Peptide synthesis: solid-phase method, solution-phase method, and production methods

Peptide synthesis is a controlled process of creating amino acid chains of a specific sequence. In chemical synthesis, selected amino acids are linked using peptide bonds, while simultaneously controlling other reactive groups present in these molecules. The aim is therefore not merely to connect amino acids, but to obtain a specific molecular structure with a defined sequence, stereochemistry, chain-end architecture and – if required – additional structural features.

The main methods include solid-phase peptide synthesis (SPPS), classical solution-phase synthesis, and newer liquid-phase peptide synthesis (LPPS) methods using soluble supports or molecular tags. Research laboratories and manufacturing plants may also combine different approaches, prepare shorter fragments separately and then join them together, or use biological expression if it is more suitable for a given sequence.

Comparison of chemical synthesis with bacterial expression of polypeptides consisting of 20–40 amino acid residues demonstrates an important principle: the practical choice of production method depends on the specific molecule, its sequence, propensity for aggregation, required modifications, scale of production, and analytical requirements. [1]

Obtaining the intended amino acid sequence is only one of the stages of peptide production.

The whole process may include:

sequence design → preparation of building blocks → protection of functional groups → coupling → deprotection → chain assembly → cleavage or fragment condensation → final structural shaping → purification → identity confirmation → impurity analysis → content assay → final characterisation.

For some peptides, additional steps such as cyclisation, the formation of disulphide bridges, folding, conjugation or other chemical modifications are also necessary.

Because of this, correct assembly of the chain does not automatically mean that the final material possesses the correct identity, purity, stereochemistry, conformation, stability or other required characteristics.

What is peptide synthesis?

Creation of a specific amino acid sequence

Peptide it consists of amino acid residues joined by peptide bonds.

The aim of peptide synthesis is to form these bonds in a predetermined order, often while simultaneously controlling:

• types of amino acids;
• amino acid sequences in the sequence;
• stereochemistry;
• N-terminal structures;
• C-terminal structures;
• side-chain modification;
• disulphide bridge distributions;
• cyclisation;
• other required structural characteristics.

The sequence is of fundamental importance, because an identical amino acid composition can lead to the formation of different molecules if the amino acids occur in a different order.

The statement that a given molecule is a „peptide composed of five amino acids” therefore provides very little information about its actual structure. To describe a molecule correctly, one needs to know the types of amino acids, their sequence, stereochemistry, terminal groups and any additional modifications.

Two peptides can therefore have the exact same amino acid composition and yet be different molecular structures.

Additional differences may arise from chain end modifications, the presence of disulphide bridges, a cyclic backbone, side-chain alterations or a different stereochemical configuration.

Chemical peptide synthesis usually employs amino acids whose reactive groups have been selectively protected.

Temporary protecting groups enable the creation of a specific intended chemical bond while limiting competing reactions at other sites on the molecule.

In the subsequent planned stages of the synthesis, these groups are removed or altered.

A controlled reaction rather than spontaneous combination

In basic chemical descriptions, the formation of a peptide bond is often presented as a condensation reaction in which two amino acids combine with the net release of a water molecule.

Such an equation describes the overall reaction balance.

However, it should not be equated with the way in which controlled peptide synthesis is carried out in practice.

Simply mixing free amino acids would not allow one to reliably determine:

• which amino group is to react;
• which carboxylic group is to react;
• which amino acid should come first;
• which is to be the second;
• will sidechains start to react;
• whether the stereochemistry will be preserved;
• will numerous unwanted products not be formed.

Therefore, in practical chemical synthesis, controlled strategies for activation and protection of functional groups are applied.

The group derived from the carboxyl group is usually activated in such a way that it can react effectively with the chosen amino group. Protecting groups, the order of reactions, carrier chemistry, coupling reagents, the solvent environment and isolation methods jointly help to direct the reaction towards the intended product.

Effective peptide synthesis therefore requires control over both the chemistry that is intended to take place and that which must be prevented.

A good example is the formation of aspartimide.

Researchers have developed an alternative method for carboxyl group protection, aimed at limiting this problematic side reaction during peptide assembly. [2]

The study reveals a broader principle of peptide chemistry: preventing undesirable molecular transformations can be just as important as forming the correct peptide bond.

When can a peptide be considered ready?

The completion of the linear peptide backbone does not always signify the end of the entire synthesis.

Depending on the target molecule, additional steps may be needed after chain assembly.

These may include:

• complete removal of protective groups;
• cleavage of the peptide from the solid support;
• removal of temporary markers;
• cyclisation;
• oxidation;
• formation of disulphide bonds;
• folding;
• conjugation;
• chain end modification;
• purification;
• analytical characteristics.

Some peptides require relatively few additional structural modifications after chain assembly.

For others, the later stages are crucial.

A good example is the synthesis of bovine pancreatic trypsin inhibitor (BPTI). The researchers assembled the linear sequence, purified the material, and then obtained a folded structure containing disulphide bridges. According to the analyses and functional comparisons presented in the study, the final material with the natural sequence corresponded to the native protein in terms of the parameters studied. [3]

This example illustrates the difference between assembling a peptide backbone and obtaining a fully characterised final molecular form.

The method that allows you to obtain the correct linear sequence does not automatically confirm:

• correct folding;
• proper arrangement of disulphide bonds;
• biological activity;
• stability;
• contamination profile;
• fitness for a particular purpose.

Each of these issues requires separate evidence.

Chemical synthesis, biological expression and combined methods

In chemical peptide synthesis, previously prepared molecular blocks are joined in controlled laboratory reactions.

Biological expression, on the other hand, uses cellular machinery to produce the encoded amino acid sequence. The material must then be recovered, purified and, if necessary, subjected to further processing.

Both methods can lead to the production of peptides or polypeptides, but they involve different possibilities and limitations.

Chemical synthesis can be particularly useful when needed are:

• selected non-standard amino acids;
• site-specific chemical modifications;
• specific chain end structures;
• isotope markers;
• peptide backbone modifications;
• other features difficult to obtain through standard biological expression.

Biological expression can be attractive in the case of appropriate longer sequences or specific labelling strategies, but it does not eliminate problems occurring at later stages of production.

Material recovery, folding, processing, host-related impurities, purification and analytical characterisation must still be taken into account.

Therefore, none of the methods is universally the best.

When comparing polypeptides consisting of 20–40 amino acid residues, both sequence length and aggregation propensity affected the outcomes of solid-phase synthesis and bacterial expression. [1]

These observations support an approach in which the production method is chosen to suit the specific molecule.

