Epimerization is one of the most important and most underestimated risks in peptide manufacturing.

A difference at just one stereocenter can be sufficient to alter a peptide’s function. Changes in receptor binding, metabolic stability, and biological activity are all possible. At the same time, epimeric impurities are often difficult to remove, increasing purification complexity and cost.

For CDMOs, epimerization is not a minor analytical issue. It is a core process and route design challenge.

Managing this risk requires a combination of chemistry understanding, process control, and early analytical strategy.

What is epimerization in peptide synthesis?

Epimerization is the change of configuration at a single stereocentre.

In peptide synthesis, this typically refers to the α-carbon of an amino acid residue: the position that defines whether the residue is in the natural L-form or the corresponding D-form.

Even low levels of epimerization can create significant challenges, including:

• Altered biological activity
• Regulatory comparability concerns
• Impurities that are difficult to separate chromatographically

Because these impurities are structurally very similar to the target peptide, standard purification approaches are not always sufficient.

Why does epimerization happen?

Epimerization in peptide synthesis is driven by three main chemical pathways. In these cases, the underlying principle is the same: destabilizing the alpha stereocentre creates an opportunity for epimerization.

Oxazolone formation

During amino acid activation, the carbonyl group can cyclize with the backbone nitrogen to form an oxazolone intermediate.

This intermediate is planar and temporarily achiral at the alpha carbon. When it is reprotonated, both L- and D-configurations can form.

Oxazolone formation is more likely when:

• Activation times are extended
• Reactive coupling reagents are used
• Activated intermediates are allowed to accumulate

Base-mediated Hα abstraction

Under basic conditions, a proton can be removed directly from the alpha carbon.

This again creates a planar intermediate that can be reprotonated without stereochemical control.

This pathway becomes more relevant when:

• Strong bases are used
• Deprotection conditions are harsh
• Exposure times are prolonged

Aspartimide/glutarimide formation and ring opening

In sequences containing Asp (or Glu), the side chain can cyclize to form a reactive imide (aspartimide or glutarimide).

Ring opening can occur at different positions, temporarily destabilizing the alpha stereocentre and allowing epimerization alongside regioisomer formation.

Aspartimides and their ring‑opened epimers are therefore classical stability-indicating impurities.

This pathway becomes more relevant when:

• Asp–Gly or similar motifs are present
• Exposure to basic conditions
• Elevated temperatures are used

When is epimerization risk highest?

Epimerization can occur at multiple stages of peptide synthesis.

Activation and coupling

This is typically the highest-risk step.

The time between amino acid activation and incorporation into the growing chain determines how long reactive intermediates are present. Longer activation times and higher temperatures increase the likelihood of oxazolone formation.

Deprotection

Strongly basic deprotection conditions can promote direct Hα abstraction, particularly if exposure times are not tightly controlled.

Conjugation of fragments

Fragment-based synthesis approaches introduce additional risk.

When two peptide fragments are joined in solution, conjugation often requires conditions that favor epimerization, including:

• Extended reaction times
• Reactive intermediates
• Non-ideal solvent environments

Purification

Epimerization is not always resolved during purification.

In some cases, chromatographic processes can concentrate epimeric impurities rather than remove them, particularly when separation between diastereomers is limited.

Sequence and process factors that increase epimerization risk

Not all peptides carry the same epimerization risk.

Sequence-related factors

Certain amino acids are more prone to epimerization, for example, phenylglycine, cysteine or histidine.

Residues with electron-withdrawing side chains or more acidic alpha protons are particularly sensitive. Sterically hindered residues can also behave unpredictably during coupling.

Process-related factors

Key variables that influence epimerization include:

• Coupling reagent and base selection
• Activation time and reaction time
• Solvent choice and polarity
• Base exposure during deprotection

Fragment-based strategies can amplify epimerization risks, as conjugation introduces additional steps where stereochemical integrity must be maintained.

What feasibility studies show about epimerization risk

Bachem’s feasibility study comparing linear vs fragment SPPS for peptide manufacturing highlights how strongly epimerization risk can influence route selection.

In this study, a representative GLP-1 receptor agonist was evaluated across multiple synthesis strategies. The peptide included a long backbone and a fatty acid-containing side chain, requiring more than 40 coupling cycles.

Linear SPPS was compared with several fragment-based approaches. The results showed clear differences:

• One fragment route exhibited ~13% epimer formation during conjugation and was discontinued
• A second showed 2–3% epimerization, increasing to ~5% after purification
• Linear SPPS delivered the highest overall yield and avoided epimerization issues

The key takeaway was that fragment strategies can introduce epimerization risks that are difficult or impossible to resolve downstream. As a result, the linear route proved to be the most suitable option in practice, confirming our prediction and demonstrating consistently low epimerization.