Peptide stability and degradation: the routes and what drives them

Two kinds of change

Chemical degradation alters the covalent structure of the molecule: an atom is added, a group is lost, or a bond is rearranged or broken. Physical degradation leaves every bond in place and changes where the molecules are: they associate with one another, or they leave the solution for a surface.

Review The two are linked. A review of the physical stability of peptide medicines counts chemical degradation among the factors that influence aggregation, next to sequence, concentration, pH, net charge, surfaces and interfaces.⁷ A review of peptide formulation in water concludes that the most practical ways to slow degradation are to optimize the pH and to choose the buffer with care.⁸

The sections below add three routes that the figure leaves out: isomerization at aspartate, diketopiperazine formation and adsorption to surfaces. Amino acids are named in full here; their three-letter and one-letter codes are set out in the guide to peptide nomenclature and modifications.

Chemical routes

Oxidation of methionine, cysteine and tryptophan

Review A review of oxidation in protein medicines names methionine, cysteine, histidine, tryptophan and tyrosine as the residues most susceptible, because they react readily with reactive oxygen species.¹ It lists three triggers during processing and storage: oxidants present as contaminants, transition metal ions acting as catalysts, and light.¹ The outcome also depends on pH, temperature and buffer composition, and oxidation may cost a protein its biological activity.¹

Review The same review cautions that the remedy depends on the mechanism. Antioxidants or radical scavengers can inhibit oxidation caused by oxidants in the solution, but metal-catalyzed oxidation happens at specific sites, and there an added antioxidant may speed the reaction up.¹

Chemistry study Methionine is oxidized to methionine sulfoxide. In small model peptides exposed to iron and ascorbate, the reaction was fastest at pH 6 to 7, and a histidine close to the methionine markedly increased both the rate and the amount of sulfoxide formed.⁹ In that system ascorbate, an antioxidant, promoted the oxidation where it might have been expected to inhibit it.⁹

Review A 2020 review of tryptophan describes it as highly susceptible to oxidation and says it degrades into multiple products during manufacturing, storage and processing, mainly by oxidation or cleavage of its indole ring.¹⁰ The causes it lists are reactive oxygen species such as hydrogen peroxide, light together with photosensitizers, metals, and heat.¹⁰

Chemistry study Cysteine chemistry centres on bonds between sulfur atoms. When oxytocin, a peptide hormone that contains a disulfide bridge, was heated in solution at pH 4.5, 7 and 9, the products included molecules with three or four sulfur atoms in the bridge and dimers joined by disulfide bonds.¹¹

Deamidation of asparagine and glutamine

Deamidation turns the amide group on the side chain of asparagine or glutamine into an acid group. Asparagine becomes aspartate, and glutamine becomes glutamate.

Chemistry study A 1987 experiment used a synthetic hexapeptide in which asparagine is followed by glycine, held in buffer at pH 7.4 and 37 °C.² The asparagine closed into a ring called a succinimide, with a half-life of 1.4 days.² Water then opened the ring within hours, and it opened two ways: to ordinary aspartate and to isoaspartate, a rearranged form.² The ring also racemized, so part of the product was the mirror-image D form.²

Chemistry study The neighbouring residue set the pace. Replacing the glycine with a bulky leucine or proline slowed degradation 33- to 50-fold.²

Chemistry study Conditions matter as well as sequence. A later study of the same hexapeptide found that deamidation depended markedly on pH, temperature and buffer composition.¹² Between pH 5 and 12 the reaction ran only through the ring and gave both the aspartate and the isoaspartate peptide.¹² In acid, the side-chain amide was hydrolyzed directly and gave the aspartate peptide alone.¹² Between pH 7 and 11 the buffer itself acted as a catalyst.¹²

Chemistry study Glutamine deamidates as well. In heated oxytocin, deamidation at a glutamine, at an asparagine and at the amide that ends the chain was found at pH 2 and again at pH 9.¹¹

Aspartate: isomerization and a weak bond

Chemistry study Aspartate can form the same ring directly, though more slowly: putting aspartate in place of asparagine in the model hexapeptide cut the rate of ring formation 34-fold.²

Chemistry study A study of that aspartate hexapeptide followed its fate across the pH scale.³ From pH 0.3 to 3 the main reaction was hydrolysis of the bond between aspartate and the next residue, which cut the chain into a tetrapeptide and a dipeptide.³ At pH 4 and 5, cutting and isomerization ran side by side.³ Above pH 6 the isoaspartate peptide was the only product observed.³

Chemistry study Isoaspartate is an isomer of aspartate: the same atoms, joined in a different order, so the change adds no mass to the peptide. The deamidation study followed the aspartate and isoaspartate peptides by HPLC.¹²

Hydrolysis of the backbone

Hydrolysis is the cutting of a peptide bond by water.