Historical data on performance, costs, equipment and production time should not be treated as universal contemporary values, as technologies, reagents, automation, purification methods and production economics change with technological development.

Solid-phase peptide synthesis

The growing chain is attached to an insoluble support

In solid-phase peptide synthesis (SPPS), the growing peptide chain remains attached to an insoluble support during successive cycles of synthesis.

The carrier is most commonly a functionalised polymer resin.

The linker, or spacer, binds the peptide to the carrier and influences, amongst other things, the manner in which the finished chain is subsequently released and the type of end group obtained.

One of the main practical advantages of SPPS is easier separation of the material.

Upon completion of a given reaction stage, soluble reagents and many soluble by-products can be removed by washing, while the growing peptide remains bound to the solid phase.

Because of this, there is no need to isolate and purify the growing peptide as a separate, free compound after the addition of each successive amino acid.

However, this convenience can lead to a significant misunderstanding.

Washing the resin does not mean that all incorrect peptides have been removed.

If the chain attached to the resin does not react during one coupling step, it may still remain on the support.

In subsequent cycles it may react again and elongate further.

As a result, a deletion sequence may be formed, which is a molecule differing from the target peptide by the absence of one or more amino acid residues.

Similarly, chemically modified or incompletely unblocked resin-bound chains can participate in subsequent synthesis steps.

SPPS therefore simplifies the separation of soluble substances from the resin-bound material.

However, I cannot guarantee perfect sequence alignment.

Repeated cycle of unblocking and engaging

A typical stepwise SPPS synthesis involves the repetition of three basic operations:

disarming → coupling → rinsing.

First, the reactive amino group at the end of the growing chain is exposed.

Next, another protected amino acid is attached to the chain.

Soluble reagents, excess amino acid and many reaction products are removed before the next cycle begins.

Repeating this process makes it possible to build up the peptide one amino acid residue at a time.

In classical SPPS, the C-terminal residue is usually attached to the support first, and then the chain is elongated towards the N-terminus.

This differs from standard ribosomal protein synthesis, during which successive amino acids are added to the C-terminus of the growing chain.

However, the final amino acid sequence can be identical, even though the direction and chemistry of the folding process are different.

The term „cycle” should not be understood as a single universal set of reaction conditions suitable for every residue and every sequence.

The course of the reaction may depend on:

• protection group strategies;
• of amino acid;
• spatial conditions around the reaction site;
• type of resin;
• Linker;
• chain lengths;
• aggregation;
• solvent;
• coupling chemistry;
• structural behaviour of the growing peptide.

A reaction that proceeds easily at the beginning of the synthesis can become significantly more difficult at a later stage.

Therefore, process monitoring remains essential even in highly automated systems.

Why do resin properties matter?

A fixed medium is not merely a passive holder.

Its physical and chemical properties can directly influence the course of the reaction.

One of the important parameters is swelling.

If the resin swells correctly in the selected solvent environment, the reagents can more easily reach the reactive sites located throughout the polymer structure.

Insufficient swelling or structural changes in the resin may restrict this access.

Also of significance may be:

• stocking density;
• polymer architecture;
• linker script;
• solvent compatibility;
• mechanical stability;
• porosity;
• diffusion properties;
• interactions between growing peptide chains.

As the peptide length increases, individual chains may also interact with each other.

Aggregation on the resin can restrict access to reactive sites and lead to incomplete coupling or incomplete deprotection.

This means that a sequence behaving correctly at the beginning of synthesis may become increasingly difficult as the chain lengthens.

Researchers developed a variable-bed flow reactor that enabled the monitoring of resin swelling based on pressure-related measurements. [4]

The study focused on resin aggregation and incomplete bonding as process issues that might otherwise be difficult to observe directly during synthesis.

This demonstrates that monitoring the physical behaviour of the medium can provide information that goes beyond simply recording the added reagents.

What is Wang resin used for?

Wang linker chemistry is widely utilised in SPPS when a C-terminal carboxylic acid is desired following peptide cleavage.

The peptide is attached via an ester-type bond, which can subsequently be cleaved under appropriate conditions.

Alternative linker systems may be selected when a different C-terminal structure is required, for example an amide.

It is worth distinguishing the linker chemistry from the entire physical vehicle.

Related Wang-type linkers can be introduced into various polymer architectures.

Such materials do not need to have identical:

• swelling properties;
• capacities;
• solvent compatibility;
• mechanical properties;
• behaviours during a reaction.

Studies using peptidyl-Wang-TentaGel resins have demonstrated a specialised alternative method for preparing peptide hydrazides, which are useful intermediates in peptide chemistry. [5]

This does not mean that standard cleavage from Wang resin usually leads to the formation of peptide hydrazides.

It does, however, demonstrate an important principle:

The solid support + linker + reaction conditions + cleavage chemistry collectively determine the resulting product.

Synthesis of SPPS in batch and flow systems

SPPS can be carried out in classical batch vessels or in systems where reagents flow through a reactor containing a solid support.

Flow systems can enable rapid reagent exchange and more precise control of the chemical environment surrounding the resin.

A 2014 study demonstrated rapid flow-based peptide synthesis with continuous monitoring and applied it to several peptides and a small protein. [6]

A later automated study described significantly faster chain assembly, yielding crude product purity and isolated yields for the analysed examples comparable to classical batch synthesis. [7]

These studies demonstrate the capabilities of specific systems under defined conditions.

They do not prove, however, that every peptide can be produced equally quickly and with the same quality.

The time required to produce the peptide also involves much more than just chain assembly.

The complete process may include:

• preparation of reagents;
• preparation of resin;
• coupling;
• disarming;
• washing;
• splitting off;
• product processing;
• purification;
• solvent removal;
• final structuring;
• laboratory tests;
• documentation.

Rapid cable assembly is therefore just one element of overall production efficiency.

Liquid-phase synthesis

What does this term mean?

The term „liquid-phase peptide synthesis” can be used in several senses.

Broadly speaking, it can simply mean peptide synthesis carried out in solution.

However, in more specialist literature, a distinction is often made between:

1. classical solution-phase peptide synthesis;
2. solid-phase peptide synthesis;
3. newer LPPS methods, in which the growing chain possesses a soluble tag or support.

This distinction is important because the method of separating and recovering the product changes.

A freely dissolved, protected peptide intermediate may require isolation utilising its own physical and chemical properties.

In the case of a labelled intermediate, the label may be designed to facilitate precipitation, extraction, membrane retention or another method of separation.

