in GLP‑1 Peptides: How Carbon Media Enhances Purification

Crude GLP-1–class peptides and other long sequences typically enter purification at 45–65% area purity. The dominant impurities present at this include peptides with:

1. Residue deletions

2. Partial deprotections

3. Sequence isomers

4. Charge variants

5. Aggregates

Residue deletion peptides, partial deprotections, and sequence isomers usually represent the significant classes of impurities immediately after solid-phase peptide synthesis (SPPS) or the "crude" stage. This majority is primarily due to the inherent chemistry and coupling errors associated with SPPS.

Charge variants and aggregates may become more significant during downstream processing, formulation, or due to inadequate purification conditions, underscoring the necessity for careful attention to the purification conditions we employ.

For long peptides, such as GLP-1 analogs, deletion and isomeric impurities are generally the most prevalent at the crude stage. However, it is equally essential to consider charge variants and aggregates when aiming to meet final purity and regulatory standards. Therefore, these impurities need to be monitored and controlled during both analysis and purification.

In this blog, we will discuss the various types of impurities, their origins, and their impact on purification processes and the final product quality. Additionally, we will explore how All Carbon microbeads, with their unique properties, can assist in effectively managing these impurities.

1. Peptides with one or two residues deleted are a modified version of the intended full-length sequence. A peptide residue refers to an individual amino acid unit in a peptide chain that remains after bonding with other amino acids. For instance, if the target peptide consists of 30 amino acids, a peptide with a -1 deletion will contain 29 amino acids, while a -2 deletion will have 28 amino acids. Both modified peptides retain the same sequence as the target peptide, except for the missing amino acids (Figure 1).

Figure 1. The image illustrates the difference between an intended full-length peptide chain and a peptide chain with a deleted residue. Here, the AA3 amino acid residue present in the full-length peptide chain has been deleted. The square blocks represent the amino acid residues. The circle on each amino acid residue is the functional group that connects with other amino acid residues to form the peptide bond. The lines that connect each amino acid residue are the polypeptide backbone.

Origin. Deletion peptides occur in solid-phase peptide synthesis (SPPS) when an amino acid is not added or is lost during sequence extension.

Impact. The truncated side products are often structurally similar to the full peptide and can share chemical properties, making their separation difficult during stage 1 bulk removal and requiring high-resolution purification.

Stage 1 bulk removal refers to the initial purification step in peptide processing. The goal is to separate and remove the majority of unwanted impurities and by-products, truncated sequences, deletion peptides, side-reaction products, and unreacted reagents from the crude peptide mixture. This step typically employs preparative reverse-phase high-performance liquid chromatography (RP-HPLC) under acidic conditions, such as using 0.1% trifluoroacetic acid (TFA) in a water/acetonitrile mix.

Stage 1 results in a partially purified product known as the “pool,” which can then undergo further high-resolution purification in subsequent stages. It is important to note that deletion peptides and shorter fragments often co-elute when strong TFA ion-pairing is used.

How can All Carbon media help? The pH of the mobile phase media can be adjusted to neutral or basic values using ammonium buffers on the same carbon media. This alteration creates subtle differences in the charge on the full-length peptide and the peptide with the deleted residue. These differences affect their interactions with the carbon media and facilitate the separation of closely related deleted residues and target peptides.

2. Partial deprotections occur when one or more protecting groups on peptides are only partially removed, often leaving an amino acid side chain or N-terminus with some protection intact (Figure 2).

Figure 2. The figure shows a peptide chain with a protecting functional group (PG). This PG has not been removed from the amino acid residue after the formation of the peptide bond.

Origin. This incomplete removal can arise from steric hindrance, insufficient time, or insufficient strength of deprotection reagents.

Impact. Remaining protecting groups may alter peptide charge, hydrophilicity, and biological activity, potentially interfering with analytical assays or causing column fouling.

How can All Carbon media help? All Carbon media can effectively clear partial deprotections and adducts, such as +42 Da acylation (where an extra chemical group adds 42 units to the mass of the peptide), during the Stage 1 bulk removal purification process.

3. Sequence isomers are peptides that have the same amino acid composition and mass as a target peptide, but their amino acids are arranged in a different order or specific residues are in different positions. Figure 3 illustrates the several types of sequence isomers.

Figure 3. Types of sequence isomers. (a) This image shows the correct sequence of amino acids. Each amino acid residue is represented by the square block A, B, and C. (b) Positional Isomers: These occur when two amino acids switch places (e.g., AB becomes BA). (c) Inversions: Here, the entire sequence or part of it is reversed (e.g., ABC becomes BCA). Misincorporations: This happens when a similar amino acid replaces the correct one (e.g., leucine substitutes for isoleucine).

