SPPS vs LPPS vs hybrid: how peptide APIs are manufactured, and why the route matters to buyers

Peptide APIs are made by one of three chemical routes: solid-phase synthesis (SPPS), liquid-phase synthesis (LPPS), or a hybrid that combines them. Some are also made by recombinant expression, or by expression followed by chemical modification. For a buyer, the route is not a detail of the supplier’s process. It determines which impurities the API can contain, how far the process can scale, how much solvent it consumes and what the regulatory file has to explain.

This article compares the three chemical routes, sets out where each is used, and explains what to ask a supplier about its route.

The three routes at a glance

	SPPS	LPPS	Hybrid (SPPS + LPPS)
Principle	Chain built on an insoluble resin; excess reagents washed away after each step	Chain built in solution; intermediates isolated	Fragments made by SPPS, then joined in solution
Purification	Usually once, after cleavage from the resin	Intermediates can be purified along the way	Fragments and final peptide purified
Development speed	Fast; readily automated	Slower; step-specific development	Moderate; more steps to develop
Typical sweet spot	Most peptide APIs; practical limit around 100 amino acids	Shorter peptides, about 5–10 amino acids	Longer peptides and large volumes
Material use	High: average PMI about 13,000	Lower per amino acid	Mostly driven by the SPPS steps
Examples	Most synthetic peptide APIs	Short peptides at large scale	Enfuvirtide (early); tirzepatide (recent)

The length ranges and examples are from the 2024 ACS Green Chemistry Institute Pharmaceutical Roundtable analysis by Kekessie and colleagues, which pooled data on 40 peptide processes from 14 member companies.

Solid-phase peptide synthesis (SPPS)

Bruce Merrifield described solid-phase synthesis in 1963 (Merrifield, 1963) and received the 1984 Nobel Prize in Chemistry “for his development of methodology for chemical synthesis on a solid matrix” (NobelPrize.org). The idea is simple: anchor the first amino acid to an insoluble resin, then repeat a cycle of deprotection, washing, coupling and washing until the sequence is complete. Because the peptide stays on the resin, excess reagents can be used to push each coupling to completion and then simply washed away. The finished peptide is cleaved from the resin, side-chain protecting groups are removed, and the crude peptide is purified, usually by preparative reversed-phase chromatography.

Fmoc versus Boc

Two protection strategies dominate. Boc chemistry needs anhydrous hydrogen fluoride for final cleavage; Fmoc chemistry uses a base to remove the temporary protecting group and acid for final cleavage, giving an orthogonal scheme. A 2016 review states that “the majority of synthetic peptides are now prepared by Fmoc” SPPS and that Boc is “now generally only used for specialist applications”, partly because many modifications are not stable to HF cleavage (Behrendt et al., 2016).

Why length is the limiting factor

Every coupling and deprotection must go almost to completion, because small losses compound. If each of 30 cycles achieves 99%, about 74% of chains are complete (0.99³⁰); at 98% per cycle the figure falls to about 55%. The incomplete chains become impurities that are similar to the product and difficult to separate. EMA’s synthetic peptides guideline lists the typical classes:

• Deletion sequences, missing one or more residues after incomplete coupling or deprotection, often limited by repeating couplings and by capping unreacted sites.
• Truncated sequences, the acetylated fragments produced by that capping.
• Insertion sequences, where an amino acid is coupled twice.
• Diastereomers, from epimerisation during activation and coupling.
• Cleavage by-products, where reactive species released from protecting groups modify sensitive residues such as Trp, Tyr and Met unless scavenged.

The guideline also notes tools used to reduce aggregation on the resin, such as pseudoproline dipeptides and Dmb-glycine (EMA, 2025). The Kekessie analysis puts the practical limit for efficient SPPS manufacture at approximately 100 amino acids, and notes that approved synthetic peptides range up to about 60.

Liquid-phase peptide synthesis (LPPS)

LPPS, the classical approach, builds the peptide in solution. Each intermediate is isolated, and can be purified and characterised before the next step, so impurities can be removed early rather than carried to the end. It uses smaller excesses of reagents, and it allows different protecting-group strategies, such as Boc and Cbz, for complex targets. The cost is development time: each step needs its own work-up and isolation conditions.

The Kekessie analysis notes that LPPS “for the manufacture of shorter peptides, containing 5–10 AAs for example, offers some advantages”, and its small LPPS data set showed lower material use per amino acid than SPPS.

Tag-assisted LPPS

Newer LPPS variants attach the growing chain to a soluble tag that makes it easy to precipitate or extract after each step, combining solution-phase chemistry with simple isolation. A 2022 review describes tags based on polyethylene glycol, fluorous groups, ionic liquids, hydrophobic polymers and group-assisted purification (GAP) chemistry (Sharma et al., 2022; An et al., 2015). The Kekessie analysis reports lipophilic-tag LPPS being applied at 15–20 kg scale.