The review on LPPS clearly distinguishes between classical solution synthesis, SPPS and methods utilising soluble tags. [8]

The publication is useful for ordering terminology and methodology.

However, it should not be interpreted as a single experiment proving that one method is the best for every peptide.

Classical solution-phase peptide synthesis

In classical solution-phase synthesis, protected amino acids, peptide intermediates or fragments remain dissolved in the reaction medium rather than being covalently attached to an insoluble resin.

Following completion of the reaction, the corresponding intermediate can be isolated, purified and, if necessary, characterised before starting the next step.

This may bring certain benefits.

Researchers can:

• characterise the individual semi-finished products;
• choose the cleaning method according to the specific semi-product;
• optimize the individual transformations;
• prepare the fragments independently;
• modify the synthesis route when a specific intermediate causes problems.

Synthesis in solution can, however, also involve significant difficulties.

With the elongation of the peptide chain, they can:

• become poorly soluble;
• aggregate;
• be difficult to crystallize;
• to create closely related pollutants;
• be difficult to separate;
• requires repeated isolation;
• cause material losses during the subsequent processing stages.

Synthesis in a solution is therefore not automatically simpler or easier to scale than SPPS.

Its usefulness depends on the specific sequence, the stability of the intermediates, solubility, the length of the synthesis route, the retention during purification, and the efficiency of the subsequent isolation steps.

The development of labeled LPPS strategies is partly due to attempts to solve precisely these problems related to separation and material handling.[8]

Soluble markers combine reaction design with separation design

In the marked LPPS, the growing peptide has a temporary molecular attachment designed in such a way as to affect its physical properties.

The marker can enable the peptide to remain sufficiently soluble during the reaction and then facilitate its selective recovery.

Depending on the technology, the separation can use:

• drilling;
• extraction;
• membrane retention;
• phase behavior;
• other properties resulting from the presence of the marker.

In one of the experimental strategies, a long-chain protective group at the C-terminus was used to prepare peptide amides.

Repeated reaction and elution steps allowed the researchers to prepare a hydrophobic peptide that, in the comparison they used, was difficult to obtain satisfactorily using the SPPS method. [9]

This shows the usefulness of this particular strategy for the analyzed sequence.

However, it does not prove the universal advantage of marked synthesis.

The marker becomes an element of the production process and raises its own questions:

• How effectively is it connected?
• Does it change the course of the reaction?
• How reliably can the marked semi-product be recovered?
• Does it lead to the formation of new pollutants?
• How is it removed?
• Can the complete removal be confirmed?
• What analytical methods confirm its absence in the final product?

Therefore, soluble markers should be treated as functional elements of the process design, and not simply as simple labels.

Comparison of the main methods

The comparison presents the general principles of the operation of the individual methods.

It does not specify which of them will provide the best performance, cleanliness, cost, environmental sustainability, or scalability for a specific sequence.

These parameters should be evaluated based on the actual molecule and the specific production process.[4][8][9]

Merging fragments and hybrid production

A peptide does not always have to be formed in a single continuous process involving the addition of successive amino acid residues.

An alternative strategy is to prepare separate shorter peptide fragments and then combine them.

The hybrid process can, for example, use SPPS to obtain individual fragments and chemistry in solution to combine them.

Fragmentary strategies can limit some of the problems associated with the very long step-wise synthesis.

However, they introduce new challenges, such as:

• selection of the appropriate connection point;
• dissolubility of fragments;
• activation of the connection point;
• chemoselective selectivity;
• the preservation of stereotyping;
• side effects;
• cleaning up fragments;
• cleaning after their connection.

An important example of controlled fragment joining is native chemical ligation.

In the classical version, the peptide thioester reacts with the second peptide containing the N-terminal cysteine. Subsequent rearrangement leads to the formation of a proper peptide bond at the ligation site in the backbone.

Initial studies showed the possibility of obtaining after ligation and folding of a protein containing disulfide bridges.[10]

This confirms the importance of this strategy of combining fragments.

However, this does not mean that any two unmodified peptide fragments can spontaneously combine.

Equipment and security groups

The combination creates a purposeful connection

Coupling is a reaction step during which a new peptide bond is formed between the selected units.

Usually, a group derived from the carboxyl group is activated so that it can react effectively with the appropriate amino group.

The activation chemistry must comply with:

• a growing sequence;
• temporary protective groups;
• protection of the side chains;
• stereochemistry;
• a carrier or a marker;
• solvent environment;
• other functional groups present in the molecule.

Effective coupling is particularly important in the stepwise synthesis because incomplete reaction can lead to the formation of deletion sequences.

If one step of amino acid addition does not work for some growing chains, such chains can remain in the system and participate in subsequent cycles.

As a result, molecules that are very similar to the target peptide can be formed, but they are devoid of one of the remaining residues.

Even small inefficiencies can therefore accumulate during many stages of synthesis.

A simple mathematical example illustrates this problem well.

If each of the 50 independent stages of submission had a 99% probability of success, the percentage of molecules for which all stages would end successfully would be:

0,99^50 ≈ 0,605

that is about 61%.

This is purely a mathematical illustration.

It does not provide a forecast of the actual purity or yield of the peptide synthesis containing 50 residues.

Actual production includes:

• different coupling efficiencies at individual positions;
• stages of deactivation;
• side effects;
• dependent on the difficulty sequence;
• reintegration strategies;
• losses during de-coupling;
• losses during cleaning;
• other procedural variables.

The calculation only shows why even seemingly minor inefficiencies in a single step can become very significant in a multi-step synthesis.

Security groups are controlling the reaction area

An amino acid can have several chemically reactive groups.

Without selective control, binding reactions could occur in undesirable places.

Security groups are temporarily blocking selected reactive areas.

The amino group used to extend the chain usually requires temporary protection, whereas the reactive side chains may require protection that remains in place for many synthesis cycles.

The connection to the carrier must also survive these stages until the planned disconnection occurs.

The choice of protective groups therefore affects the entire synthesis strategy.

It is necessary to take into account:

• the method of their introduction;
• stability;
• method of disposal;
• compatibility with other protective groups;
• compatibility with the carrier;
• side effects;
• the influence of a specific sequence;
• final conditions for de-insurance.

Studies using cyanide sulfonylide-based protection concerned the formation of aspartimide through a change in the way the carboxyl group is protected.[2]

The results show that the strategy of protective groups not only influences whether the intended reaction can occur, but also which undesirable reactions become possible.