Origin. Sequence isomers often occur due to errors in automated solid-phase peptide synthesis (SPPS), such as incorrect attachment of amino acids. They can also form from chemical changes during synthesis or storage. For example, aspartic acid (Asp) may rearrange in the peptide chain, creating a different bond with nearby amino acids.

Impact. Isomeric impurities create significant problems because they have the same mass and often look similar, making it hard to tell them apart and remove them without using very high-resolution chromatography or mass spectrometry.

How can All Carbon media help? All carbon media can help switch from trifluoracetic acid (TFA) to formic acid (FA) or change the pH to between 7.5 and 8.5. This change improves the separation of similar molecules through changes in how they pair and interact with charges.

TFA is a strong acid that forms tight pairs with peptides, especially with basic amino acids like lysine and arginine. FA is a weaker acid that forms looser pairs, allowing peptides to come out at different times and helping to separate similar forms.

Adjusting the pH to 7.5–8.5 enhances separation even more. At a lower pH, peptides are usually protonated and strongly bind with the column. At a higher pH, some amino acids lose their charge, which reduces these connections. This difference helps to separate various peptides more effectively.

4. Charge Variants. These peptides have the same basic sequence as the intended product but differ in charge due to chemical modifications. Some variants may have increased positive or negative charges, or blocked charges, which can affect their purification, biological activity, and safety.

Figure 4. Depiction of charge variants. Peptide chains have the sequence of amino acids, but differ in charge.

Common modifications include:

• Deamidation: Converts neutral asparagine or glutamine into negatively charged aspartic acid or glutamic acid, adding an extra negative charge.

• Oxidation: Changes neutral methionine to methionine sulfoxide, which can alter charge or reactivity.

• Cyclization: Links the ends or side chains of the peptide, potentially trapping or modifying charges.

• Incomplete deprotection.: A side chain in the peptide still has part of a protecting group attached, which can block or introduce extra charge.

Origin. Charge variants can occur during synthesis, storage, or handling due to pH changes, oxidation, or incomplete deprotection of charged groups.

Impact. These charge differences can affect a peptide's solubility, stability, and functionality, potentially reducing its effectiveness and altering how the body processes it.

How can All Carbon media help? Carbon media can effectively remove most charge variants of GLP-1 during the initial RP-HPLC stage (Stage 1 bulk removal).

5. Aggregates. Aggregates are clumps of peptide molecules that stick together. They can join loosely through forces like water avoidance (hydrophobic) or charge attraction, or they can link tightly by forming actual chemical bonds (such as disulfide bridges).

Origin. Peptides can aggregate at high concentrations, improper buffer conditions, or due to an

Figure 5. Depiction of peptide aggregates. Peptide chains clump together through physical and chemical forces.

imbalance of hydrophobic and hydrophilic properties. This aggregation may occur during synthesis, handling, or purification. Additionally, chromatography media can contribute to clumping, especially if the peptide interacts strongly with or sticks to the surface of the media.

Impact. Aggregates can affect solubility, dosing accuracy, and bioavailability, and may trigger adverse immune responses upon injection.

How can All Carbon media help? The surface of carbon microbeads interacts with peptides to enable effective separation, but this interaction is not strong enough to cause sticking. It allows for careful control of loading and flow rates. This approach minimizes the residence time of peptides and helps prevent crowding during the purification process.

In summary, what makes All Carbon phases so powerful in long peptide purification is their versatility:

• Operate under both strong ion-pairing (TFA) and mild conditions (FA, neutral pH)

• Enable differential charge-based separation at higher pH ranges

• Provide surfaces that minimize peptide aggregation compared to silica phases

• Offer robust impurity clearance during both bulk removal (Stage 1) and fine polishing (Stage 2)

By leveraging these properties, carbon media can address deletion impurities, partial deprotections, sequence isomers, charge variants, and aggregates collectively—ensuring a cleaner path toward regulatory-grade purity.

Get in touch with us to discuss how we can support your chromatography separation needs. For more information or to request samples, please email us at inquiry@millennialscientific.com, call us at 855 388 2800, or fill in our online form.

Reference

Michael Jack Parente, Balaji Sitharaman, Liquid Chromatography Evaluation of a Novel Graphitic Carbon Stationary Phase Using Glucagon-Like Peptide-1 Analogs, ACS Omega, 2025.