Hybrid processes: fragments plus solution-phase coupling

For longer peptides and large volumes, the sequence can be divided into fragments that are made separately by SPPS and then joined in solution. The Kekessie analysis describes this convergent approach, with fragments produced in parallel, and cites enfuvirtide, a 36-amino-acid peptide, as an early production-scale example and tirzepatide as a recent one. Bray’s 2003 review describes the route selection behind enfuvirtide’s large-scale chemical manufacture (Bray, 2003).

Hybrid routes add steps, and fragment condensation is itself a source of process impurities that EMA expects to be characterised and controlled. EMA also distinguishes between short peptide fragments, which may be accepted as starting materials when justified, and longer or modified fragments, which are treated as intermediates made under GMP.

Recombinant and semi-synthetic routes

Chemical synthesis is not the only option. The US prescribing information for the reference semaglutide product states that its peptide backbone “is produced by yeast fermentation” and then chemically modified (DailyMed). By contrast, a generic liraglutide injection approved in the US states that its liraglutide “is manufactured by chemical synthesis” (DailyMed).

That difference has regulatory consequences. FDA’s July 2026 draft guidance for semaglutide injection applies to products made by “recombinant, synthetic or semi-synthetic processes” and sets impurity comparisons against the reference product (FDA). In the EU, the biosimilar pathway is not available for chemically synthesised peptides, so a synthetic version of a recombinant reference product needs a full analytical comparison, including the peptide-related impurity profile at 0.1–0.5% (EMA, 2025).

Solvents and sustainability

Peptide synthesis uses far more material per kilogram of product than small-molecule manufacture. The ACS Green Chemistry Institute analysis found:

Measure	Value
Average process mass intensity (PMI), SPPS	About 13,000 kg input per kg API
Commercial and Phase 3 SPPS processes	PMI 1,684 to 34,585 (average 13,063) for 6–43-mers
Small molecules, for comparison	Median PMI 168–308
Biopharmaceuticals, for comparison	PMI about 8,300
Largest organic solvent in SPPS	DMF, 39% of solvent use

Source: Kekessie et al., 2024. The ACS Roundtable had earlier identified greener peptide processes as a critical need (Isidro-Llobet et al., 2019).

Regulation is adding pressure. Since 12 December 2023, REACH Annex XVII entry 76 has restricted DMF in the EU: it may only be placed on the market or used at 0.3% or more if worker exposure is kept below derived no-effect levels of 6 mg/m³ by inhalation and 1.1 mg/kg/day by skin contact (Regulation (EU) 2021/2030). The Kekessie analysis also flags safety concerns with common coupling chemistry, including hydrogen cyanide formation from DIC/Oxyma under normal reaction conditions and reports of anaphylaxis linked to some coupling reagents.

How the choice is usually made

Factor	Points towards
Short sequence, very large volume	LPPS or tag-assisted LPPS
Typical therapeutic length (the commercial and Phase 3 SPPS processes in the Kekessie data set ran from 6 to 43 residues)	SPPS
Long sequence or very large volume of a long peptide	Hybrid, or recombinant for natural L-amino-acid sequences
Lipidation, unnatural amino acids, D-amino acids	Chemical synthesis (SPPS or hybrid), possibly with semi-synthetic steps
Generic of a recombinant reference product	Any route, but expect a demanding impurity comparison with the reference product

What to ask a peptide API supplier about its route

1. Which route, and where is it run? SPPS, LPPS, hybrid or recombinant, and the manufacturing site for each stage.
2. What are the registered starting materials? EMA applies ICH Q11 principles; protected L-amino acid derivatives are generally acceptable, longer fragments are usually intermediates.
3. How are route-specific impurities controlled? Ask for the impurity profile above 0.1%, with identified deletion, insertion and diastereomeric impurities.
4. Which counter-ion is delivered? Reversed-phase purification often uses TFA; EMA expects counter-ion and residual TFA to be controlled in the specification.
5. Is the route fixed for your filing? A change of route after registration is a regulated variation or supplement and can require new comparability data.

Our article on peptide API impurities and specifications covers the analytical side in detail, and how to qualify a peptide API supplier covers documents and audits.

Winhycare Pharma

Winhycare Pharma supplies Semaglutide API on a B2B basis and offers peptide CRDMO services, including process and analytical development and technology transfer, from Hyderabad, India. After synthesis and purification, our lyophilisation CMO service can take client-supplied peptide API into vials for research and development applications. For research-grade material, we also offer custom peptide synthesis, for research use only. For the regulatory side of sourcing, see our buyer guides for Europe and the USA.