Fmoc, Boc and orthogonal protection

Fmoc and Boc belong to widely used groups protecting amino groups.

They do indeed differ in the way they are removed.

The Fmoc is usually removed under basic conditions, whereas the Boc is sensitive to acidic conditions.

These properties allow them to be used as elements of broader protection strategies.

In the popular Fmoc-based SPPS, the temporary N-terminal protection of the Fmoc is repeatedly removed, while the selected side-chain protecting groups and the bond to the resin remain intact until later stages.

Other strategies may use different combinations.

The orthogonal protection is particularly important.

Two protective groups are considered orthogonal if one can selectively remove it under conditions in which the other remains sufficiently stable to allow further synthesis to proceed.

Orthogonality is therefore a practical relationship of chemical consistency.

This does not mean that every protected group will remain completely unchanged after unlimited exposure to all reaction conditions.

Side effects may still occur.

In the Fmoc synthesis studies, both the formation of aspartimide and undesirable stereochemical changes were described despite the overall selective nature of the protective strategy assumed.[11]

Stereochemistry is part of the identity of a molecule

Many amino acids have a chiral center.

This means that their atoms can adopt different spatial arrangements corresponding to different stereochemical forms.

Changing the configuration of a single stereogenic center can lead to the formation of another peptide structure without the need to change its nominal mass.

Therefore, the mass alone cannot confirm the full identity of the peptide.

Epimerization usually means a change in the configuration of one stereogenic center in a molecule containing several such centers.

Racemization is also often discussed in peptide chemistry in the context of the loss of stereochemical integrity of the amino acid residues.

These changes are significant because molecular recognition can strongly depend on the spatial arrangement of the atoms.

Two molecules with the same elemental composition and nominal mass can therefore behave differently if they differ in stereochemistry.

In a study of microwave-assisted Fmoc synthesis, it was found that some residues were susceptible to undesirable stereochemical changes under the conditions employed. Modifying the method reduced the occurrence of these reactions.[11]

This does not mean that heating, by definition, damages all peptides.

The correct conclusion is that the acceleration of the reaction should be evaluated together with the sequence-dependent chemical selectivity.

Aspartame and other related pollutants

The formation of aspartimide is a known side reaction related to certain sequences containing aspartic acid.

An undesirable cyclic intermediate may be formed, from which subsequently compounds with altered backbone connectivity or stereochemistry are formed.

The probability of this reaction depends on the sequence and the conditions of the process.

Other impurities associated with synthesis can result from:

• incomplete integration;
• incomplete deactivation;
• the formation of deletion sequences;
• reaction of side chains;
• stereotypical changes;
• reactions during deattachment;
• oxidation;
• reorganisations;
• conditions of product processing;
• incomplete removal of protective groups.

Some pollutants cause a readily noticeable change in the molecular mass.

Others are much more difficult to distinguish.

In analytical studies of a synthetic peptide that is a substrate of renin, many impurities have been identified, including components that exhibit the same mass change but contain modifications at different positions.[12]

An additional structural analysis was needed to distinguish them.

The researchers also considered the proposed mechanisms for the formation of these pollutants as hypotheses.

This is an important distinction:

Determining what pollution is and proving the exact mechanism of its formation are two separate scientific questions.

The probable mechanism should not be presented as confirmed solely because it corresponds to the observed molecular mass.

Purification, scaling, and quality of production

Raw product, purified product and yield

The crude peptide is the material obtained before the completion of the appropriate purification steps.

May contain:

• target peptide;
• deletion sequences;
• material not completely secured;
• modified sequences;
• residues of reagents;
• products related to de-pollution;
• salt;
• solvents;
• other components formed during the process.

Purification aims to separate the appropriate peptide from this mixture.

It is necessary to clearly distinguish three concepts:

purity of the raw product — measured composition of the material before final purification;

the yield of the purified or isolated product — the amount of recovered target material relative to a specific initial or theoretical value;

final purity — the composition of the isolated material determined using a specific analytical method.

These concepts are not interchangeable.

The process can lead to the formation of a large amount of raw material, but it allows the recovery of a relatively small amount of purified product.

Another process may yield less raw material, but it will be characterized by high selectivity in creating the final product.

Similarly, high analytical purity does not necessarily mean high production efficiency.

In accelerated flow synthesis studies, the purity of the raw material and the yield after isolation were correctly reported separately [7].

Chromatography separates components, but it does not answer every question.

High-performance reverse-phase liquid chromatography (RP-PLHPLC) is widely used for the analysis and purification of peptides.

Different molecules interact in different ways with the stationary and moving phases.

Under appropriate conditions, these differences allow the components of the mixture to be separated.

Analytical and preparative chromatography have related but different applications.

Analytical HPLC is primarily used to analyze the composition of the sample.

Precipitative HPLC is used to physically collect the separated components.

The chromatogram can provide many valuable information.

However, a large main peak or a high percentage of its surface area should not be considered as a complete molecular characteristic.

The results of chromatography depend, among other things, on:

• type of column;
• the moving phase;
• gradient;
• temperatures;
• detection methods;
• wavelength;
• sample preparation;
• the way pixels are integrated;
• resolution;
• properties of pollutants.

Two compounds can potentially elute at the same time.

Some sample components may also react differently to the detector used.

Therefore, high surface purity in HPLC is not synonymous with full confirmation of molecular identity.

Isotopic studies of peptide impurities and chiral analyses show why additional analytical methods may be needed. [12] [13]

The analytical method must correspond to a specific structural question.

Identity, purity, and content mean different things

In the description of peptides, three concepts are particularly important.

Identity answers the question:

Is the intended molecule present in the sample?

Cleanliness answers the question:

What other components are present in the sample or which part of the measured signal corresponds to the main component?

The content answers the question:

How much real peptide is there in the sample?

These questions cannot always be answered with a single measurement.

The material containing the peptide may also contain:

• water;
• ion counter;
• salt;
• solvent residues;
• other components related to the process;
• pollution.

The total mass of the bold material does not therefore have to correspond to the mass of the peptide itself.

Similarly, the agreement between the measured molecular mass and the theoretical value does not confirm:

• complete amino acid sequence;
• proper connection of the individual parts;
• stereochemical integrity;
• disulphide bridge distributions;
• folding;
• absence of isobaric impurities.

Mass spectrometry can provide strong evidence regarding the molecular mass.

Tandem mass spectrometry and other structural techniques can provide additional information about the sequence.

Chiral methods allow us to analyze stereochemical issues that cannot be resolved by conventional mass measurements.