Chemistry study The bond after aspartate is one weak point, as the acid results above show.³ Asparagine can lead to a cut too: when leucine or proline followed the asparagine in the model peptides, cleavage products appeared.²

Review Cutting is not confined to solutions. A review of solid-state chemistry lists peptide bond cleavage among the reactions that take place in dry peptide and protein preparations.⁶

Diketopiperazine formation

Chemistry study A peptide with a free amino group at the start of its chain can lose its first two residues as a small ring, a diketopiperazine. In a model compound in which phenylalanine is followed by proline, that amino group attacked a bond further along its own chain, and the first two residues left as a ring.¹³ The reaction depended strongly on pH, because the amino group was more reactive in its unprotonated form.¹³ Phosphate and glycine buffers catalyzed it, and changing the residue in front of the proline changed the rate significantly at pH 7.¹³

Physical routes

Aggregation

Review Aggregation is the association of peptide molecules with one another. A review of peptide medicines describes two outcomes, amorphous aggregates and highly structured fibrils, and says there are many cases in which they limit the use of a biological medicine.⁷ The factors it reviews include sequence, concentration, pH, net charge, added ingredients, surfaces and interfaces, and impurities, together with physical conditions such as temperature, agitation and freeze-drying.⁷

Chemistry study Chemical and physical change can arrive together. The oxytocin study found covalently linked dimers and larger aggregates next to the deamidated and sulfur-modified forms.¹¹

Adsorption to surfaces

Chemistry study A peptide can leave a solution without reacting at all. In one HPLC study of three cationic, membrane-active peptides, 90% or more could be lost from solution at typical experimental concentrations through rapid adsorption to the walls of glass and plastic sample containers.⁴

That study, and how the choice of container enters bench work, are covered in reconstitution concepts.

What drives the rate

Five conditions recur in the studies above. ICH guideline Q1A(R2), the 2003 international text on stability testing of new drug substances, asks for stress tests on most of them: temperature, humidity, oxidation, light, and hydrolysis across a wide range of pH.¹⁴

Temperature

Chemistry study When oxytocin solutions were held at 40 to 80 °C, the loss of intact peptide followed first-order kinetics, and its dependence on temperature fitted the Arrhenius equation, the standard relation between temperature and reaction rate.¹¹

pH and buffer

Chemistry study In the model peptides, pH decided which route ran. The aspartate hexapeptide was cut in strong acid and isomerized above pH 6.³ The asparagine hexapeptide deamidated through the ring from pH 5 upward.¹² Oxytocin degraded fastest at pH 9 and slowest at pH 4.5 among the four values tested.¹¹ Buffer salts are not bystanders either: they catalyzed both deamidation and diketopiperazine formation in the model studies.¹²¹³

Review The formulation review cited earlier ranks pH optimization and buffer selection as the most practical stabilizing measures, and lists co-solvents, exclusion of air and added polyols among the others.⁸

Moisture

Review In a dry peptide, water content is a variable in its own right. The solid-state review names temperature, moisture content, added ingredients and the physical state of the solid, amorphous or crystalline, as the factors that influence the reactions.⁶

The practical side, including why a cold vial is brought to room temperature before it is opened, is in the peptide storage guide.

Light and oxygen

Review Light can induce oxidation, and light together with photosensitizers is among the causes of tryptophan breakdown.¹¹⁰ Oxygen is a reagent in its own right: excluding air is one of the practical strategies listed for peptide solutions.⁸

The ICH guideline lists photolysis among its stresses and makes photostability testing an integral part of stress testing.¹⁴

Freezing and thawing

Review A review of protein stability during freezing names three stresses: low temperature itself, freeze-concentration and the formation of ice.¹⁵ As ice forms, everything dissolved is crowded into the liquid that remains, which can speed reactions between molecules, make buffer components crystallize and redistribute the solutes.¹⁵ The review concerns proteins, which are larger than most research peptides, so it describes a mechanism and not a measured effect on any one peptide.

Each cycle of freezing and thawing puts a solution through those stresses again. Why solutions are divided into single-use portions is explained in reconstitution concepts.

Why the dry state is more stable

Several of the routes above need water, as a reagent, as the medium in which molecules move and meet, or as both. A lyophilized, or freeze-dried, peptide holds much less of it.

Review Peptide and protein drugs are often formulated as solids to stabilize them during storage.⁶ The formulation review says that peptides are often unstable in aqueous solution, and that a stable dry preparation, dissolved when needed, might be designed in place of a liquid one.⁸

Chemistry study One study measured the difference. Two model peptides were held at 50 °C, either in pH 7 buffer with or without sucrose or mannitol, or as solids freeze-dried from those sugar solutions and kept at 30% relative humidity.⁵ They degraded 2 to 80 times more slowly in the solids than in solutions of the same sugars.⁵ The solid mattered too: sucrose stabilized the peptides against deamidation more than mannitol did.⁵

Review Dry does not mean inert. The solid-state review lists deamidation, peptide bond cleavage, oxidation, the Maillard reaction, beta-elimination, and dimerization or aggregation as reactions that still take place in solids.⁶

How the water is removed is described in why peptides are lyophilized.

How degradation is detected

Chemistry study The kinetic studies cited here followed the disappearance of the starting peptide and the appearance of its products by HPLC.¹²¹³ The oxytocin study identified its products by mass spectrometry.¹¹ Both techniques are explained in the guide to analytical methods for peptides.

Human evidence

None of the studies cited on this page was carried out in people or in animals. They are laboratory studies of peptides in buffer, in sample containers and as dry solids, together with reviews of such work on peptides and proteins. They describe what happens to a material in its container and say nothing about what any compound does in a living body.