One analytical study even included the racemization that occurs during sample preparation. This illustrates another important principle: the analytical procedure itself can produce artifacts if it is not properly designed. [13]

Twisting and activity may require separate confirmation

In the case of structurally limited peptides and small proteins, the correct sequence and molecular mass may still be insufficient to confirm the intended final structure.

Additional questions may include:

• Have the appropriate disulfide bridges been formed?
• Has the molecule adopted the expected conformation?
• Is the cyclization complete?
• Are there structural isomers present?
• Does the synthetic molecule exhibit the expected activity in a specific experimental test?

Several analytical and biophysical methods were used in the BPTI synthesis study, along with an assessment of trypsin inhibition.[3]

The combined results supported the consistency of the synthetic material with a natural sequence in terms of the criteria examined.

The activity test, however, answers a specific functional question.

It does not replace the analysis of pollutants.

Similarly, similar activity in one test does not confirm full equivalence of all biological, structural, or production properties.

Scaling up production changes more than just the amount of material

Increasing the scale of production does not solely consist in a proportional increase in the quantity of all the reagents.

Physical processes can behave differently on a larger scale.

They may have significance:

• mixing;
• heat exchange;
• distribution of reagents;
• diffusion;
• thickening of the resin;
• pressure;
• filtration;
• the solvent economy;
• drilling;
• solubility of semi-products;
• mass transport;
• the capabilities of the purification system.

These factors can influence both the course of the reaction and the formation of pollutants.

Cleaning at later stages can also become a bottleneck for the entire production.

Increasing the synthesis rate is of limited significance if the purification system is unable to effectively separate the contaminants that are produced or to recover the product of the required quality.

In an industrial analysis covering 40 peptide synthesis processes from 14 pharmaceutical companies, the consumption of materials during synthesis, purification, and isolation was assessed.[14]

The study shows why the evaluation of production should be conducted for the entire process.

However, this does not mean that every factory, sequence, or production technology achieves identical results.

The quality of the production is the result of many control elements

Reliable production of peptides requires more than just obtaining a single satisfactory analytical result.

A controlled process usually involves several related elements:

Defined target molecule → appropriate starting materials → controlled synthesis → controlled purification → proper sampling → validated or target-appropriate analysis → batch documentation.

The party’s documentation combines analytical results with material that was actually produced.

The Certificate of Analysis (COA) summarizes the selected test results of a specific batch.

Its informational value depends on:

• the type of tests performed;
• the methods used;
• the usefulness of these methods;
• specification;
• identification of the sample;
• the possibility of tracing the party;
• confirmation that the reported results actually apply to the material in question.

A single value defined as „purity” cannot by itself confirm:

• complete molecular identity;
• the content of the peptide;
• stereochemical integrity;
• level of solvent residues;
• water content;
• the composition of the counterions;
• microbiological properties;
• folding;
• biological activity;
• suitability for a specific purpose.

The appropriate set of analyses depends on the intended use of the material.

Peptides used as an analytical reference material, research reagents used in a cellular experiment, and materials in the pharmaceutical development phase may require answers to other qualitative questions.

Analytical evidence should therefore be interpreted within a clearly defined context, and not as universal proof of quality.

The environmental balance assessment must cover the entire process

The synthesis of peptides may require significant amounts of solvents and reagents.

The environmental impact assessment should therefore take into account much more than just the coupling stage itself.

Important elements may include:

• preparation of building blocks;
• coupling agents;
• deactivator reagents;
• washing;
• splitting off;
• product processing;
• chromatography;
• replacement of solvents;
• isolation;
• drying;
• waste management.

Reducing the environmental impact may require a simultaneous improvement of several stages.

The published analysis concerning peptide production discusses the problems associated with both synthesis and purification, and indicates the need to improve the entire process design.[15]

As a prospective publication, it does not constitute a controlled, direct comparison proving that one specific technology is always more environmentally friendly.

Similarly:

faster synthesis does not automatically mean more environmentally friendly synthesis,

and the lower consumption of solvents during the synthesis itself does not automatically mean a smaller impact of the entire production process.

Environmental comparisons require the use of consistent analytical limits and should ideally relate the consumption of resources to a specific amount of the isolated and characterized final product.

Frequently asked questions

1. How is the synthesis of peptides at the solid phase different from the synthesis in the liquid phase?

In continuous phase synthesis, the growing peptide remains attached to the insoluble carrier, whereas in classical synthesis, the peptide intermediate remains dissolved and usually has to be recovered based on its physical and chemical properties. In a narrower sense, the dissolved growing chain of an LPPS has an additional soluble marker or carrier that facilitates subsequent separation. Studies of labeled synthesis show one strategy of combining the solution chemistry with recovery through elution, whereas the literature on LPPS distinguishes such solutions from both classical synthesis in solution and SPPS. Neither method is by definition optimal for every peptide. The fundamental difference concerns the physical handling and separation of the growing chain during the subsequent stages of synthesis. [8] [9]

2. Why can impurities form during peptide synthesis?

Contaminations can arise because intended reactions do not always occur quantitatively, and other functional groups may participate in undesirable reactions. Incomplete coupling can lead to deletion sequences, incomplete deprotection can disrupt further chain assembly, and some sequences are susceptible to stereochemical changes, the formation of aspartimides, oxidation, rearrangement, or byproducts associated with deprotection. Studies of Fmoc synthesis and structural analyses of synthetic peptide contaminants document several such examples.[11][12] The frequency and significance of individual contaminants depend on the sequence and process, therefore automation of synthesis should not be equated with a complete absence of chemical errors.

3. What role does the resin play in the synthesis of peptides?

The resin maintains the growing peptide, while soluble reagents and many byproducts can be removed between successive synthesis steps. This greatly simplifies the repeated assembly of the chain, but the resin is not solely a mechanical hold. Its swelling, the degree of occupancy, the polymer architecture, the compatibility with solvents, and the availability of reaction sites can influence the effectiveness of the reagents reaching the attached peptide. In studies using a variable-complex reactor, the behavior of the resin during aggregation analysis and the efficiency of binding were monitored. [4] Such monitoring does not eliminate sequence errors, but it shows why the physical state of the resin can be an important process variable.

4. What is the purpose of Wanga resin in peptide synthesis?

Wang-type carriers are commonly associated with the preparation of peptides released after appropriate cleavage from the C-terminus in the form of a carboxylic acid. However, the exact final product depends on the linker, the carrier architecture, and the applied cleavage chemistry, and not just on the name of the resin. The researchers also presented a specialized method for converting the peptide-Wang-TentaGel material into peptide hydrazides, which can serve as useful synthetic intermediates. [5] The alternative reaction does not change the significance of the standard Wanga-resin cleavage. It does, however, show why the entire carrier system and reaction conditions must be analyzed when predicting the product.

5. Does SPPS build a peptide from the N-terminus or from the C-terminus?

Classical stepwise SPPS usually begins with the attachment of the C-terminal residue to the carrier, after which the chain is extended towards the N-terminus. This is the opposite direction to the standard chain extension on the ribosome, where the protein grows by adding consecutive amino acids to the C-terminus. The synthesis of BPTI is an example of using protected building blocks and the carrier to prevent subsequent release, purification, and folding of the product.[3] However, there are specialized peptide chemistry strategies that utilize other solutions, so building from the C-terminus to the N-terminus should be considered a classic SPPS approach rather than an absolute rule for all synthesis methods.

6. Why are security groups needed?

Amino acids often contain several reactive functional groups. Protective groups temporarily prevent the reaction at selected sites, leaving the appropriate site available at a specific stage of synthesis. This helps to control the order and manner in which residues are joined together. Without proper protection, competing reactions could lead to the formation of complex mixtures or products with a modified structure. Studies on the formation of aspartimide showed that a change in the chemistry of the carboxyl group protecting group can limit an important side reaction in the analyzed sequences.[2] Protective groups do not guarantee the ideal course of synthesis, but they provide selective chemical control that must remain consistent with all the important stages of the process.

7. What are the differences between Fmoc and Boc?

Fmoc and Boc are widely used temporary groups protecting the amino group, but they react to other chemical conditions. Fmoc is usually removed in an alkaline environment, whereas Boc is sensitive to acids. This difference affects the way they are combined with side chain protection, linkers, and other synthesis steps. Fmoc-based synthesis is widely used in modern SPPS, but studies have also shown the possibility of sequence-dependent side reaction pathways under such conditions.[11] In practice, therefore, it is more useful to compare entire protection strategies and their compatibility with a given sequence than to consider only the names of the two protective groups.

8. What does orthogonal protection mean?

Orthogonal protection means that one protective group can be removed under conditions where another necessary protective group remains sufficiently stable to allow continued synthesis. This allows different reactive sites to be revealed at specific stages. This concept is fundamental during controlled synthesis of complex peptides. However, orthogonality is dependent on conditions, not on absolute values: repeated exposure, sequence characteristics, and reaction parameters can still lead to side reactions. Research into alternative methods of protecting carboxyl groups shows how the protection strategy can be modified to control difficult peptide chemistry.[2]

9. What is a delection sequence?

A deleuxin sequence is a peptide lacking one or more residues present in the intended target sequence. It can be formed when the amino acid binding step does not occur completely, but the unreacted chain remains attached to the carrier and participates in subsequent cycles. Because the incorrect chain remains bound to the substrate, conventional washing does not necessarily remove it. Real-time SPPS monitoring studies indicate incomplete amide bond formation as an important process problem.[4] Therefore, a deleuxin sequence is a problem related to the structure and identity of the product, and not merely the residue of the reagent.

10. Can two peptide impurities have the same molecular mass?

Yes. Different molecular structures can have identical or indistinguishable nominal mass. For this reason, mass similarity alone does not confirm the full identity of a molecule. In studies of a synthetic peptide that is a substrate for renin, isobarytic impurities were identified, with modifications occurring at different positions. Derivatization and tandem mass spectrometry provided additional structural information. [12] This example shows why the observed mass's consistency with the expected cannot by itself confirm the correct sequence, connectivity, stereochemistry, or absence of structurally similar isomers.

11. What are epimerization and racemization in peptide synthesis?

Epimerization and racemization involve undesirable changes in stereochemical configuration. They can occur without changing the nominal amino acid sequence or molecular mass, therefore conventional mass measurements may not show a structural difference. In studies of microwave-assisted Fmoc synthesis, residues susceptible to stereochemical changes were identified and it was shown that modifying reaction conditions can limit such processes.[11] The risk depends on the building block, sequence, and process conditions. Stereochemical quality therefore requires analytical methods capable of evaluating the configuration, and cannot be inferred solely from molecular mass.

12. What is the formation of aspartimide?

The formation of aspartimide is an undesirable reaction associated with specific sequences containing aspartic acid. The formation of the cyclic intermediate can lead to subsequent products with altered backbone or stereochemistry, reducing yield and making purification difficult. Researchers analyzed alternative protection strategies designed to reduce this reaction in selected peptides and proteins.[2] The results do not indicate a single universal solution for all sequences containing aspartic acid. However, they show why the design of synthesis and contamination control strategies must take into account the specific chemical behavior of a given sequence.

13. Does high purity of HPLC confirm the correct identity of the peptide?

No. The purity of the HPLC describes the sample within the context of a specific chromatographic separation, the detector used, and the method of data analysis. A high proportion of the main peak area can be valuable evidence that the sample is dominated by one chromatographically separated component, but does not by itself confirm the complete molecular identity. Co-occurring impurities, stereoisomers, and other variants may require additional techniques. In studies of the chiral analysis of peptides, a specially designed process was used that also accounted for the stereochemical changes that occur during sample preparation. [13] Identity and purity should therefore be treated as related but separate analytical questions.

14. Does the performance of the purified product mean the same thing as purity?

No. The yield of a purified or isolated product determines the amount of target material recovered relative to a defined initial or theoretical amount. Purity, on the other hand, describes the composition of the recovered material according to a specific analytical method. Synthesis can lead to the production of a relatively small amount of product of very high purity or a large amount of raw material containing significant amounts of impurities. In studies of automatic flow synthesis, the purity of the raw product and the yield after isolation are reported separately.[7] Separating these parameters prevents the erroneous treatment of a high value of one of them as evidence of a high value of the other.

15. Can long peptides be produced by joining shorter fragments?

Yes. In a fragmental strategy, shorter peptide segments can be prepared separately and then combined using appropriate chemistry. Nuclease chemical ligation was an important example where unprotected peptide fragments were combined in a way that led to the formation of a natural peptide backbone bond, and then a folded protein was obtained. [10] This does not mean that any peptide segments can spontaneously bind together. The structure of the fragments, the ligation chemistry, the choice of ligation site, solubility, stereochemistry, and purification requirements must be taken into account. Therefore, fragment assembly is an alternative strategy for designing synthesis rather than an automatic solution to the problems associated with long peptides.

16. Does faster automated synthesis eliminate the need for purification?

No. Faster automated or flow-based synthesis reduces the time required for certain synthesis steps, but it does not prove that each resulting molecule has the intended structure. In studies of fast flow synthesis, the raw material is still characterized and subsequent purification is applied, and in subsequent works on automation, the purity of the raw material and the yield after isolation are compared separately.[6][7] Good system performance for the investigated sequences does not guarantee identical results for each new target. Accelerated synthesis still needs to be combined with appropriate purification, identity confirmation, impurity analysis, and final characterization.

17. Why is increasing the scale of production more complicated than preparing a larger batch?

Increasing scale changes physical and chemical conditions, not just the amount of material. Mixing, heat exchange, polymer swelling, the distribution of reagents, pressure, filtration, solvent handling, the solubility of semi-products, and the capabilities of the purification system may behave differently at higher production volumes. In an industrial analysis, 40 peptide processes were evaluated separately for synthesis, purification, and isolation during material consumption analysis. [14] The results show why production must be considered as a complete process. A scalable method must consistently lead to the production of a characterized material at all stages, and not just a larger amount of crude peptide.

18. Is the peptide ready immediately after the correct sequence is assembled?

Not necessarily. The assembly of a chain creates the backbone of the sequence only to the extent that the process and analytical data confirm it. Depending on the molecule, deprotection, dephosphorylation, cyclization, oxidation, formation of disulfide bridges, folding, conjugation, purification, and characterization may still be required. The synthesis of BPTI provides a good example of a process in which after the linear sequence is assembled, purification and the formation of a folded structure containing disulfide bridges followed. [3] The term „ready peptide” should therefore refer to the molecular form required in a given scientific context, and not just to the completion of the sequential addition of amino acids.

19. Does the correct molecular mass confirm the correct peptide sequence?

No. Mass consistency is a valuable evidence, but it cannot independently confirm the complete sequence or molecular structure. Isobaric structures can have the same mass, and differences in stereochemistry may not at all lead to a change in the molecular mass. Studies of synthetic peptide impurities have shown that distinguishing structurally different components may require tandem mass spectrometry and additional analyses. [12] In cases involving the structural integrity of stereochemically distinct components, chiral methods may also be needed. [13] Mass consistency should therefore be considered as one of the elements of identity assessment, rather than its full confirmation.

20. Why should purification be considered as part of the peptide production process, and not just an end-product addition?

Purification determines whether the target peptide can be effectively separated from the sequencing products, by-products, residue of reagents, structurally similar impurities, and other components formed during synthesis. A method that allows the very rapid production of large quantities of crude material may still be impractical if the target product of required quality cannot be effectively recovered. For this reason, industrial analyses of peptide processes include synthesis, purification, and isolation together. [14] The efficiency of production must ultimately be evaluated based on the quantity and quality of the isolated final material, rather than solely on the speed of synthesis.

Limitations when comparing peptide synthesis methods

When interpreting studies comparing peptide production technologies, several important limitations should be kept in mind.

First, the sequence strongly influences the difficulty of synthesis. A method that works for one peptide may not work the same way for another. Aggregation, hydrophobicity, amino acid composition, length, tendency to form secondary structures, and susceptibility to side reactions can all change the behavior of the process.[1][4]

Secondly, the purity of the raw material, the yield after isolation, the final purity and the content of peptide are different parameters. Comparing methods solely based on one of them may give an incomplete picture of the production efficiency. [7]

Thirdly, rapid chain assembly does not mean rapid production of the entire material. Debinding, cleaning, isolation, final structuring, analytical testing, and documentation also affect the overall production time. [6] [7]

Fourthly, analytical purity depends on the method used. HPLC, mass spectrometry, tandem mass spectrometry, and chiral analysis address different questions. One analytical result does not necessarily confirm the full structural identity. [12] [13]

Fifthly, scale affects the behavior of the process. Laboratory synthesis results cannot be automatically transferred to industrial production without considering mixing, mass transport, purification capabilities, material consumption, and other scale-dependent factors.[14]

Sixthly, historical economic comparisons may lose their relevance over time. The costs of reagents, the level of automation, expression technologies, purification equipment, solvent recovery, and production infrastructure are constantly evolving. Older cost analyses may still be of scientific significance, but they do not necessarily reflect the current economics of the market.[1]

Seventhly, environmental comparisons require consistent limits for analysis. Assessing solvent consumption during the actual chain synthesis while ignoring purification and isolation can lead to misleading conclusions regarding the equilibrium of the process.[14][15]

Finally, the name of the method itself does not determine the quality of the product. SPPS, solution synthesis, LPPS, fragment ligation, and biological expression can all lead to the production of high-quality material if the process is properly designed and controlled. However, none of these methods guarantees automatically the correct identity, purity, stereochemistry, folding, or suitability for a specific application.

Summary

The synthesis of peptides is best understood as a controlled process of molecule production, rather than simply the formation of peptide bonds.

The goal is to build a specific amino acid sequence while controlling the way the residues are joined, the chemistry of the protecting groups, stereochemistry, side reactions, and any additional structural requirements.

The synthesis of peptides at the solid phase maintains the growing chain on an insoluble carrier, making the repeated coupling and washing steps well suited for automation. However, the advantages associated with repeated assembly do not eliminate problems such as incomplete reactions, aggregation, sequence-dependent reactions, or sequence-dependent side reactions.[4][6][7]

Classical synthesis in solution maintains the peptide intermediates in a liquid phase and can provide greater flexibility in the isolation and characterization of individual intermediates. However, multiple separations and deteriorating solubility can become serious limitations.

Methods of liquid phase synthesis with soluble markers attempt to combine solution chemistry with designed material recovery strategies. They can therefore be an additional option in the case of sequences and production problems for which conventional SPPS is not optimal.[8][9]

Longer or particularly complex molecules can also be prepared by assembling fragments, including native chemical ligation, rather than relying solely on the continuous addition of successive amino acid residues.[10]

Regardless of the method chosen for the synthesis, the efficiency of the coupling and the chemistry of the protecting groups remain crucial, because minor inefficiencies and side reactions can accumulate during subsequent stages of the synthesis. The formation of aspartimide, the delection sequences, and the stereochemical changes show why the intended sequence alone does not describe everything that can be formed during the process.[2][11][12]

The work does not end even with the placing of the chain.

Purification determines whether the target product can be separated from other components.

Subsequently, appropriately selected analytical methods address specific questions regarding identity, purity, content, stereochemistry, conformation, and – if relevant – functional activity.[3][12][13]

Increasing the scale of production introduces further issues regarding mass transport, resin retention, solvent consumption, filtration, purification capabilities, process repeatability, and its environmental impact. For this reason, the industrial evaluation of the process should encompass synthesis, purification, and isolation together, rather than focusing solely on the rate of coupling.[14][15]

The most important principle is the following:

Effective peptide production requires control of the entire process – from the molecular design and bond formation to purification and structural characterization. Obtaining a chain with the expected nominal mass or a high peak size in the chromatogram represents valuable evidence, but none of these parameters alone can confirm the full molecular identity or the quality of the production process.

Disclaimer

The article is purely educational and scientific-informative in nature. It describes the general principles of peptide chemistry, solid-phase synthesis, solution-phase synthesis, protective group strategies, coupling, purification, analytical characterization, production processes, and examples derived from published studies. It is not a laboratory protocol, a manufacturing instruction, medical advice, a guide to dosage or administration, or a quality certificate for any particular peptide product. Experimental results should be interpreted in the context of the sequence, the methods used, the scale of the process, the analytical techniques, and the limitations of individual studies.

References

1. Stráner, P., Taricska, N., Szabó, M., Tóth, G. K., & Perczel, A. (2016). Bacterial expression and/or solid phase peptide synthesis of 20-40 amino acid long polypeptides and miniproteins, the case study of Class B GPCR ligands. Current Protein & Peptide Science, 17(2), 147–155. https://doi.org/10.2174/1389203716666151102105215
2. Neumann, K., Farnung, J., Baldauf, S., & Bode, J. W. (2020). Prevention of aspartimide formation during peptide synthesis using cyanosulfurylides as carboxylic acid-protecting groups. Nature Communications, 11(1), Article 982. https://doi.org/10.1038/s41467-020-14755-6
3. Ferrer, M., Woodward, C., & Barany, G. (1992). Solid-phase synthesis of bovine pancreatic trypsin inhibitor (BPTI) and two analogues. A chemical approach for evaluating the role of disulfide bridges in protein folding and stability. International Journal of Peptide and Protein Research, 40(3–4), 194–207. PubMed record
4. Sletten, E. T., Nuño, M., Guthrie, D., & Seeberger, P. H. (2019). Real-time monitoring of solid-phase peptide synthesis using a variable bed flow reactor. Chemical Communications, 55(97), 14598–14601. https://doi.org/10.1039/c9cc08421e
5. Bello, C., Kikul, F., & Becker, C. F. W. (2015). Efficient generation of peptide hydrazides via direct hydrazinolysis of Peptidyl-Wang-TentaGel resins. Journal of Peptide Science, 21(3), 201–207. https://doi.org/10.1002/psc.2747
6. Simon, M. D., Heider, P. L., Adamo, A., Vinogradov, A. A., Mong, S. K., Li, X., Berger, T., Policarpo, R. L., Zhang, C., Zou, Y., Liao, X., Spokoyny, A. M., Jensen, K. F., & Pentelute, B. L. (2014). Rapid flow-based peptide synthesis. ChemBioChem, 15(5), 713–720. https://doi.org/10.1002/cbic.201300796
7. Mijalis, A. J., Thomas, D. A., III, Simon, M. D., Adamo, A., Beaumont, R., Jensen, K. F., & Pentelute, B. L. (2017). A fully automated flow-based approach for accelerated peptide synthesis. Nature Chemical Biology, 13(5), 464–466. https://doi.org/10.1038/nchembio.2318
8. Sharma, A., Kumar, A., de la Torre, B. G., & Albericio, F. (2022). Liquid-phase peptide synthesis (LPPS): A third wave for the preparation of peptides. Chemical Reviews, 122(16), 13516–13546. https://doi.org/10.1021/acs.chemrev.2c00132
9. Takahashi, D., Yano, T., & Fukui, T. (2012). Novel diphenylmethyl-derived amide protecting group for efficient liquid-phase peptide synthesis: AJIPHASE. Organic Letters, 14(17), 4514–4517. https://doi.org/10.1021/ol302002g
10. Dawson, P. E., Muir, T. W., Clark-Lewis, I., & Kent, S. B. (1994). Synthesis of proteins by native chemical ligation. Science, 266(5186), 776–779. https://doi.org/10.1126/science.7973629
11. Palasek, S. A., Cox, Z. J., & Collins, J. M. (2007). Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis. Journal of Peptide Science, 13(3), 143–148. https://doi.org/10.1002/psc.804
12. Mathews, W. R., Runge, T. A., Haroldsen, P. E., & Gaskell, S. J. (1989). Characterization of impurities in a synthetic renin substrate peptide by fast-atom bombardment mass spectrometry and hybrid tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 3(9), 314–319. https://doi.org/10.1002/rcm.1290030912
13. Goodlett, D. R., Abuaf, P. A., Savage, P. A., Kowalski, K. A., Mukherjee, T. K., Tolan, J. W., Corkum, N., Goldstein, G., & Crowther, J. B. (1995). Determination of peptide chiral purity: Hydrolysis in deuterated acid, derivatization with Marfey’s reagent and analysis using high-performance liquid chromatography-electrospray ionization-mass spectrometry. Journal of Chromatography A, 707(2), 233–244. https://doi.org/10.1016/0021-9673(95)00352-n
14. Kekessie, I., Wegner, K., Martinez, I., Kopach, M. E., White, T. D., Tom, J. K., Kenworthy, M. N., Gallou, F., Lopez, J., Koenig, S. G., Payne, P. R., Eissler, S., Arumugam, B., Li, C., Mukherjee, S., Isidro-Llobet, A., Ludemann-Hombourger, O., Richardson, P., Kittelmann, J., … van den Bos, L. J. (2024). Process mass intensity (PMI): A holistic analysis of current peptide manufacturing processes informs sustainability in peptide synthesis. The Journal of Organic Chemistry, 89(7), 4261–4282. https://doi.org/10.1021/acs.joc.3c01494
15. Isidro-Llobet, A., Kenworthy, M. N., Mukherjee, S., Kopach, M. E., Wegner, K., Gallou, F., Smith, A. G., & Roschangar, F. (2019). Sustainability challenges in peptide synthesis and purification: From R&D to production. The Journal of Organic Chemistry, 84(8), 4615–4628. https://doi.org/10.1021/acs.joc.8b03001